Alginate as a carrier for mechanically isolated Stromal Vascular Fraction

Alginate as a carrier for mechanically isolated Stromal Vascular Fraction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Alginate as a carrier for mechanically isolated Stromal Vascular Fraction Gregory Reid, Ann-Kathrin Seitz, Mauro Vasella, Luzie Hofmann, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7520241/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Human adipose tissue-derived mechanically isolated stromal vascular fraction (mSVF) is a heterogeneous cell population containing mesenchymal stromal and progenitor cells known to have immense regenerative potential. Current research aims to enhance mSVF delivery by improving cell retention and survival, with hydrogels emerging as promising scaffolds. Among them, alginate stands out due to its biocompatibility, cost-effectiveness, and established use in wound healing and tissue engineering. In this study, mSVF was cultured in varying concentrations of alginate for 21 days and tested for hydrogel degradation, cell viability, as well as protein and growth factor release. Alginate encapsulated mSVF was co-cultured with human dermal fibroblasts and analyzed via immunohistochemical and immunofluorescence imaging. After 21 days, all hydrogel samples of different concentrations maintained the original size and shape. Cell viability and protein release was comparable to the positive control of mSVF only. Fibroblast growth factor (FGF) release, as quantified by growth factor analysis, increased in the co-culture. In addition, the co-culture exhibited increased fibroblast viability as compared with negative controls as well as increased CD31 and CD73 expression. As a long-term proof-of-concept, we demonstrate alginate is an attractive carrier for mSVF. Our findings support its potential for enhancing cell-based therapies in regenerative medicine. Biological sciences/Biotechnology Biological sciences/Cell biology Physical sciences/Materials science Health sciences/Medical research Biological sciences/Stem cells Tissue engineering Wound healing biomaterials Cellular therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Skin defects due to chronic wounds, acute trauma or large area burns can pose significant challenges in clinical practice. Patients experience a reduced quality of life, high morbidity and extended healing times, coupled with a high financial burden. Delayed wound healing is defined by a disruption in the physiological healing process. It is invariably can be caused by various reasons, primarily leading to an altered microenvironment and lack of blood and nutrient supply 1 , 2 . In addition, pre-existing systemic diseases, such as malnutrition, obesity 3 or diabetes 4 , 5 can impede the local wound healing process 6 . To battle these challenges, regenerative medicine and tissue engineering aim to provide novel management options, by using cellular and biomaterial-based therapies. Among regenerative medical approaches, the use of human adipose tissue-derived stromal vascular fraction (SVF) has emerged as a highly promising cell source 7 . SVF is a heterogeneous cell population composed of mesenchymal stromal cells (MSCs), endothelial cells and their precursors (ECs), pericytes (PCs) and immune cells. Common methods for isolating SVF from adipose tissue involve either enzymatic or mechanical digestion 8 . Mechanical digestion of adipose tissue requires multiple passes through a sharp filter, hereby micro fragmenting the tissue. This can be done either using a handheld Luer lock filter 9 or by means of an automated isolation device 10 . Enzymatic digestion on the other hand is performed by sequential digestion of extracellular matrix proteins and subsequent filtration 11 . The main drawback of enzymatic digestion is the extracorporeal enzymatic processing steps, potentially conflicting with regional ethical regulations and requiring good manufacturing practice (GMP) processing. The resulting cellular SVF mixture from both methods exhibits robust regenerative potential, capable of promoting angiogenesis, tissue repair 12 and positive modulation of the inflammatory response 13 . In clinical applications, SVF has been investigated extensively for its efficacy in various tissue engineering contexts 14 , including wound healing 15 , muscle- and bone regeneration 16 , 17 , and extensive scar treatment 18 . The ease of isolation, abundant availability, and multipotent nature of SVF cells make them particularly attractive for therapeutic use 19 . Additionally, the anti-aging effects of SVF on photoaged skin 20 and the comparison of different isolation methods 8 , 21 for SVF have been investigated, underscoring the versatility 22 and therapeutic potential 13 . Current clinical research focuses on enhancing the delivery of mSVF to target tissues, addressing major challenges, such as poor cell retention and cell survival. One such method to overcome this limitation, is to use a hydrogel as a scaffold 23 , 24 . A hydrogel can encapsulate cells and deliver bioactive molecules 25 making them ideal candidates for mSVF transfer and maintenance in wound healing applications. Among proposed hydrogels, alginate has garnered great attention 26 , 27 . It is a naturally occurring polysaccharide derived from brown algae and boasts high biocompatibility and availability at a low cost 28 . Importantly, a number of alginate-based hydrogels are already approved for wound healing purposes 29 . It is capable of forming stable hydrogels under physiological conditions and previous studies have demonstrated the utility of alginate to promote cell survival 30 and facilitate controlled release of growth factors 31 . It maintains tissue integrity in various tissue engineering applications, including 3D bioprinting, drug delivery and other functionalization strategies 28 , 32 – 34 . Therefore, this study aims to evaluate the potential of alginate hydrogels in retaining cell survival and function of mSVF. Various concentrations of alginate (1.5%, 2%, 2.5%, 3%) were examined for hydrogel degradation over 21 days, along with cell viability, total protein release, and the release of the growth factors FGF and Vascular endothelial growth factor (VEGF). Additionally, co-culture experiments with human dermal fibroblasts were performed to assess the impact of mSVF-hydrogel mixtures on fibroblast viability. Results Patient sample characteristics: Lipoaspirate was collected from a total of 11 patients. Mean age was 41 (Range 18–51) and 9 patients (81.8%) were female. Mean weight was 84 ± 14 kg equaling a calculated mean BMI of 29 ± 4.3 kg/m 2 . The patient data is summarized in Table 1 . Table 1 Patient characteristics from tissue samples collected. Patient Gender Age (years) Weight (kg) Height (cm) BMI (kg/m 2 ) 1 f 51 93 166 33.7 2 f 51 85 168 30.1 3 f 47 82 164 30.4 4 m 18 95 190 26.3 5 f 43 67 165 24.6 6 f 19 78 156 32.1 7 f 49 94 183 28.1 8 f 46 60 170 20.8 9 f 32 86 170 29.8 10 f 45 75 170 26.0 11 m 46 110 175 35.9 Alginate hydrogel culture and degradation: Long term maintenance of hydrogel stability is crucial for clinical applications. mSVF boasts an intrinsic vasculogenic potential, as well as an extrinsic angiogenic potential. In order to capitalize on both these attributes, structural maintenance throughout an extended period is crucial. We therefore investigated varying concentrations of alginate in regard to size reduction over the 21-day culture period. Alginate hydrogels were formed using varying concentrations (1.5%, 2%, 2.5%, 3%) and mean size was estimated on day 21 in relation to day 1 (Fig. 1 ). Alginate 1.5% showed a mean size retention of 86.6 ± 16.1%, alginate 2% of 86.5 ± 16.7%, alginate 2.5% of 85.6 ± 17.1% and alginate 3% of 89.1 ± 10.8% without statistical difference. Representative macroscopic images and exemplary measurement images can be seen in (supplementary files). In between concentrations, there was no statistical difference and the size retained was over 80% of the starting size, indicating such structural stability. mSVF-Hydrogel cellular viability and total protein release In order to determine whether a higher alginate concentration in the hydrogel mixture might inhibit mSVF function, both overall cell viability and total protein release was measured at regular intervals until day 21, taking day 0 as a reference (100%). Cell viability is a crucial indicator for cell survival; protein release an important indicator for cell function. If transferred cells can survive long-term, then this would allow sufficient time for the ingrowth of host vasculature and maintenance of new blood vessels. All alginate concentrations demonstrated a similar viability curve (supplementary files), differing only from the positive control. Exemplary concentration plot of alginate 2% is seen in (Fig. 2 ). After an initial drop below baseline on day 1, cell viability increased over the baseline until day 7. The positive control of only mSVF maintained a lower cell viability until day 7, where it crosses the baseline and maintains a higher cell viability until day 21, where it merges with the alginate test groups, slightly below baseline. No significant differences were seen between any alginate concentrations or the positive control. This suggests that alginate does not impede cell survival but rather provides a cytocompatible cell environment. Furthermore, alginate concentration can therefore be chosen based on suitable mechanical factors for the designated use. Total protein release and growth factor release from incorporated cells is a critical aspect of their therapeutic potential. When analyzing the total protein release, only on day 0 a significant difference between the positive control and all alginate test groups was found ( p < 0.0001) (Fig. 3 ). This difference evened out and stayed without any difference throughout the culture period of 21 days. Similar to the cell viability analysis, a large initial drop off could be seen in the positive control. Relative to day 1, at the end of the culture period, an overall increase in protein concentration found in the well supernatant was found to be over 20%. The total protein present in the culture media of the positive control on the other hand, was found to be unchanged compared to day 1. This could certainly be explained by an increasing degradation of the alginate hydrogel. Any increase in mSVF protein release seems therefore negligible, pointing towards a synergistic effect of the hydrogel on the cells present within. Growth factor release To further determine if the increase in protein released in the cell culture media might correlate with growth factor release, determining the significance for the regenerative potential, VEGF and FGF were analyzed on day 21 by means of ELISA (Fig. 4 ). Compared to all mSVF-hydrogel concentrations, there was a considerably greater release of VEGF by the positive control of only mSVF ( p < 0.0001). FGF release on the other hand was similar for all groups, showing a trend towards higher expression compared to the positive control, albeit without any statistical significance. VEGF release was established to be significantly lower than the positive control, with an inverse trend in regard to increasing alginate concentration. This suggests that while alginate can support certain aspects of SVF function, the release of specific growth factors may vary depending on the cell-carrier interaction. Co-culture with human dermal fibroblasts Dermal fibroblasts play a significant role in wound healing and scar formation, leading the dermal closure from the edges. The complex interaction and effect of SVF on fibroblast migration and viability has been previously described 35 . We therefore chose to co-culture Human dermal fibroblasts were with varying concentrations of alginate mSVF-hydrogel and mSVF as a positive control in order to determine if the positive effect of mSVF can be retained in the alginate hydrogel. After 5 days, there was a significant increase in cell viability of present cells in all groups including mSVF, compared to the negative fibroblast control. The cell viability for is exemplified for 2% mSVF-hydrogel in Fig. 5 . This trend continued and after 7 days all alginate groups demonstrated a higher effect on cell-viability compared to the negative fibroblast control (supplementary files). Immunofluorescent, immunohistochemical and histological analysis To further determine ultrastructural differences and growth patterns of mSVF in alginate hydrogel, H&E and Masson's trichrome staining was performed. After 21 days, the alginate architecture was maintained. A qualitative increase in cell numbers, cell clustering and extracellular matrix deposition was observed compared to day 7 (Fig. 6 a, b). Trichrome staining visualized an increase in collagen fiber presence (supplementary files). Relative differences in alginate concentration demonstrated equal overall hydrogel thickness, cell distribution, cell clustering and adipocyte presence. In comparison to 1.5% w/v alginate, the denser hydrogel was clearly visible in the 3% w/v alginate (supplementary files). The expression of perilipin2, CD31 and CD73 was further analyzed by means of IHC staining. Perilipin2 antibody expression, a marker for adipocytes, remained constant after 21 days of culture. The total area stained was found to be 0.37 ± 0.2% on day 7 and 0.37 ± 0.29% on day 21 (Fig. 7 a). CD31, an endothelial cell marker was found to be present in twice the stained area on day 21, compared to day 7 (Day 7: 0.09 ± 0.9; Day 21: 0.2 ± 0.12; p = 0.0002, Fig. 7 b). CD73, a marker for endothelial cells and MSCs also increased significantly after 21 days of culture (Day 7: 0.84 ± 0.56; Day 21: 1.7 ± 0.89; p = 0.001, (Fig. 7 c). The Immunofluorescent staining for cell nuclei (DAPI) and cytoskeleton (phalloidin) was performed. Image analysis of the alginate hydrogels demonstrated viable cell nuclei with more pronounced actin filaments after 21 days. Cell spreading and elongation as sign of tissue maturation was observed (Fig. 8 a, b). Discussion The extrinsic potential of SVF and adipose derived mesenchymal stromal cells in vitro and in vivo has been extensively described in the past. For instance, Hongsen Bi et al. 5 recently showed that SVF and human adipose-derived stem cells (hADSCs) significantly improved wound healing in hyperglycemic mice by promoting angiogenesis and matrix remodeling. Other studies have examined the protective effects of SVF, such as to mitigate hypoxic damage 36 and the combination of SVF with hyaluronic acid and gelatin calcium phosphate for bone regeneration 17 . Beccia et al. 15 demonstrated the formation of cell clusters and vessel structures using SVF and Integra®, a commonly used dermal substitute in full thickness skin defects. Certainly, a benefit of using mSVF is the speed and also possible navigation of regulatory issues 37 . Enzymatic dissociation of fat tissue is yet to be cleared by western national drug monitoring agencies for human use, as the long-term effects of non-autologous collagenase are not yet known. The process takes multiple hours before SVF cells can be isolated, posing another challenge. When comparing both isolation methods, enzymatically dissociated SVF (eSVF) has been found to deliver a higher cell yield, whereas mSVF shows greater wound healing properties 8 . This is thought to be attributed to the presence of ECM and other cellular proteins still present. Previous studies have investigated the positive effect of the acellular components of fat tissue 38 , further underlining this assumption. In this study, the potential of alginate hydrogel as a carrier for mSVF from human adipose tissue was investigated. The long-term compatibility of mSVF-alginate complex is crucial when conceptualizing possible treatments of cutaneous defects 39 . Taking chronic diabetic ulcers as an example, traditional methods of cell delivery into wound sites, such as injection or spraying 40 , might lead to fast material dislocation, lower survival rate and overall reduction of reparative functions 41 . To overcome the limitation of the semi-liquid nature of mSVF, maintaining a more stable shape without excessive deformation of the carrier is paramount. Various methods of cellular transfer have been proposed in the past, ranging from synthetic polymers and natural hydrogels to non-hydrogel-based carriers such as microspheres, decellularized extracellular human or animal matrices. Hydrogels offer an easy and quick encapsulation of cells, rapid application, and good on-site molding to three-dimensional tissue defects. Furthermore, an ideal hydrogel would allow cellular remodeling and slow integration into the host tissue and degradation over time 26 . Synthetic polymers, such as poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG)-based scaffolds, offer tunable mechanical properties and controlled degradation rates but often lack the tested bioactivity necessary for optimal cell survival and integration. Naturally occurring hydrogels, including collagen, fibrin, and gelatin-based matrices have been extensively described in literature, lack however easy handling and are inherently expensive. Alginate further provides a low-cost, easy to handle and also sustainable 42 alternative. In our study, alginate maintained its shape and diameter throughout the 21-day culture period. This would mean that transplanted cells suspended in the hydrogel can kept in the designated place. Extended structural integrity may on the other hand also impede cell viability and interaction. We therefore investigated whether cell viability differed according to alginate concentration in the hydrogel and also throughout the culture period, using mSVF as a positive control. The cell viability assays revealed that mSVF encapsulated in alginate hydrogels exhibited a similar viability curve to the positive control group of mSVF alone. This suggests that alginate does not impede cell survival but rather provides a cytocompatible cell environment. These findings are in line with previous studies, showing that alginate can support the growth and survival of various cell types, including human dermal fibroblasts and MSCs 43 , 44 . The standard laboratory culture conditions are however not typically representative of in vivo circumstances. In vivo, the hydrogel might be prone to desiccation, heat fluctuations, altered degradation by a competent immune system and a surely more complex cellular cross-talk. The maintenance of in vitro cell viability levels near baseline are however a promising reference. In comparison to the positive cell control of only mSVF, mSVF-hydrogels demonstrated an increase of cell viability to nearly 110% at day 3, followed by a decrease again to around 100% at day 21. This trend was mirrored in all hydrogel concentrations (supplementary files). This might be explained by the initial response of certain cell types to the favourable alginate microenvironment. The cellular encapsulation might also restrict certain inhibitory cross-talk of from apoptotic or dead cells following cell harvest and transfer. After an initial cell proliferation stage, hydrogel remodeling might allow for a certain migration throughout and even outside the alginate gel. Representative histological analysis (Fig. 6 ; supplementary files) would give the impression of a cellular distribution throughout the hydrogel. mSVF controls on the other hand showed an opposite curve, with an initial drop to around 95% viability at day 3, followed by an increase above 100% at day 7, finishing with a final viability just below 100%. A possible explanation here may be the initial cell death of susceptible cell populations after the cell harvest, that directly inhibit neighboring cell populations. These cells would wash out at media changes, allowing for an increase in cell proliferation of other more robust cellular subgroups. Total protein release into the surrounding environment acts an indicator for reactivity of the mSVF-hydrogel. The higher initial protein levels in the mSVF group might result from cell stress, apoptosis, or rapid secretion upon seeding, aligning with documented early drop in cell viability. In contrast, the alginate hydrogel appears to modulate this response, possibly mechanically by encapsulating the cells. This prevents a sharp decline in viability and enables sustained protein release. This controlled release, is influenced certainly by hydrogel degradation. When analyzing two important growth factors for tissue repair, VEGF and FGF, all mSVF-hydrogel groups fell behind the positive control at 21days for VEGF release. A non-significant difference between all groups for the FGF release profile was demonstrated. It is surely a timepoint analysis, not reflective of the whole culture period. A possible explanation might be, that certain cellular subgroups responsible for VEGF release are not stimulated to do so. Encapsulation and lack of stimulatory cellular cross-talk, or even a more trivial reason such as a delayed release of the larger protein VEGF. To determine a potential effect of mSVF-hydrogel complexes on cells that are crucial for wound repair, a co-culture with dermal fibroblasts assay performed. After 7 days, all alginate groups and the mSVF control significantly increased overall cell viability compared to the negative control. This enhancement in co-culture conditions indicates that alginate hydrogels allow for sufficient cellular paracrine cross-talk. This indicates that the stimulatory effect of SVF is maintained, underlining its relevance for wound healing applications. This finding is in line with previous studies on alginate for protein encapsulation and tissue engineering. For example, Moay et al. 45 demonstrated the use of keratin-alginate composite sponges for supporting human dermal fibroblasts, while Haq et al. 1 reported improved stem cell survival in alginate hydrogels pre-treated with caffeic acid. Immunohistochemical and immunofluorescent analyses provided deeper insights into the cellular maturation and activity within the alginate hydrogels. The expression of CD31 and CD73, markers for endothelial cells and mesenchymal stromal cells, respectively, increased significantly over the 21-day culture period. This suggests that alginate hydrogels support not only the survival but also the functional differentiation of SVF cells. The presence of perilipin2, a critical regulator of lipid droplet dynamics, lipid metabolism and lipolysis remained constant, indicating that the health of SVF cells is maintained within the alginate hydrogels. Additionally, the formation of actin filaments and cell elongation observed through immunofluorescent staining indicated ongoing tissue maturation and cellular activity within the hydrogels. In contrast to eSVF, mSVF is composed of cell ECM proteins that interact with alginate and increase the functional integration of the mSVF-hydrogel complex into the surrounding tissue. The use of alginate as a scaffold material offers several advantages in regenerative medicine. Its biocompatibility and ability to form hydrogels with tunable mechanical properties make it a versatile candidate for various tissue engineering applications. Components can be readily added, tuning desired properties such as anti-microbial or for drug delivery 27 , 46 . Moreover, the development of composite alginate hydrogels, such as a recently proposed fibrinogen–nisin–alginate composite gel 47 further expands the wide range of potential applications of alginate in fine tuning the application design, specifically for patient-specific tissue regeneration. Wang et al. 48 explored the incorporation of magnesium-containing poly(lactic-co-glycolic acid) microspheres into alginate hydrogels, which enhanced their mechanical strength and supported better cell proliferation and differentiation. Further research is certainly warranted to find the ideal mechanical properties of alginate hydrogels to either mimic the native tissue environment or increase the regenerative potential of mSVF. Since the dawn of 3D bioprinting, alginate was proven to be a versatile, widely biocompatible and commonly used ink. This approach presents another exciting avenue for future research and could enable the precise fabrication of complex or tissue structures 34 , 46 . Furthermore, studies have shown that the addition of bioactive molecules or modifications to the alginate structure can enhance cell behavior and function. For example, the integration of nanocellulose and anti-inflammatory extracts in alginate-based dressings has been shown to promote cell adhesion and reduce inflammation, thereby supporting tissue regeneration 33 . In comparison, we previously investigated the use of methacrylate gelatin (GelMa) as a possible hydrogel carrier for mSVF 49 . Whereas GelMA requires a short ultraviolet radiation time during photocross-linking, alginate does not. The associated loss of cell viability can therefore be avoided by means of ionic cross-linking with divalent ion such as calcium solution. While the co-culture analysis with mSVF–GelMa and a fibroblast monolayer did maintain a certain degree of total cell viability in starvation medium compared with mSVF-free controls, mSVF-alginate could demonstrate a significantly greater increase in cell viability than the control groups, albeit in normal medium (Fig. 5 ). Therefore, while both mSVF-containing hydrogels investigated by our group support cell viability, alginate would certainly be superior in terms of production and costs. To further investigate the clinical translatability of alginate-based SVF therapies, in vivo studies are required to confirm their safety and efficacy. Firstly, investigating the optimal alginate concentration and formulation for maximizing cell viability, growth factor release and therapeutic efficacy in vivo is essential. Exogenous factors such as tissue desiccation or dislocation, parallel wound healing, complex intercellular communication and immune cell response are only a few of such processes. A higher concentration of alginate or greater average molecular weight could certainly be postulated to have an inhibitory effect 50 . Moreover, integrating and correlating advanced imaging techniques and biomolecular assays could provide deeper insights into the mechanisms underlying mSVF-alginate interactions and tissue integration dynamics. The presence and accessibility of matrix proteins, as well as the perifocal stimulatory effect are further areas worth investigating. Techniques such as live-cell imaging and transcriptomic analysis could elucidate how alginate influences mSVF behavior and phenotype change over time. Conclusion This in vitro study demonstrates that the combination of mSVF with alginate hydrogels leverages the advantages of both components to create a potent therapeutic tool. The results suggest that alginate hydrogels can effectively maintain cell viability, protein, and growth factor release without compromising cell function, while positively influencing adjacent cell viability. These findings warrant further investigation of alginate hydrogels as a delivery system for SVF, providing a readily available, cost-effective, and biocompatible treatment option. Methods Sample Collection Lipoaspirates from subcutaneous fat depots and skin samples from discarded healthy tissue were harvested and directly transferred to the laboratory for further processing. All patients were older than 18 years of age with no serious comorbidities, and all surgeries were elective. Proper surgical technique was performed according to international standard operating procedures (SOP). All reagents and laboratory materials were purchased from Sigma-Aldrich (Merck Millipore, Burlington, MA, USA) unless otherwise stated. Isolation of mSVF Mechanical SVF isolation was executed immediately after harvest, according to a previously established protocol 51 . In brief, lipoaspirates were emulsified using a 1.4µm Luer lock connector (Tulip Aesthetics®, San Diego, CA, USA) 52 and centrifuged at 500 relative centrifugal force (RCF) for 10 min. The oily and watery fractions were removed by aspiration and the central fraction of purified fat resuspended in culture media. A subsequent second centrifugation at 500 RCF for 5 min was performed. After removal by aspiration of the upper and lower layers, the resulting mSVF was immediately used for further experiments. Alginate hydrogel production Alginate in powder form was dissolved in Dulbecco's phosphate-buffered saline (DPBS) overnight at 37°C in a shaking water bath set to 150 rpm, to make 3, 4, 5, or 6% weight/volume (w/v). The resulting alginate mixture was combined at a volume ratio of 1:1 with mSVF to create the final concentrations of 1.5, 2, 2.5, or 3% w/v, respectively, and seeded directly into the designated 24-well plates. At ambient room temperature of 21°C, 1 ml of 50 mM CaCl 2 was added to the well for 15 min and the gels were then rinsed three times with 500 µl Hanks' Balanced Salt Solution (HBSS) for 3 min. Standard culture medium consisting of high glucose Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum and 1% penicillin-streptomycin was used for subsequent culture. A minimum of 9 hydrogels were made from 3 separate donors per experiment and used in a subsequent fashion. Dermal Fibroblast Isolation and Co-Culture Assay Human dermal fibroblasts were isolated from patient skin samples according to established protocols 53 , 54 . In brief, fibroblast isolation was executed immediately after intraoperative harvest of skin samples. By means of sharp dissection, both the epidermis and the subcutis was removed and the dermis minced. It was followed by digestion with 1,5% bovine serum albumin (BSA) and 0,2% collagenase in collagenase buffer (1 mM CaCl 2 , 5 mM Glucose, 50 mM KCl, 100 mM HEPES and 120 mM NaCl) for 45 minutes. Extracellular remnants were removed by filtering the digest through a 100 µm and a 70µm nylon filter (Thermoscientific, Waltham, MA, USA). The cells were subsequently washed and expanded in standard cell culture medium, then used between passages 3–5. Co-culture assay was performed in a 12-well plate by seeding 20,000 cells/cm 2 (70,000 fibroblasts per well). After 2h at standard incubator conditions (37°C, 5% CO 2 , 95% humidity), alginate gels were added, separated from fibroblasts through a 0.4µm well plate insert (Thermoscientific Nunc Cell Culture Insert System). An empty well insert was used as a negative fibroblast control. Alginate hydrogel dimensional degradation Directly after seeding and after 21 days culture, culture media was aspirated and the wells rinsed with 37°C phosphate-buffered saline (PBS). Photos of each gel were acquired alongside a calibration tab and the total surface area was determined using Imitocam AI software (Imito AG, Zürich, ZH, Switzerland). Measurements were provided in cm 2 . Viability Assay Cell viability within mSVF-hydrogel mixtures was measured at regular intervals on day 0, 1, 3, 7, 14 until day 21 using AlamarBlue® assay (Thermoscientific) according to the manufacturer’s instructions. At each interval, well supernatants were collected and transferred to a 96-well plate, maintaining cell culture conditions. Absorbance was measured in triplicate using a microplate reader (Cytation 5, BioTek Instruments, Winooski, VT, USA). Normalization of absorbance was achieved by using supernatant of alginate hydrogel without cells as a negative control, cell culture medium as a blank and mSVF in suspension without hydrogel served as a positive control (PC) 55 . Total protein Assay Total protein concentration present in well supernatant from mSVF-hydrogel samples was measured at day 1, 7 and 21 using Pierce™ BCA Protein Assay Kits according to the manufacturer’s instructions (Thermoscientific). At each time interval, well supernatant was diluted 1:10 and transferred to a 96-well plate. After adding reagent and 30 min of incubation, absorbance was measured in triplicate using a microplate reader, mentioned above. Normalization of absorbance was achieved by means of a manufacturer’s standard curve. Furthermore, mSVF in suspension served as a positive control and culture medium as a blank. ELISA Culture medium was collected from all samples at day 0, 7 and 21. The concentration of FGF and VEGF in the culture medium was quantified by ELISA using a commercial ABTS ELISA development kit and ABTS ELISA buffer kit (PeproTech, Cranbury, NJ, USA) according to the manufacturer’s instructions. Absorbance was measured using the microplate reader mentioned above. Immunohistochemistry (IHC) and Immunofluorescence (IF) Tissue samples from all sample constructs were collected on days 7 and 21, fixed in paraformaldehyde for 24h at 4°C and rinsed in. Staining was performed by an automated IHC system, Autostainer Link48 and PT Link device (Agilent Dako, Santa Clara, CA, USA) on previously paraffin-embedded, 5 µm tissue slices. The following antigens were targeted in addition to staining of hematoxylin-eosin (H&E) and Masson’s trichrome staining: Perilipin-2 (Novus Biologicals, Centennial, CO, USA), CD73 and CD31 (Abcam, Cambridge, Cambridgeshire, United Kingdom). Using FIJI software 56 to determine the antigen area stained, firstly the total area of alginate was designated as a region of interest (ROI). From this area, the area stained was measured using a threshold method set to 200 hue, 80 saturation and 50 brightness and the relative area was calculated. Staining of cell nuclei and actin filaments was performed by IF using DAPI and phalloidin (Cat.-Nr. ab176753, Abcam) which was diluted in PBS in a ratio of 1:1000. Using a cryostat (Leica Biosystems, Wetzlar, Hessen, Germany), previously fixed samples were cut at 10µm sections and left to dry. Slides were incubated with staining solution at room temperature for 90 min protected from light. Gels were then washed 3 times with PBS for 5 min at a time. Microscopy was performed using the Slidescanner Zeiss Axio Scan.Z1 (Carl Zeiss, Jena, Thuringia, Germany). Statistical Analysis All values are presented as means with standard error of mean (SEM) or standard deviation (SD). Normal distribution was tested by a Shapiro–Wilk test followed by an unpaired t-test or one-way ANOVA with GraphPad Prism V10.0 (GraphPad Software, San Diego, CA, USA). p values < 0.05 were accepted as statistically significant: * = p < 0.05; ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Declarations Credit authorship contribution statement GR: Writing–Original draft, Methodology, Investigation, Data curation, Conceptualization. AKS: Data curation, Validation, Software, Investigation. MV: Investigation, Visualization, Data curation, Writing–review & editing. LH: Data curation, Writing–Review & editing. DD: Data curation, Conceptualization, Methodology. JAW: Investigation, Writing–Review & editing. GR6: Resources, Writing–Review & editing, Visualization. MT: Resources, Methodology, Writing–Review & editing, Project administration. BSK: Conceptualization, Supervision, Resources, Methodology, Writing–Review & editing, Project administration. All authors have read and approved the final manuscript draft. Data availability The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Ethics declaration This study was conducted in compliance with the Declaration of Helsinki. All patients signed a general informed consent of the Zurich University Hospital prior to operation and tissue harvest. Dermal samples were fully anonymized after harvest. The collection and usage of human samples was approved by the regional ethics committee of the Canton of Zurich, Switzerland (Date: 02.04.2019, BASEC 2019-00389). The study protocol adhered to the guidelines established by the journal. All material was handled according to biosecurity guidelines established by the competent ethics committee. Competing interests The authors declare no competing interests. Funding statement This project did not receive specific funding. It was performed as part of the employment at the University hospital of Zürich, Rämistrasse 100, 8091 Zürich, Switzerland. References Shifa ul Haq, H. M. et al. Priming with caffeic acid enhances the potential and survival ability of human adipose-derived stem cells to counteract hypoxia. Regen Ther 22 , 115–127 (2023). Broughton, G., Janis, J. E. & Attinger, C. E. Wound healing: An overview. Plastic and Reconstructive Surgery 117-1e-S-32e-S Preprint at https://doi.org/10.1097/01.prs.0000222562.60260.f9 (2006). Bender, R. et al. Human Adipose Derived Cells in Two- And Three-Dimensional Cultures: Functional Validation of an in Vitro Fat Construct. Stem Cells Int 2020 , (2020). Nilforoushzadeh, M. A. et al. Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin–collagen hydrogel: A clinical study for diabetic wound healing. J Tissue Eng Regen Med 14 , 424–440 (2020). Bi, H. et al. Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process. Stem Cell Res Ther 10 , (2019). Deptuła, M., Brzezicka, A., Skoniecka, A., Zieliński, J. & Pikuła, M. Adipose-derived stromal cells for nonhealing wounds: Emerging opportunities and challenges. Medicinal Research Reviews vol. 41 2130–2171 Preprint at https://doi.org/10.1002/med.21789 (2021). Busato, A. et al. Simple and rapid non-enzymatic procedure allows the isolation of structurally preserved connective tissue micro-fragments enriched with svf. Cells 10 , 1–14 (2021). Tiryaki, K. T., Cohen, S., Kocak, P., Canikyan Turkay, S. & Hewett, S. In-vitro comparative examination of the effect of stromal vascular fraction isolated by mechanical and enzymatic methods on wound healing. Aesthet Surg J 40 , 1232–1240 (2020). Kim, B. S. et al. In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt. Pharmaceutics 14 , (2022). Saxer, F. et al. Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First-in-Man Study. Stem Cells 34 , 2956–2966 (2016). Mytsyk, M. et al. Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function. Biotechnol Bioeng 116 , 132–142 (2019). Balko, S., Kerr, E., Buchel, E., Logsetti, S. & Raouf, A. Paracrine signalling between keratinocytes and SVF cells results in a new secreted cytokine profile during wound closure. Stem Cell Res Ther 14 , (2023). van Boxtel, J., Vonk, L. A., Stevens, H. P. & van Dongen, J. A. Mechanically Derived Tissue Stromal Vascular Fraction Acts Anti-inflammatory on TNF Alpha-Stimulated Chondrocytes In Vitro. Bioengineering 9 , (2022). Freitas-Ribeiro, S. et al. Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue. Mar Drugs 20 , (2022). Beccia, E. et al. Adipose Stem Cells and Platelet-Rich Plasma Induce Vascular-Like Structures in a Dermal Regeneration Template. Tissue Eng Part A 27 , 631–641 (2021). Oskarsdotter, K. et al. Autologous endothelialisation by the stromal vascular fraction on laminin-bioconjugated nanocellulose-alginate scaffolds. Biomedical Materials (Bristol) 18 , (2023). Park, S. su, Park, M. & Lee, B. T. Autologous stromal vascular fraction-loaded hyaluronic acid/gelatin-biphasic calcium phosphate scaffold for bone tissue regeneration. Biomaterials Advances 132 , (2022). Velier, M. et al. Paracrine Effects of Adipose-Derived Cellular Therapies in an in Vitro Fibrogenesis Model of Human Vocal Fold Scarring. Journal of Voice (2022) doi:10.1016/j.jvoice.2022.05.012. Malekzadeh, H., Tirmizi, Z., Arellano, J. A., Egro, F. M. & Ejaz, A. Application of Adipose-Tissue Derived Products for Burn Wound Healing. Pharmaceuticals vol. 16 Preprint at https://doi.org/10.3390/ph16091302 (2023). Wang, J. et al. Anti-Aging Effect of the Stromal Vascular Fraction/Adipose-Derived Stem Cells in a Mouse Model of Skin Aging Induced by UVB Irradiation. Front Surg 9 , (2022). Gentile, P. et al. Impact of the different preparation methods to obtain human adipose-derived stromal vascular fraction cells (AD-SVFs) and human adipose-derived mesenchymal stem cells (AD-MSCs): Enzymatic digestion versus mechanical centrifugation. International Journal of Molecular Sciences vol. 20 Preprint at https://doi.org/10.3390/ijms20215471 (2019). Guillaume, O. et al. Stromal vascular fraction cells as biologic coating of mesh for hernia repair. Hernia 24 , 1233–1243 (2020). Cao, H., Duan, L., Zhang, Y., Cao, J. & Zhang, K. Current hydrogel advances in physicochemical and biological response-driven biomedical application diversity. Signal Transduction and Targeted Therapy vol. 6 Preprint at https://doi.org/10.1038/s41392-021-00830-x (2021). Catoira, M. C., Fusaro, L., Di Francesco, D., Ramella, M. & Boccafoschi, F. Overview of natural hydrogels for regenerative medicine applications. J Mater Sci Mater Med 30 , (2019). Shafei, S. et al. Exosome loaded alginate hydrogel promotes tissue regeneration in full-thickness skin wounds: An in vivo study. J Biomed Mater Res A 108 , 545–556 (2020). Farshidfar, N., Iravani, S. & Varma, R. S. Alginate-Based Biomaterials in Tissue Engineering and Regenerative Medicine. Marine Drugs vol. 21 Preprint at https://doi.org/10.3390/md21030189 (2023). Abourehab, M. A. S. et al. Alginate as a Promising Biopolymer in Drug Delivery and Wound Healing: A Review of the State-of-the-Art. International Journal of Molecular Sciences vol. 23 Preprint at https://doi.org/10.3390/ijms23169035 (2022). Wang, J. et al. Alginate: Microbial production, functionalization, and biomedical applications. Int J Biol Macromol 242 , (2023). Aderibigbe, B. A. & Buyana, B. Alginate in wound dressings. Pharmaceutics vol. 10 Preprint at https://doi.org/10.3390/pharmaceutics10020042 (2018). Hemadi, M. et al. Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: Stemness gene expression and structural features. Zygote 1–7 (2021) doi:10.1017/S0967199421000551. Kostenko, A., Swioklo, S. & Connon, C. J. Alginate in corneal tissue engineering. Biomedical Materials (Bristol) vol. 17 Preprint at https://doi.org/10.1088/1748-605X/ac4d7b (2022). Porter, G. C. et al. AgNP/Alginate Nanocomposite hydrogel for antimicrobial and antibiofilm applications. Carbohydr Polym 251 , (2021). Gilljam, K. M. et al. Alginate and Nanocellulose Dressings With Extract From Salmon Roe Reduce Inflammation and Accelerate Healing of Porcine Burn Wounds. Journal of Burn Care & Research 44 , 1140–1149 (2023). Rastogi, P. & Kandasubramanian, B. Review of alginate-based hydrogel bioprinting for application in tissue engineering. Biofabrication 11 , (2019). Bi, H. et al. Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process. Stem Cell Res Ther 10 , (2019). Mytsyk, M. et al. Long-term severe in vitro hypoxia exposure enhances the vascularization potential of human adipose tissue-derived stromal vascular fraction cell engineered tissues. Int J Mol Sci 22 , (2021). Bora, P. & Majumdar, A. S. Adipose tissue-derived stromal vascular fraction in regenerative medicine: A brief review on biology and translation. Stem Cell Research and Therapy vol. 8 Preprint at https://doi.org/10.1186/s13287-017-0598-y (2017). Jiang, X. et al. Decellularized adipose tissue: A key factor in promoting fat regeneration by recruiting and inducing mesenchymal stem cells. Biochem Biophys Res Commun 541 , 63–69 (2021). Qin, J., Chen, F., Wu, P. & Sun, G. Recent Advances in Bioengineered Scaffolds for Cutaneous Wound Healing. Frontiers in Bioengineering and Biotechnology vol. 10 Preprint at https://doi.org/10.3389/fbioe.2022.841583 (2022). Zimmerlin, L. et al. Human adipose stromal vascular cell delivery in a fibrin spray. Cytotherapy 15 , 102–108 (2013). Ding, Y., Wang, Y. & Hu, Q. Recent advances in overcoming barriers to cell-based delivery systems for cancer immunotherapy. Exploration vol. 2 Preprint at https://doi.org/10.1002/EXP.20210106 (2022). Wawszczak, A., Kocki, J. & Kołodyńska, D. Alginate as a Sustainable and Biodegradable Material for Medical and Environmental Applications—The Case Studies. Journal of Biomedical Materials Research - Part B Applied Biomaterials vol. 112 1–23 Preprint at https://doi.org/10.1002/jbm.b.35475 (2024). Jahandideh, A. et al. Alginate scaffolds improve functional recovery after spinal cord injury. European Journal of Trauma and Emergency Surgery vol. 48 1711–1721 Preprint at https://doi.org/10.1007/s00068-021-01760-7 (2022). Shuai, Q. et al. Sodium alginate hydrogel integrated with type III collagen and mesenchymal stem cell to promote endometrium regeneration and fertility restoration. Int J Biol Macromol 253 , (2023). Moay, Z. K. et al. Keratin‐alginate sponges support healing of partial‐thickness burns. Int J Mol Sci 22 , (2021). Cleetus, C. M. et al. Alginate hydrogels with embedded zno nanoparticles for wound healing therapy. Int J Nanomedicine 15 , 5097–5111 (2020). Soleimanpour, M. et al. Designing a new alginate-fibrinogen biomaterial composite hydrogel for wound healing. Sci Rep 12 , (2022). Wang, L. et al. Alginate hydrogels containing different concentrations of magnesium-containing poly(lactic-co-glycolic acid) microspheres for bone tissue engineering. Biomedical Materials (Bristol) 18 , (2023). Vasella, M. et al. Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair. Int J Mol Sci 24 , (2023). Kaijzel, E. L., Koolwijk, P., van Erck, M. G. M., van Hinsbergh, V. W. M. & de Maat, M. P. M. Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo. J Thromb Haemost 4 , 1975–81 (2006). Pallua, N., Grasys, J. & Kim, B. S. Enhancement of progenitor cells by two-step centrifugation of emulsified lipoaspirates. Plast Reconstr Surg 142 , 99–109 (2018). Tonnard, P. et al. Nanofat grafting: Basic research and clinical applications. Plast Reconstr Surg 132 , 1017–1026 (2013). Koch, C. M. et al. Specific age-associated DNA methylation changes in human dermal fibroblasts. PLoS One 6 , (2011). He, Y. et al. Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling. Cells 11 , (2022). Kim, B. S. et al. The Effect of Antiseptics on Adipose-Derived Stem Cells. Plast Reconstr Surg 139 , 625–637 (2017). Schindelin, J. et al. Fiji: An open-source platform for biological-image analysis. Nature Methods vol. 9 676–682 Preprint at https://doi.org/10.1038/nmeth.2019 (2012). Additional Declarations No competing interests reported. Supplementary Files AlginateSupplementaryfiles.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Mar, 2026 Reviews received at journal 17 Mar, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviews received at journal 16 Feb, 2026 Reviewers agreed at journal 13 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor invited by journal 23 Jan, 2026 Editor assigned by journal 29 Sep, 2025 Submission checks completed at journal 23 Sep, 2025 First submitted to journal 23 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7520241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":591021199,"identity":"c69ddd53-8373-46f5-a880-3baf4a2e0063","order_by":0,"name":"Gregory Reid","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Gregory","middleName":"","lastName":"Reid","suffix":""},{"id":591021200,"identity":"e47b623c-890d-4d2d-a779-cd2d027d2a8a","order_by":1,"name":"Ann-Kathrin Seitz","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Ann-Kathrin","middleName":"","lastName":"Seitz","suffix":""},{"id":591021201,"identity":"3fbbfe39-0d1d-4977-ba55-3225954a4307","order_by":2,"name":"Mauro Vasella","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Mauro","middleName":"","lastName":"Vasella","suffix":""},{"id":591021207,"identity":"bdf5b695-c69a-47af-932d-1d6543de3b11","order_by":3,"name":"Luzie Hofmann","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Luzie","middleName":"","lastName":"Hofmann","suffix":""},{"id":591021208,"identity":"14a75317-3c18-4ccd-b8f8-1aa97517f41c","order_by":4,"name":"Dalia Dranseike","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"","lastName":"Dranseike","suffix":""},{"id":591021209,"identity":"283506ed-e7bc-4e5c-b2e4-dc4df18674b9","order_by":5,"name":"Jennifer A. Watson","email":"","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"A.","lastName":"Watson","suffix":""},{"id":591021210,"identity":"f5bb7c89-f686-4ad0-88f5-547f9e8323c0","order_by":6,"name":"Gavin Reid","email":"","orcid":"","institution":"Addenbrooke's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gavin","middleName":"","lastName":"Reid","suffix":""},{"id":591021211,"identity":"7d60fa4d-49e8-4b50-8abf-2fa023208113","order_by":7,"name":"Mark W Tibbitt","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"W","lastName":"Tibbitt","suffix":""},{"id":591021212,"identity":"2a45d0b7-f099-48b0-8081-bfb691cd8e60","order_by":8,"name":"Bong-Sung Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACAwSTjeEAQwWURYKWM6RqYWBsI0KLOXvv4RcMfw7nyTuwJR66Oe+wvXwD87MH+LRY9pxLs2BsO1xseIDtwOHcbYcTGxvYzA3waTG4kWNm/LfhcOLGBvYGkJYEZgYeNglCWgyADoNqmXPYno0ILcYPGNgOJ85nADms4TBjDyEtlj1nzIABlZ64gZkt4XDOsfTEGcxsZni1mLP3GH9g+GOdOL+9zfhzTo21vXx78zO8WoAA4gyDwzA+MwH1ICUfQKR8A2GVo2AUjIJRMEIBADhWR+Ym+f5/AAAAAElFTkSuQmCC","orcid":"","institution":"University Hospital of Zurich","correspondingAuthor":true,"prefix":"","firstName":"Bong-Sung","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-09-02 17:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7520241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7520241/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-50191-0","type":"published","date":"2026-04-25T15:58:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":102962788,"identity":"04449221-9302-4c9d-bd91-f2d5aab926b9","added_by":"auto","created_at":"2026-02-19 04:11:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":783439,"visible":true,"origin":"","legend":"\u003cp\u003eAlginate hydrogel degradation after 21 days in culture, relative to day 0. No significant difference was found in between experimental conditions. Data shown as Mean \u003cu\u003e+\u003c/u\u003e SEM, from a minimum of 5 independent samples per group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/0ceabb0b62f7a92957990216.png"},{"id":102839386,"identity":"661a7356-9d3c-4a52-8b51-89880001c6cd","added_by":"auto","created_at":"2026-02-17 11:45:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":920417,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability analysis for Alginate 2% hydrogels compared to positive control of only mSVF. Baseline was determined as cell viability on day 0. ns = no significant difference determined by means of multiple unpaired student’s T-Tests. All data is showed as mean \u003cu\u003e+\u003c/u\u003e SEM. A minimum of 9 measurements per timepoint from 6 independent experiments were acquired.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/64f7dea6a2dd5566f90d9cdb.png"},{"id":102839388,"identity":"67fc3695-b6c3-40f8-964b-c1f8002092b8","added_by":"auto","created_at":"2026-02-17 11:45:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1077347,"visible":true,"origin":"","legend":"\u003cp\u003eTotal protein concentrations of varying alginate concentrations, 1.5%; 2%; 2.5%; 3% and positive control of only mSVF. **** \u003cem\u003ep\u003c/em\u003e = \u0026lt;0.0001 determined by means of a one-way ANOVA. All data is showed as mean \u003cu\u003e+\u003c/u\u003e SEM. A minimum of 6 measurements per timepoint from 6 independent experiments were acquired.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/c38b0ca79d60023337f4a286.png"},{"id":102839393,"identity":"0a2381ea-eb9c-403e-a73d-02f16a778eba","added_by":"auto","created_at":"2026-02-17 11:45:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":777271,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth factor release for \u003cstrong\u003ea \u003c/strong\u003eVEGF and \u003cstrong\u003eb \u003c/strong\u003eFGF for varying alginate concentrations: 1.5%; 2%; 2.5%; 3% and positive control of only mSVF. **** \u003cem\u003ep\u003c/em\u003e= \u0026lt;0.0001 determined by means of a one-way ANOVA. All data is showed as mean \u003cu\u003e+\u003c/u\u003e SEM. A minimum of 3 measurements in duplicate from 6 independent experiments were acquired.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/f1ca47c812bb34f197e2a1c6.png"},{"id":102839394,"identity":"ce315b17-6138-400b-8e32-296ee0110465","added_by":"auto","created_at":"2026-02-17 11:45:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1347938,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability analysis for alginate 2% hydrogel in co-culture with fibroblast monolayer. Fibroblast monolayer as negative control and positive control of only mSVF. * \u003cem\u003ep\u003c/em\u003e = \u0026lt;0.05 determined by means of a one-way ANOVA. All data is showed as mean \u003cu\u003e+\u003c/u\u003e SD. A minimum of 3 measurements per timepoint from 3 independent experiments were acquired.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/5ad81d6c14ca9664e519fc19.png"},{"id":102839391,"identity":"1b880fd2-fd0e-47ba-ac3c-3a90011ceac0","added_by":"auto","created_at":"2026-02-17 11:45:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30955472,"visible":true,"origin":"","legend":"\u003cp\u003eExemplary \u003cstrong\u003ea \u003c/strong\u003eH\u0026amp;E and \u003cstrong\u003eb \u003c/strong\u003emasson’s trichrome staining on day 21. Scale bar reference at 200 micrometers. Close-up magnification inset in the top right corner.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/3593eaf2da4f96742dfb244b.png"},{"id":102962981,"identity":"d5c1a715-bf7d-4eb9-ab3e-335900ca6f3b","added_by":"auto","created_at":"2026-02-19 04:12:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10949309,"visible":true,"origin":"","legend":"\u003cp\u003eIHC analysis of area stained for \u003cstrong\u003ea\u003c/strong\u003e Perilipin2, \u003cstrong\u003eb\u003c/strong\u003e CD31 and \u003cstrong\u003ec\u003c/strong\u003eCD73. Comparisons were made between day 7 and day 21. Scale reference at 100 micrometers. ** \u003cem\u003ep \u003c/em\u003e= \u0026lt;0.01; *** \u003cem\u003ep\u003c/em\u003e = \u0026lt;0.001 determined by means of an unpaired students t-test. All data is showed as mean \u003cu\u003e+\u003c/u\u003e SD. A minimum of 19 measurements per timepoint from 4 independent experiments were acquired.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/ec412392117e4506371d8fea.png"},{"id":102839392,"identity":"3d986342-11a0-492a-83e5-60028d4d975a","added_by":"auto","created_at":"2026-02-17 11:45:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":13926668,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining of alginate 2% hydrogels for DAPI in blue and Phalloidin in green.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eImage acquired on day 7, \u003cstrong\u003eb\u003c/strong\u003e Image acquired day 21. Scale bar reference at 200 micrometers. White dashed lines border the hydrogel. Exemplary Close-up magnification inset in the top right Corner.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/48d86a8035bcb10e00d310f8.png"},{"id":107927758,"identity":"0cd716fb-f2f6-4861-a9b6-9527a8180849","added_by":"auto","created_at":"2026-04-27 16:03:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":57738136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/769fa484-cddc-4659-8913-880b6c982fe3.pdf"},{"id":102839389,"identity":"511ac6d8-dc14-44a7-87f2-584983c984d4","added_by":"auto","created_at":"2026-02-17 11:45:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6552843,"visible":true,"origin":"","legend":"","description":"","filename":"AlginateSupplementaryfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7520241/v1/62939233b0c846d6e71e550f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alginate as a carrier for mechanically isolated Stromal Vascular Fraction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin defects due to chronic wounds, acute trauma or large area burns can pose significant challenges in clinical practice. Patients experience a reduced quality of life, high morbidity and extended healing times, coupled with a high financial burden. Delayed wound healing is defined by a disruption in the physiological healing process. It is invariably can be caused by various reasons, primarily leading to an altered microenvironment and lack of blood and nutrient supply \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In addition, pre-existing systemic diseases, such as malnutrition, obesity \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e or diabetes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e can impede the local wound healing process \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To battle these challenges, regenerative medicine and tissue engineering aim to provide novel management options, by using cellular and biomaterial-based therapies.\u003c/p\u003e \u003cp\u003eAmong regenerative medical approaches, the use of human adipose tissue-derived stromal vascular fraction (SVF) has emerged as a highly promising cell source \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. SVF is a heterogeneous cell population composed of mesenchymal stromal cells (MSCs), endothelial cells and their precursors (ECs), pericytes (PCs) and immune cells.\u003c/p\u003e \u003cp\u003eCommon methods for isolating SVF from adipose tissue involve either enzymatic or mechanical digestion \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Mechanical digestion of adipose tissue requires multiple passes through a sharp filter, hereby micro fragmenting the tissue. This can be done either using a handheld Luer lock filter \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e or by means of an automated isolation device \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Enzymatic digestion on the other hand is performed by sequential digestion of extracellular matrix proteins and subsequent filtration \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The main drawback of enzymatic digestion is the extracorporeal enzymatic processing steps, potentially conflicting with regional ethical regulations and requiring good manufacturing practice (GMP) processing. The resulting cellular SVF mixture from both methods exhibits robust regenerative potential, capable of promoting angiogenesis, tissue repair \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and positive modulation of the inflammatory response \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In clinical applications, SVF has been investigated extensively for its efficacy in various tissue engineering contexts \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, including wound healing \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, muscle- and bone regeneration \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and extensive scar treatment \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The ease of isolation, abundant availability, and multipotent nature of SVF cells make them particularly attractive for therapeutic use \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, the anti-aging effects of SVF on photoaged skin \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and the comparison of different isolation methods \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e for SVF have been investigated, underscoring the versatility \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and therapeutic potential \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent clinical research focuses on enhancing the delivery of mSVF to target tissues, addressing major challenges, such as poor cell retention and cell survival. One such method to overcome this limitation, is to use a hydrogel as a scaffold \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. A hydrogel can encapsulate cells and deliver bioactive molecules \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e making them ideal candidates for mSVF transfer and maintenance in wound healing applications. Among proposed hydrogels, alginate has garnered great attention \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is a naturally occurring polysaccharide derived from brown algae and boasts high biocompatibility and availability at a low cost \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Importantly, a number of alginate-based hydrogels are already approved for wound healing purposes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It is capable of forming stable hydrogels under physiological conditions and previous studies have demonstrated the utility of alginate to promote cell survival \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and facilitate controlled release of growth factors \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. It maintains tissue integrity in various tissue engineering applications, including 3D bioprinting, drug delivery and other functionalization strategies \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, this study aims to evaluate the potential of alginate hydrogels in retaining cell survival and function of mSVF. Various concentrations of alginate (1.5%, 2%, 2.5%, 3%) were examined for hydrogel degradation over 21 days, along with cell viability, total protein release, and the release of the growth factors FGF and Vascular endothelial growth factor (VEGF). Additionally, co-culture experiments with human dermal fibroblasts were performed to assess the impact of mSVF-hydrogel mixtures on fibroblast viability.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatient sample characteristics:\u003c/h2\u003e\n \u003cp\u003eLipoaspirate was collected from a total of 11 patients. Mean age was 41 (Range 18\u0026ndash;51) and 9 patients (81.8%) were female. Mean weight was 84\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;14 kg equaling a calculated mean BMI of 29\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;4.3 kg/m\u003csup\u003e2\u003c/sup\u003e. The patient data is summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePatient characteristics from tissue samples collected.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHeight (cm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e166\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e33.7\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e85\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e168\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e30.1\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e47\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e82\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e164\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e30.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e95\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e190\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e26.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e43\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e67\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e165\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e24.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e78\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e156\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e32.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e94\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e183\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e170\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e86\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e170\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e29.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e45\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e75\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e170\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e26.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003em\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e110\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e175\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\n\u003c/div\u003e\n\u003ch3\u003eAlginate hydrogel culture and degradation:\u003c/h3\u003e\n\u003cp\u003eLong term maintenance of hydrogel stability is crucial for clinical applications. mSVF boasts an intrinsic vasculogenic potential, as well as an extrinsic angiogenic potential. In order to capitalize on both these attributes, structural maintenance throughout an extended period is crucial. We therefore investigated varying concentrations of alginate in regard to size reduction over the 21-day culture period. Alginate hydrogels were formed using varying concentrations (1.5%, 2%, 2.5%, 3%) and mean size was estimated on day 21 in relation to day 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Alginate 1.5% showed a mean size retention of 86.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;16.1%, alginate 2% of 86.5\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;16.7%, alginate 2.5% of 85.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;17.1% and alginate 3% of 89.1\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;10.8% without statistical difference. Representative macroscopic images and exemplary measurement images can be seen in (supplementary files). In between concentrations, there was no statistical difference and the size retained was over 80% of the starting size, indicating such structural stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003emSVF-Hydrogel cellular viability and total protein release\u003c/h3\u003e\n\u003cp\u003eIn order to determine whether a higher alginate concentration in the hydrogel mixture might inhibit mSVF function, both overall cell viability and total protein release was measured at regular intervals until day 21, taking day 0 as a reference (100%). Cell viability is a crucial indicator for cell survival; protein release an important indicator for cell function. If transferred cells can survive long-term, then this would allow sufficient time for the ingrowth of host vasculature and maintenance of new blood vessels.\u003c/p\u003e \u003cp\u003eAll alginate concentrations demonstrated a similar viability curve (supplementary files), differing only from the positive control. Exemplary concentration plot of alginate 2% is seen in (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After an initial drop below baseline on day 1, cell viability increased over the baseline until day 7. The positive control of only mSVF maintained a lower cell viability until day 7, where it crosses the baseline and maintains a higher cell viability until day 21, where it merges with the alginate test groups, slightly below baseline. No significant differences were seen between any alginate concentrations or the positive control. This suggests that alginate does not impede cell survival but rather provides a cytocompatible cell environment. Furthermore, alginate concentration can therefore be chosen based on suitable mechanical factors for the designated use.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTotal protein release and growth factor release from incorporated cells is a critical aspect of their therapeutic potential. When analyzing the total protein release, only on day 0 a significant difference between the positive control and all alginate test groups was found (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This difference evened out and stayed without any difference throughout the culture period of 21 days. Similar to the cell viability analysis, a large initial drop off could be seen in the positive control. Relative to day 1, at the end of the culture period, an overall increase in protein concentration found in the well supernatant was found to be over 20%. The total protein present in the culture media of the positive control on the other hand, was found to be unchanged compared to day 1. This could certainly be explained by an increasing degradation of the alginate hydrogel. Any increase in mSVF protein release seems therefore negligible, pointing towards a synergistic effect of the hydrogel on the cells present within.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGrowth factor release\u003c/h3\u003e\n\u003cp\u003eTo further determine if the increase in protein released in the cell culture media might correlate with growth factor release, determining the significance for the regenerative potential, VEGF and FGF were analyzed on day 21 by means of ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared to all mSVF-hydrogel concentrations, there was a considerably greater release of VEGF by the positive control of only mSVF (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). FGF release on the other hand was similar for all groups, showing a trend towards higher expression compared to the positive control, albeit without any statistical significance. VEGF release was established to be significantly lower than the positive control, with an inverse trend in regard to increasing alginate concentration. This suggests that while alginate can support certain aspects of SVF function, the release of specific growth factors may vary depending on the cell-carrier interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCo-culture with human dermal fibroblasts\u003c/h3\u003e\n\u003cp\u003eDermal fibroblasts play a significant role in wound healing and scar formation, leading the dermal closure from the edges. The complex interaction and effect of SVF on fibroblast migration and viability has been previously described \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We therefore chose to co-culture Human dermal fibroblasts were with varying concentrations of alginate mSVF-hydrogel and mSVF as a positive control in order to determine if the positive effect of mSVF can be retained in the alginate hydrogel.\u003c/p\u003e \u003cp\u003eAfter 5 days, there was a significant increase in cell viability of present cells in all groups including mSVF, compared to the negative fibroblast control. The cell viability for is exemplified for 2% mSVF-hydrogel in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. This trend continued and after 7 days all alginate groups demonstrated a higher effect on cell-viability compared to the negative fibroblast control (supplementary files).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescent, immunohistochemical and histological analysis\u003c/h2\u003e \u003cp\u003eTo further determine ultrastructural differences and growth patterns of mSVF in alginate hydrogel, H\u0026amp;E and Masson's trichrome staining was performed. After 21 days, the alginate architecture was maintained. A qualitative increase in cell numbers, cell clustering and extracellular matrix deposition was observed compared to day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Trichrome staining visualized an increase in collagen fiber presence (supplementary files). Relative differences in alginate concentration demonstrated equal overall hydrogel thickness, cell distribution, cell clustering and adipocyte presence. In comparison to 1.5% w/v alginate, the denser hydrogel was clearly visible in the 3% w/v alginate (supplementary files).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of perilipin2, CD31 and CD73 was further analyzed by means of IHC staining. Perilipin2 antibody expression, a marker for adipocytes, remained constant after 21 days of culture. The total area stained was found to be 0.37\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.2% on day 7 and 0.37\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.29% on day 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). CD31, an endothelial cell marker was found to be present in twice the stained area on day 21, compared to day 7 (Day 7: 0.09\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.9; Day 21: 0.2\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.12; p\u0026thinsp;=\u0026thinsp;0.0002, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). CD73, a marker for endothelial cells and MSCs also increased significantly after 21 days of culture (Day 7: 0.84\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.56; Day 21: 1.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.89; p\u0026thinsp;=\u0026thinsp;0.001, (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Immunofluorescent staining for cell nuclei (DAPI) and cytoskeleton (phalloidin) was performed. Image analysis of the alginate hydrogels demonstrated viable cell nuclei with more pronounced actin filaments after 21 days. Cell spreading and elongation as sign of tissue maturation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe extrinsic potential of SVF and adipose derived mesenchymal stromal cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e has been extensively described in the past. For instance, \u003cem\u003eHongsen Bi et al.\u003c/em\u003e \u003csup\u003e5\u003c/sup\u003e recently showed that SVF and human adipose-derived stem cells (hADSCs) significantly improved wound healing in hyperglycemic mice by promoting angiogenesis and matrix remodeling. Other studies have examined the protective effects of SVF, such as to mitigate hypoxic damage \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and the combination of SVF with hyaluronic acid and gelatin calcium phosphate for bone regeneration \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eBeccia et al.\u003c/em\u003e \u003csup\u003e15\u003c/sup\u003e demonstrated the formation of cell clusters and vessel structures using SVF and Integra\u0026reg;, a commonly used dermal substitute in full thickness skin defects. Certainly, a benefit of using mSVF is the speed and also possible navigation of regulatory issues \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Enzymatic dissociation of fat tissue is yet to be cleared by western national drug monitoring agencies for human use, as the long-term effects of non-autologous collagenase are not yet known. The process takes multiple hours before SVF cells can be isolated, posing another challenge. When comparing both isolation methods, enzymatically dissociated SVF (eSVF) has been found to deliver a higher cell yield, whereas mSVF shows greater wound healing properties \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This is thought to be attributed to the presence of ECM and other cellular proteins still present. Previous studies have investigated the positive effect of the acellular components of fat tissue \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, further underlining this assumption.\u003c/p\u003e \u003cp\u003eIn this study, the potential of alginate hydrogel as a carrier for mSVF from human adipose tissue was investigated. The long-term compatibility of mSVF-alginate complex is crucial when conceptualizing possible treatments of cutaneous defects \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Taking chronic diabetic ulcers as an example, traditional methods of cell delivery into wound sites, such as injection or spraying \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, might lead to fast material dislocation, lower survival rate and overall reduction of reparative functions \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To overcome the limitation of the semi-liquid nature of mSVF, maintaining a more stable shape without excessive deformation of the carrier is paramount. Various methods of cellular transfer have been proposed in the past, ranging from synthetic polymers and natural hydrogels to non-hydrogel-based carriers such as microspheres, decellularized extracellular human or animal matrices. Hydrogels offer an easy and quick encapsulation of cells, rapid application, and good on-site molding to three-dimensional tissue defects. Furthermore, an ideal hydrogel would allow cellular remodeling and slow integration into the host tissue and degradation over time \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Synthetic polymers, such as poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG)-based scaffolds, offer tunable mechanical properties and controlled degradation rates but often lack the tested bioactivity necessary for optimal cell survival and integration. Naturally occurring hydrogels, including collagen, fibrin, and gelatin-based matrices have been extensively described in literature, lack however easy handling and are inherently expensive. Alginate further provides a low-cost, easy to handle and also sustainable \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e alternative.\u003c/p\u003e \u003cp\u003eIn our study, alginate maintained its shape and diameter throughout the 21-day culture period. This would mean that transplanted cells suspended in the hydrogel can kept in the designated place. Extended structural integrity may on the other hand also impede cell viability and interaction. We therefore investigated whether cell viability differed according to alginate concentration in the hydrogel and also throughout the culture period, using mSVF as a positive control.\u003c/p\u003e \u003cp\u003eThe cell viability assays revealed that mSVF encapsulated in alginate hydrogels exhibited a similar viability curve to the positive control group of mSVF alone. This suggests that alginate does not impede cell survival but rather provides a cytocompatible cell environment. These findings are in line with previous studies, showing that alginate can support the growth and survival of various cell types, including human dermal fibroblasts and MSCs \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The standard laboratory culture conditions are however not typically representative of \u003cem\u003ein vivo\u003c/em\u003e circumstances.\u003c/p\u003e \u003cp\u003eIn vivo, the hydrogel might be prone to desiccation, heat fluctuations, altered degradation by a competent immune system and a surely more complex cellular cross-talk. The maintenance of \u003cem\u003ein vitro\u003c/em\u003e cell viability levels near baseline are however a promising reference. In comparison to the positive cell control of only mSVF, mSVF-hydrogels demonstrated an increase of cell viability to nearly 110% at day 3, followed by a decrease again to around 100% at day 21. This trend was mirrored in all hydrogel concentrations (supplementary files). This might be explained by the initial response of certain cell types to the favourable alginate microenvironment. The cellular encapsulation might also restrict certain inhibitory cross-talk of from apoptotic or dead cells following cell harvest and transfer. After an initial cell proliferation stage, hydrogel remodeling might allow for a certain migration throughout and even outside the alginate gel. Representative histological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; supplementary files) would give the impression of a cellular distribution throughout the hydrogel.\u003c/p\u003e \u003cp\u003emSVF controls on the other hand showed an opposite curve, with an initial drop to around 95% viability at day 3, followed by an increase above 100% at day 7, finishing with a final viability just below 100%. A possible explanation here may be the initial cell death of susceptible cell populations after the cell harvest, that directly inhibit neighboring cell populations. These cells would wash out at media changes, allowing for an increase in cell proliferation of other more robust cellular subgroups.\u003c/p\u003e \u003cp\u003eTotal protein release into the surrounding environment acts an indicator for reactivity of the mSVF-hydrogel. The higher initial protein levels in the mSVF group might result from cell stress, apoptosis, or rapid secretion upon seeding, aligning with documented early drop in cell viability. In contrast, the alginate hydrogel appears to modulate this response, possibly mechanically by encapsulating the cells. This prevents a sharp decline in viability and enables sustained protein release. This controlled release, is influenced certainly by hydrogel degradation. When analyzing two important growth factors for tissue repair, VEGF and FGF, all mSVF-hydrogel groups fell behind the positive control at 21days for VEGF release. A non-significant difference between all groups for the FGF release profile was demonstrated. It is surely a timepoint analysis, not reflective of the whole culture period. A possible explanation might be, that certain cellular subgroups responsible for VEGF release are not stimulated to do so. Encapsulation and lack of stimulatory cellular cross-talk, or even a more trivial reason such as a delayed release of the larger protein VEGF.\u003c/p\u003e \u003cp\u003eTo determine a potential effect of mSVF-hydrogel complexes on cells that are crucial for wound repair, a co-culture with dermal fibroblasts assay performed. After 7 days, all alginate groups and the mSVF control significantly increased overall cell viability compared to the negative control. This enhancement in co-culture conditions indicates that alginate hydrogels allow for sufficient cellular paracrine cross-talk. This indicates that the stimulatory effect of SVF is maintained, underlining its relevance for wound healing applications. This finding is in line with previous studies on alginate for protein encapsulation and tissue engineering. For example, \u003cem\u003eMoay et al.\u003c/em\u003e \u003csup\u003e45\u003c/sup\u003e demonstrated the use of keratin-alginate composite sponges for supporting human dermal fibroblasts, while \u003cem\u003eHaq et al.\u003c/em\u003e \u003csup\u003e1\u003c/sup\u003e reported improved stem cell survival in alginate hydrogels pre-treated with caffeic acid.\u003c/p\u003e \u003cp\u003eImmunohistochemical and immunofluorescent analyses provided deeper insights into the cellular maturation and activity within the alginate hydrogels. The expression of CD31 and CD73, markers for endothelial cells and mesenchymal stromal cells, respectively, increased significantly over the 21-day culture period. This suggests that alginate hydrogels support not only the survival but also the functional differentiation of SVF cells.\u003c/p\u003e \u003cp\u003eThe presence of perilipin2, a critical regulator of lipid droplet dynamics, lipid metabolism and lipolysis remained constant, indicating that the health of SVF cells is maintained within the alginate hydrogels. Additionally, the formation of actin filaments and cell elongation observed through immunofluorescent staining indicated ongoing tissue maturation and cellular activity within the hydrogels. In contrast to eSVF, mSVF is composed of cell ECM proteins that interact with alginate and increase the functional integration of the mSVF-hydrogel complex into the surrounding tissue.\u003c/p\u003e \u003cp\u003eThe use of alginate as a scaffold material offers several advantages in regenerative medicine. Its biocompatibility and ability to form hydrogels with tunable mechanical properties make it a versatile candidate for various tissue engineering applications. Components can be readily added, tuning desired properties such as anti-microbial or for drug delivery \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Moreover, the development of composite alginate hydrogels, such as a recently proposed fibrinogen\u0026ndash;nisin\u0026ndash;alginate composite gel \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e further expands the wide range of potential applications of alginate in fine tuning the application design, specifically for patient-specific tissue regeneration. \u003cem\u003eWang et al.\u003c/em\u003e \u003csup\u003e48\u003c/sup\u003e explored the incorporation of magnesium-containing poly(lactic-co-glycolic acid) microspheres into alginate hydrogels, which enhanced their mechanical strength and supported better cell proliferation and differentiation. Further research is certainly warranted to find the ideal mechanical properties of alginate hydrogels to either mimic the native tissue environment or increase the regenerative potential of mSVF.\u003c/p\u003e \u003cp\u003eSince the dawn of 3D bioprinting, alginate was proven to be a versatile, widely biocompatible and commonly used ink. This approach presents another exciting avenue for future research and could enable the precise fabrication of complex or tissue structures \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Furthermore, studies have shown that the addition of bioactive molecules or modifications to the alginate structure can enhance cell behavior and function. For example, the integration of nanocellulose and anti-inflammatory extracts in alginate-based dressings has been shown to promote cell adhesion and reduce inflammation, thereby supporting tissue regeneration \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In comparison, we previously investigated the use of methacrylate gelatin (GelMa) as a possible hydrogel carrier for mSVF \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Whereas GelMA requires a short ultraviolet radiation time during photocross-linking, alginate does not. The associated loss of cell viability can therefore be avoided by means of ionic cross-linking with divalent ion such as calcium solution. While the co-culture analysis with mSVF\u0026ndash;GelMa and a fibroblast monolayer did maintain a certain degree of total cell viability in starvation medium compared with mSVF-free controls, mSVF-alginate could demonstrate a significantly greater increase in cell viability than the control groups, albeit in normal medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Therefore, while both mSVF-containing hydrogels investigated by our group support cell viability, alginate would certainly be superior in terms of production and costs.\u003c/p\u003e \u003cp\u003eTo further investigate the clinical translatability of alginate-based SVF therapies, \u003cem\u003ein vivo\u003c/em\u003e studies are required to confirm their safety and efficacy. Firstly, investigating the optimal alginate concentration and formulation for maximizing cell viability, growth factor release and therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e is essential. Exogenous factors such as tissue desiccation or dislocation, parallel wound healing, complex intercellular communication and immune cell response are only a few of such processes. A higher concentration of alginate or greater average molecular weight could certainly be postulated to have an inhibitory effect \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Moreover, integrating and correlating advanced imaging techniques and biomolecular assays could provide deeper insights into the mechanisms underlying mSVF-alginate interactions and tissue integration dynamics. The presence and accessibility of matrix proteins, as well as the perifocal stimulatory effect are further areas worth investigating. Techniques such as live-cell imaging and transcriptomic analysis could elucidate how alginate influences mSVF behavior and phenotype change over time.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis \u003cem\u003ein vitro\u003c/em\u003e study demonstrates that the combination of mSVF with alginate hydrogels leverages the advantages of both components to create a potent therapeutic tool. The results suggest that alginate hydrogels can effectively maintain cell viability, protein, and growth factor release without compromising cell function, while positively influencing adjacent cell viability. These findings warrant further investigation of alginate hydrogels as a delivery system for SVF, providing a readily available, cost-effective, and biocompatible treatment option.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eSample Collection\u003c/h2\u003e\u003cp\u003eLipoaspirates from subcutaneous fat depots and skin samples from discarded healthy tissue were harvested and directly transferred to the laboratory for further processing. All patients were older than 18 years of age with no serious comorbidities, and all surgeries were elective. Proper surgical technique was performed according to international standard operating procedures (SOP). All reagents and laboratory materials were purchased from Sigma-Aldrich (Merck Millipore, Burlington, MA, USA) unless otherwise stated.\u003c/p\u003e\u003ch2\u003eIsolation of mSVF\u003c/h2\u003e\u003cp\u003eMechanical SVF isolation was executed immediately after harvest, according to a previously established protocol \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In brief, lipoaspirates were emulsified using a 1.4µm Luer lock connector (Tulip Aesthetics®, San Diego, CA, USA) \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e and centrifuged at 500 relative centrifugal force (RCF) for 10 min. The oily and watery fractions were removed by aspiration and the central fraction of purified fat resuspended in culture media. A subsequent second centrifugation at 500 RCF for 5 min was performed. After removal by aspiration of the upper and lower layers, the resulting mSVF was immediately used for further experiments.\u003c/p\u003e\u003ch2\u003eAlginate hydrogel production\u003c/h2\u003e\u003cp\u003eAlginate in powder form was dissolved in Dulbecco's phosphate-buffered saline (DPBS) overnight at 37°C in a shaking water bath set to 150 rpm, to make 3, 4, 5, or 6% weight/volume (w/v). The resulting alginate mixture was combined at a volume ratio of 1:1 with mSVF to create the final concentrations of 1.5, 2, 2.5, or 3% w/v, respectively, and seeded directly into the designated 24-well plates. At ambient room temperature of 21°C, 1 ml of 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e was added to the well for 15 min and the gels were then rinsed three times with 500 µl Hanks' Balanced Salt Solution (HBSS) for 3 min. Standard culture medium consisting of high glucose Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum and 1% penicillin-streptomycin was used for subsequent culture.\u003c/p\u003e\u003cp\u003eA minimum of 9 hydrogels were made from 3 separate donors per experiment and used in a subsequent fashion.\u003c/p\u003e\u003ch2\u003eDermal Fibroblast Isolation and Co-Culture Assay\u003c/h2\u003e\u003cp\u003eHuman dermal fibroblasts were isolated from patient skin samples according to established protocols \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In brief, fibroblast isolation was executed immediately after intraoperative harvest of skin samples. By means of sharp dissection, both the epidermis and the subcutis was removed and the dermis minced. It was followed by digestion with 1,5% bovine serum albumin (BSA) and 0,2% collagenase in collagenase buffer (1 mM CaCl\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, 5 mM Glucose, 50 mM KCl, 100 mM HEPES and 120 mM NaCl) for 45 minutes. Extracellular remnants were removed by filtering the digest through a 100 µm and a 70µm nylon filter (Thermoscientific, Waltham, MA, USA). The cells were subsequently washed and expanded in standard cell culture medium, then used between passages 3–5.\u003c/p\u003e\u003cp\u003eCo-culture assay was performed in a 12-well plate by seeding 20,000 cells/cm\u003csup\u003e2\u003c/sup\u003e (70,000 fibroblasts per well). After 2h at standard incubator conditions (37°C, 5% CO\u003csup\u003e2\u003c/sup\u003e, 95% humidity), alginate gels were added, separated from fibroblasts through a 0.4µm well plate insert (Thermoscientific Nunc Cell Culture Insert System). An empty well insert was used as a negative fibroblast control.\u003c/p\u003e\u003ch2\u003eAlginate hydrogel dimensional degradation\u003c/h2\u003e\u003cp\u003eDirectly after seeding and after 21 days culture, culture media was aspirated and the wells rinsed with 37°C phosphate-buffered saline (PBS). Photos of each gel were acquired alongside a calibration tab and the total surface area was determined using Imitocam AI software (Imito AG, Zürich, ZH, Switzerland). Measurements were provided in cm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eViability Assay\u003c/h2\u003e\u003cp\u003eCell viability within mSVF-hydrogel mixtures was measured at regular intervals on day 0, 1, 3, 7, 14 until day 21 using AlamarBlue® assay (Thermoscientific) according to the manufacturer’s instructions. At each interval, well supernatants were collected and transferred to a 96-well plate, maintaining cell culture conditions. Absorbance was measured in triplicate using a microplate reader (Cytation 5, BioTek Instruments, Winooski, VT, USA). Normalization of absorbance was achieved by using supernatant of alginate hydrogel without cells as a negative control, cell culture medium as a blank and mSVF in suspension without hydrogel served as a positive control (PC) \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eTotal protein Assay\u003c/h2\u003e\u003cp\u003eTotal protein concentration present in well supernatant from mSVF-hydrogel samples was measured at day 1, 7 and 21 using Pierce™ BCA Protein Assay Kits according to the manufacturer’s instructions (Thermoscientific). At each time interval, well supernatant was diluted 1:10 and transferred to a 96-well plate. After adding reagent and 30 min of incubation, absorbance was measured in triplicate using a microplate reader, mentioned above. Normalization of absorbance was achieved by means of a manufacturer’s standard curve. Furthermore, mSVF in suspension served as a positive control and culture medium as a blank.\u003c/p\u003e\u003ch2\u003eELISA\u003c/h2\u003e\u003cp\u003eCulture medium was collected from all samples at day 0, 7 and 21. The concentration of FGF and VEGF in the culture medium was quantified by ELISA using a commercial ABTS ELISA development kit and ABTS ELISA buffer kit (PeproTech, Cranbury, NJ, USA) according to the manufacturer’s instructions. Absorbance was measured using the microplate reader mentioned above.\u003c/p\u003e\u003ch2\u003eImmunohistochemistry (IHC) and Immunofluorescence (IF)\u003c/h2\u003e\u003cp\u003eTissue samples from all sample constructs were collected on days 7 and 21, fixed in paraformaldehyde for 24h at 4°C and rinsed in. Staining was performed by an automated IHC system, Autostainer Link48 and PT Link device (Agilent Dako, Santa Clara, CA, USA) on previously paraffin-embedded, 5 µm tissue slices. The following antigens were targeted in addition to staining of hematoxylin-eosin (H\u0026amp;E) and Masson’s trichrome staining: Perilipin-2 (Novus Biologicals, Centennial, CO, USA), CD73 and CD31 (Abcam, Cambridge, Cambridgeshire, United Kingdom). Using FIJI software \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e to determine the antigen area stained, firstly the total area of alginate was designated as a region of interest (ROI). From this area, the area stained was measured using a threshold method set to 200 hue, 80 saturation and 50 brightness and the relative area was calculated. Staining of cell nuclei and actin filaments was performed by IF using DAPI and phalloidin (Cat.-Nr. ab176753, Abcam) which was diluted in PBS in a ratio of 1:1000. Using a cryostat (Leica Biosystems, Wetzlar, Hessen, Germany), previously fixed samples were cut at 10µm sections and left to dry. Slides were incubated with staining solution at room temperature for 90 min protected from light. Gels were then washed 3 times with PBS for 5 min at a time. Microscopy was performed using the Slidescanner Zeiss Axio Scan.Z1 (Carl Zeiss, Jena, Thuringia, Germany).\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll values are presented as means with standard error of mean (SEM) or standard deviation (SD). Normal distribution was tested by a Shapiro–Wilk test followed by an unpaired t-test or one-way ANOVA with GraphPad Prism V10.0 (GraphPad Software, San Diego, CA, USA). \u003cem\u003ep\u003c/em\u003e values \u0026lt; 0.05 were accepted as statistically significant: * = \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** = \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** = \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** = \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGR: Writing\u0026ndash;Original draft, Methodology, Investigation, Data curation, Conceptualization.\u003c/p\u003e\n\u003cp\u003eAKS: Data curation, Validation, Software, Investigation.\u003c/p\u003e\n\u003cp\u003eMV: Investigation, Visualization, Data curation, Writing\u0026ndash;review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eLH: Data curation, Writing\u0026ndash;Review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eDD: Data curation, Conceptualization, Methodology.\u003c/p\u003e\n\u003cp\u003eJAW: Investigation, Writing\u0026ndash;Review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eGR6: Resources, Writing\u0026ndash;Review \u0026amp; editing, Visualization.\u003c/p\u003e\n\u003cp\u003eMT: Resources, Methodology, Writing\u0026ndash;Review \u0026amp; editing, Project administration.\u003c/p\u003e\n\u003cp\u003eBSK: Conceptualization, Supervision, Resources, Methodology, Writing\u0026ndash;Review \u0026amp; editing, Project administration.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in compliance with the Declaration of Helsinki. All patients signed a general informed consent of the Zurich University Hospital prior to operation and tissue harvest. Dermal samples were fully anonymized after harvest. The collection and usage of human samples was approved by the regional ethics committee of the Canton of Zurich, Switzerland (Date: 02.04.2019, BASEC 2019-00389). The study protocol adhered to the guidelines established by the journal. All material was handled according to biosecurity guidelines established by the competent ethics committee.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project did not receive specific funding. It was performed as part of the employment at the University hospital of Z\u0026uuml;rich, R\u0026auml;mistrasse 100, 8091 Z\u0026uuml;rich, Switzerland.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShifa ul Haq, H. M. \u003cem\u003eet al.\u003c/em\u003e Priming with caffeic acid enhances the potential and survival ability of human adipose-derived stem cells to counteract hypoxia. \u003cem\u003eRegen Ther\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 115\u0026ndash;127 (2023).\u003c/li\u003e\n\u003cli\u003eBroughton, G., Janis, J. E. \u0026amp; Attinger, C. E. Wound healing: An overview. \u003cem\u003ePlastic and Reconstructive Surgery\u003c/em\u003e 117-1e-S-32e-S Preprint at https://doi.org/10.1097/01.prs.0000222562.60260.f9 (2006).\u003c/li\u003e\n\u003cli\u003eBender, R. \u003cem\u003eet al.\u003c/em\u003e Human Adipose Derived Cells in Two- And Three-Dimensional Cultures: Functional Validation of an in Vitro Fat Construct. \u003cem\u003eStem Cells Int\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eNilforoushzadeh, M. A. \u003cem\u003eet al.\u003c/em\u003e Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin\u0026ndash;collagen hydrogel: A clinical study for diabetic wound healing. \u003cem\u003eJ Tissue Eng Regen Med\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 424\u0026ndash;440 (2020).\u003c/li\u003e\n\u003cli\u003eBi, H. \u003cem\u003eet al.\u003c/em\u003e Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eDeptuła, M., Brzezicka, A., Skoniecka, A., Zieliński, J. \u0026amp; Pikuła, M. Adipose-derived stromal cells for nonhealing wounds: Emerging opportunities and challenges. \u003cem\u003eMedicinal Research Reviews\u003c/em\u003e vol. 41 2130\u0026ndash;2171 Preprint at https://doi.org/10.1002/med.21789 (2021).\u003c/li\u003e\n\u003cli\u003eBusato, A. \u003cem\u003eet al.\u003c/em\u003e Simple and rapid non-enzymatic procedure allows the isolation of structurally preserved connective tissue micro-fragments enriched with svf. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1\u0026ndash;14 (2021).\u003c/li\u003e\n\u003cli\u003eTiryaki, K. T., Cohen, S., Kocak, P., Canikyan Turkay, S. \u0026amp; Hewett, S. In-vitro comparative examination of the effect of stromal vascular fraction isolated by mechanical and enzymatic methods on wound healing. \u003cem\u003eAesthet Surg J\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, 1232\u0026ndash;1240 (2020).\u003c/li\u003e\n\u003cli\u003eKim, B. S. \u003cem\u003eet al.\u003c/em\u003e In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eSaxer, F. \u003cem\u003eet al.\u003c/em\u003e Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First-in-Man Study. \u003cem\u003eStem Cells\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 2956\u0026ndash;2966 (2016).\u003c/li\u003e\n\u003cli\u003eMytsyk, M. \u003cem\u003eet al.\u003c/em\u003e Paracrine potential of adipose stromal vascular fraction cells to recover hypoxia-induced loss of cardiomyocyte function. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 132\u0026ndash;142 (2019).\u003c/li\u003e\n\u003cli\u003eBalko, S., Kerr, E., Buchel, E., Logsetti, S. \u0026amp; Raouf, A. Paracrine signalling between keratinocytes and SVF cells results in a new secreted cytokine profile during wound closure. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003evan Boxtel, J., Vonk, L. A., Stevens, H. P. \u0026amp; van Dongen, J. A. Mechanically Derived Tissue Stromal Vascular Fraction Acts Anti-inflammatory on TNF Alpha-Stimulated Chondrocytes In Vitro. \u003cem\u003eBioengineering\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eFreitas-Ribeiro, S. \u003cem\u003eet al.\u003c/em\u003e Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue. \u003cem\u003eMar Drugs\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eBeccia, E. \u003cem\u003eet al.\u003c/em\u003e Adipose Stem Cells and Platelet-Rich Plasma Induce Vascular-Like Structures in a Dermal Regeneration Template. \u003cem\u003eTissue Eng Part A\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 631\u0026ndash;641 (2021).\u003c/li\u003e\n\u003cli\u003eOskarsdotter, K. \u003cem\u003eet al.\u003c/em\u003e Autologous endothelialisation by the stromal vascular fraction on laminin-bioconjugated nanocellulose-alginate scaffolds. \u003cem\u003eBiomedical Materials (Bristol)\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003ePark, S. su, Park, M. \u0026amp; Lee, B. T. Autologous stromal vascular fraction-loaded hyaluronic acid/gelatin-biphasic calcium phosphate scaffold for bone tissue regeneration. \u003cem\u003eBiomaterials Advances\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eVelier, M. \u003cem\u003eet al.\u003c/em\u003e Paracrine Effects of Adipose-Derived Cellular Therapies in an in Vitro Fibrogenesis Model of Human Vocal Fold Scarring. \u003cem\u003eJournal of Voice\u003c/em\u003e (2022) doi:10.1016/j.jvoice.2022.05.012.\u003c/li\u003e\n\u003cli\u003eMalekzadeh, H., Tirmizi, Z., Arellano, J. A., Egro, F. M. \u0026amp; Ejaz, A. Application of Adipose-Tissue Derived Products for Burn Wound Healing. \u003cem\u003ePharmaceuticals\u003c/em\u003e vol. 16 Preprint at https://doi.org/10.3390/ph16091302 (2023).\u003c/li\u003e\n\u003cli\u003eWang, J. \u003cem\u003eet al.\u003c/em\u003e Anti-Aging Effect of the Stromal Vascular Fraction/Adipose-Derived Stem Cells in a Mouse Model of Skin Aging Induced by UVB Irradiation. \u003cem\u003eFront Surg\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eGentile, P. \u003cem\u003eet al.\u003c/em\u003e Impact of the different preparation methods to obtain human adipose-derived stromal vascular fraction cells (AD-SVFs) and human adipose-derived mesenchymal stem cells (AD-MSCs): Enzymatic digestion versus mechanical centrifugation. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e vol. 20 Preprint at https://doi.org/10.3390/ijms20215471 (2019).\u003c/li\u003e\n\u003cli\u003eGuillaume, O. \u003cem\u003eet al.\u003c/em\u003e Stromal vascular fraction cells as biologic coating of mesh for hernia repair. \u003cem\u003eHernia\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 1233\u0026ndash;1243 (2020).\u003c/li\u003e\n\u003cli\u003eCao, H., Duan, L., Zhang, Y., Cao, J. \u0026amp; Zhang, K. Current hydrogel advances in physicochemical and biological response-driven biomedical application diversity. \u003cem\u003eSignal Transduction and Targeted Therapy\u003c/em\u003e vol. 6 Preprint at https://doi.org/10.1038/s41392-021-00830-x (2021).\u003c/li\u003e\n\u003cli\u003eCatoira, M. C., Fusaro, L., Di Francesco, D., Ramella, M. \u0026amp; Boccafoschi, F. Overview of natural hydrogels for regenerative medicine applications. \u003cem\u003eJ Mater Sci Mater Med\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eShafei, S. \u003cem\u003eet al.\u003c/em\u003e Exosome loaded alginate hydrogel promotes tissue regeneration in full-thickness skin wounds: An in vivo study. \u003cem\u003eJ Biomed Mater Res A\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 545\u0026ndash;556 (2020).\u003c/li\u003e\n\u003cli\u003eFarshidfar, N., Iravani, S. \u0026amp; Varma, R. S. Alginate-Based Biomaterials in Tissue Engineering and Regenerative Medicine. \u003cem\u003eMarine Drugs\u003c/em\u003e vol. 21 Preprint at https://doi.org/10.3390/md21030189 (2023).\u003c/li\u003e\n\u003cli\u003eAbourehab, M. A. S. \u003cem\u003eet al.\u003c/em\u003e Alginate as a Promising Biopolymer in Drug Delivery and Wound Healing: A Review of the State-of-the-Art. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e vol. 23 Preprint at https://doi.org/10.3390/ijms23169035 (2022).\u003c/li\u003e\n\u003cli\u003eWang, J. \u003cem\u003eet al.\u003c/em\u003e Alginate: Microbial production, functionalization, and biomedical applications. \u003cem\u003eInt J Biol Macromol\u003c/em\u003e \u003cstrong\u003e242\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eAderibigbe, B. A. \u0026amp; Buyana, B. Alginate in wound dressings. \u003cem\u003ePharmaceutics\u003c/em\u003e vol. 10 Preprint at https://doi.org/10.3390/pharmaceutics10020042 (2018).\u003c/li\u003e\n\u003cli\u003eHemadi, M. \u003cem\u003eet al.\u003c/em\u003e Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: Stemness gene expression and structural features. \u003cem\u003eZygote\u003c/em\u003e 1\u0026ndash;7 (2021) doi:10.1017/S0967199421000551.\u003c/li\u003e\n\u003cli\u003eKostenko, A., Swioklo, S. \u0026amp; Connon, C. J. Alginate in corneal tissue engineering. \u003cem\u003eBiomedical Materials (Bristol)\u003c/em\u003e vol. 17 Preprint at https://doi.org/10.1088/1748-605X/ac4d7b (2022).\u003c/li\u003e\n\u003cli\u003ePorter, G. C. \u003cem\u003eet al.\u003c/em\u003e AgNP/Alginate Nanocomposite hydrogel for antimicrobial and antibiofilm applications. \u003cem\u003eCarbohydr Polym\u003c/em\u003e \u003cstrong\u003e251\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eGilljam, K. M. \u003cem\u003eet al.\u003c/em\u003e Alginate and Nanocellulose Dressings With Extract From Salmon Roe Reduce Inflammation and Accelerate Healing of Porcine Burn Wounds. \u003cem\u003eJournal of Burn Care \u0026amp; Research\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 1140\u0026ndash;1149 (2023).\u003c/li\u003e\n\u003cli\u003eRastogi, P. \u0026amp; Kandasubramanian, B. Review of alginate-based hydrogel bioprinting for application in tissue engineering. \u003cem\u003eBiofabrication\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eBi, H. \u003cem\u003eet al.\u003c/em\u003e Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eMytsyk, M. \u003cem\u003eet al.\u003c/em\u003e Long-term severe in vitro hypoxia exposure enhances the vascularization potential of human adipose tissue-derived stromal vascular fraction cell engineered tissues. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eBora, P. \u0026amp; Majumdar, A. S. Adipose tissue-derived stromal vascular fraction in regenerative medicine: A brief review on biology and translation. \u003cem\u003eStem Cell Research and Therapy\u003c/em\u003e vol. 8 Preprint at https://doi.org/10.1186/s13287-017-0598-y (2017).\u003c/li\u003e\n\u003cli\u003eJiang, X. \u003cem\u003eet al.\u003c/em\u003e Decellularized adipose tissue: A key factor in promoting fat regeneration by recruiting and inducing mesenchymal stem cells. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e541\u003c/strong\u003e, 63\u0026ndash;69 (2021).\u003c/li\u003e\n\u003cli\u003eQin, J., Chen, F., Wu, P. \u0026amp; Sun, G. Recent Advances in Bioengineered Scaffolds for Cutaneous Wound Healing. \u003cem\u003eFrontiers in Bioengineering and Biotechnology\u003c/em\u003e vol. 10 Preprint at https://doi.org/10.3389/fbioe.2022.841583 (2022).\u003c/li\u003e\n\u003cli\u003eZimmerlin, L. \u003cem\u003eet al.\u003c/em\u003e Human adipose stromal vascular cell delivery in a fibrin spray. \u003cem\u003eCytotherapy\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 102\u0026ndash;108 (2013).\u003c/li\u003e\n\u003cli\u003eDing, Y., Wang, Y. \u0026amp; Hu, Q. Recent advances in overcoming barriers to cell-based delivery systems for cancer immunotherapy. \u003cem\u003eExploration\u003c/em\u003e vol. 2 Preprint at https://doi.org/10.1002/EXP.20210106 (2022).\u003c/li\u003e\n\u003cli\u003eWawszczak, A., Kocki, J. \u0026amp; Kołodyńska, D. Alginate as a Sustainable and Biodegradable Material for Medical and Environmental Applications\u0026mdash;The Case Studies. \u003cem\u003eJournal of Biomedical Materials Research - Part B Applied Biomaterials\u003c/em\u003e vol. 112 1\u0026ndash;23 Preprint at https://doi.org/10.1002/jbm.b.35475 (2024).\u003c/li\u003e\n\u003cli\u003eJahandideh, A. \u003cem\u003eet al.\u003c/em\u003e Alginate scaffolds improve functional recovery after spinal cord injury. \u003cem\u003eEuropean Journal of Trauma and Emergency Surgery\u003c/em\u003e vol. 48 1711\u0026ndash;1721 Preprint at https://doi.org/10.1007/s00068-021-01760-7 (2022).\u003c/li\u003e\n\u003cli\u003eShuai, Q. \u003cem\u003eet al.\u003c/em\u003e Sodium alginate hydrogel integrated with type III collagen and mesenchymal stem cell to promote endometrium regeneration and fertility restoration. \u003cem\u003eInt J Biol Macromol\u003c/em\u003e \u003cstrong\u003e253\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eMoay, Z. K. \u003cem\u003eet al.\u003c/em\u003e Keratin‐alginate sponges support healing of partial‐thickness burns. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eCleetus, C. M. \u003cem\u003eet al.\u003c/em\u003e Alginate hydrogels with embedded zno nanoparticles for wound healing therapy. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 5097\u0026ndash;5111 (2020).\u003c/li\u003e\n\u003cli\u003eSoleimanpour, M. \u003cem\u003eet al.\u003c/em\u003e Designing a new alginate-fibrinogen biomaterial composite hydrogel for wound healing. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eWang, L. \u003cem\u003eet al.\u003c/em\u003e Alginate hydrogels containing different concentrations of magnesium-containing poly(lactic-co-glycolic acid) microspheres for bone tissue engineering. \u003cem\u003eBiomedical Materials (Bristol)\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eVasella, M. \u003cem\u003eet al.\u003c/em\u003e Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, (2023).\u003c/li\u003e\n\u003cli\u003eKaijzel, E. L., Koolwijk, P., van Erck, M. G. M., van Hinsbergh, V. W. M. \u0026amp; de Maat, M. P. M. Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo. \u003cem\u003eJ Thromb Haemost\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1975\u0026ndash;81 (2006).\u003c/li\u003e\n\u003cli\u003ePallua, N., Grasys, J. \u0026amp; Kim, B. S. Enhancement of progenitor cells by two-step centrifugation of emulsified lipoaspirates. \u003cem\u003ePlast Reconstr Surg\u003c/em\u003e \u003cstrong\u003e142\u003c/strong\u003e, 99\u0026ndash;109 (2018).\u003c/li\u003e\n\u003cli\u003eTonnard, P. \u003cem\u003eet al.\u003c/em\u003e Nanofat grafting: Basic research and clinical applications. \u003cem\u003ePlast Reconstr Surg\u003c/em\u003e \u003cstrong\u003e132\u003c/strong\u003e, 1017\u0026ndash;1026 (2013).\u003c/li\u003e\n\u003cli\u003eKoch, C. M. \u003cem\u003eet al.\u003c/em\u003e Specific age-associated DNA methylation changes in human dermal fibroblasts. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, (2011).\u003c/li\u003e\n\u003cli\u003eHe, Y. \u003cem\u003eet al.\u003c/em\u003e Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eKim, B. S. \u003cem\u003eet al.\u003c/em\u003e The Effect of Antiseptics on Adipose-Derived Stem Cells. \u003cem\u003ePlast Reconstr Surg\u003c/em\u003e \u003cstrong\u003e139\u003c/strong\u003e, 625\u0026ndash;637 (2017).\u003c/li\u003e\n\u003cli\u003eSchindelin, J. \u003cem\u003eet al.\u003c/em\u003e Fiji: An open-source platform for biological-image analysis. \u003cem\u003eNature Methods\u003c/em\u003e vol. 9 676\u0026ndash;682 Preprint at https://doi.org/10.1038/nmeth.2019 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tissue engineering, Wound healing, biomaterials, Cellular therapy","lastPublishedDoi":"10.21203/rs.3.rs-7520241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7520241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman adipose tissue-derived mechanically isolated stromal vascular fraction (mSVF) is a heterogeneous cell population containing mesenchymal stromal and progenitor cells known to have immense regenerative potential.\u003c/p\u003e \u003cp\u003eCurrent research aims to enhance mSVF delivery by improving cell retention and survival, with hydrogels emerging as promising scaffolds. Among them, alginate stands out due to its biocompatibility, cost-effectiveness, and established use in wound healing and tissue engineering.\u003c/p\u003e \u003cp\u003eIn this study, mSVF was cultured in varying concentrations of alginate for 21 days and tested for hydrogel degradation, cell viability, as well as protein and growth factor release. Alginate encapsulated mSVF was co-cultured with human dermal fibroblasts and analyzed via immunohistochemical and immunofluorescence imaging.\u003c/p\u003e \u003cp\u003eAfter 21 days, all hydrogel samples of different concentrations maintained the original size and shape. Cell viability and protein release was comparable to the positive control of mSVF only. Fibroblast growth factor (FGF) release, as quantified by growth factor analysis, increased in the co-culture. In addition, the co-culture exhibited increased fibroblast viability as compared with negative controls as well as increased CD31 and CD73 expression.\u003c/p\u003e \u003cp\u003eAs a long-term proof-of-concept, we demonstrate alginate is an attractive carrier for mSVF. Our findings support its potential for enhancing cell-based therapies in regenerative medicine.\u003c/p\u003e","manuscriptTitle":"Alginate as a carrier for mechanically isolated Stromal Vascular Fraction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 11:45:11","doi":"10.21203/rs.3.rs-7520241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-19T05:14:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T00:53:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170797719556722552893529906615475009821","date":"2026-02-25T20:53:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T13:07:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274702307487924972679578929561733324132","date":"2026-02-13T11:26:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T15:46:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-23T12:36:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-29T15:56:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-23T09:05:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-23T08:25:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"049a0fa0-0e20-46db-9121-7675ea095049","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":62898421,"name":"Biological sciences/Biotechnology"},{"id":62898422,"name":"Biological sciences/Cell biology"},{"id":62898423,"name":"Physical sciences/Materials science"},{"id":62898424,"name":"Health sciences/Medical research"},{"id":62898425,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-04-27T16:01:37+00:00","versionOfRecord":{"articleIdentity":"rs-7520241","link":"https://doi.org/10.1038/s41598-026-50191-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-25 15:58:20","publishedOnDateReadable":"April 25th, 2026"},"versionCreatedAt":"2026-02-17 11:45:11","video":"","vorDoi":"10.1038/s41598-026-50191-0","vorDoiUrl":"https://doi.org/10.1038/s41598-026-50191-0","workflowStages":[]},"version":"v1","identity":"rs-7520241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7520241","identity":"rs-7520241","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}