Development of a Low Viscosity PEGDA - Gelatin Bioink for Embedded Bioprinting of Complex Structures and Lung Adenocarcinoma (LUAD) Model

Development of a Low Viscosity PEGDA - Gelatin Bioink for Embedded Bioprinting of Complex Structures and Lung Adenocarcinoma (LUAD) Model | 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 Research Article Development of a Low Viscosity PEGDA - Gelatin Bioink for Embedded Bioprinting of Complex Structures and Lung Adenocarcinoma (LUAD) Model Aiswarya Ganapathisankarakrishnan, Dona Shaji, Amrutha Krishnamoorthy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9513495/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Lung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer (NSCLC), accounting for 40% of all lung cancer cases. The development of 3D in vitro cancer models offers better replication of the native tumour microenvironment, facilitating patient-specific drug screening and therapeutic evaluation. However, conventional extrusion printing approaches are limited in fabricating functional vascularized tissue models for transplantation, drug screening and disease modelling, owing to restrictions caused by gravity & structural complexities. In this study, we employ embedded bioprinting to fabricate self-supporting biomimetic LUAD with intricate geometries. A low-viscosity bioink comprising polyethylene glycol diacrylate (PEGDA) & gelatin was extruded into a xanthan gum support bath, which exhibited pseudoplastic and shear thinning behaviour to overcome the gravitational and overhang limitations of standard extrusion printing. Gelatin offers biomimetic cell-binding motifs, while PEGDA enhances mechanical stability and tunable tissue stiffness. Rheological analysis confirmed shear thinning and recovery properties of the support bath system. In vitro assessments further demonstrated the cytocompatibility of both the printed construct and support bath. This approach highlights the feasibility of engineering physiologically relevant in vitro LUAD models, presenting a promising alternative to animal models for preclinical screening of therapeutics and personalised medicine applications. Embedded bioprinting LUAD Model low viscosity support bath shear thinning PEGDA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Tissue & disease models emerge as promising non-animal platforms to investigate disease progression and test therapeutic drugs. Precisely recapitulating complex human physiological systems with in vitro models require biomimetic cellular organization and structural complexity due to the dependency of tissue functionality on morphological and geometric resemblance to native tissues [ 1 ]. Conventional cancer tissue models, such as 2D monolayer cultures and basic tumour spheroids or organoids, fail to replicate the three-dimensional tumour microenvironment imperative to determine disease progression & drug responses [ 2 ]. In recent times, there has been a significant shift from exclusive reliance on chemotherapy to modern therapies, such as immunotherapies and targeted therapies, which contributed to a considerable reduction in mortality rates. However, the successful clinical outcomes of these novel interventions mostly depend on precise prognosis and pathological evaluation. Unfortunately, the poor predictive accuracy of the available preclinical models remains a major bottleneck, often leading to increased attrition rates for new therapeutic drug candidates during clinical trials [ 3 ]. Although animal models may offer better insights, alternatively, they fail to fully replicate the human-specific responses due to species level genetic & physiological differences. Considering the above, there is a huge clinical demand to establish next-generation in vitro cancer models that have the potential to offer precise predictions on therapeutic outcome and enable mechanistic understanding of tumour development in a clinically relevant context comparable to humans [ 4 ]. Over the last decade, LUAD happened to be the most common subtype of lung cancer, amounting for about 40% of all cases across the globe. Typically associated with chronic smoking, incidence of LUAD has been notably rising among regular smokers and also among non-smokers underscoring its complexity in aetiology [ 5 , 6 ]. Despite considerable breakthroughs in clinical treatment strategies (targeted immunotherapies and genetically targeted therapies), LUAD continues to be a challenging global health burden. Additionally, the five year survival rate generally remains between 5% and 20% (influenced by healthcare access, geographic location and individual patient variability), highlighting the dire need for enhanced therapeutic outcomes [ 7 ]. Existing conventional models (2D monolayer cultures, tumour spheroids, and ex vivo tissue cultures) for LUAD poorly reproduce the tumour microenvironment (TME), though offer valuable insights to some extent. The TME comprises a complex vascular network around the cancer tissue, immune cells & components, fibroblasts, extracellular matrix components and other factors (soluble growth factors, signalling molecules like chemokines, cytokines) that are crucial in tumour progression and therapeutic response. Nevertheless, these conventional models have failed to capture cell-cell & cell-matrix interactions, spatial and mechanical heterogeneity that are hallmarks of tumours, reducing the predictive efficacy for drug responses and tumour behaviour [ 8 , 9 ]. In order to better mimic the TME, advanced fabrication methods have paved new ways for creating physiologically relevant LUAD models by enabling precise spatial localization of multiple cell types and matrix components [ 10 ]. Still, traditional extrusion-based 3D bioprinting techniques experience several limitations, particularly in fabricating complex, overhanging vascularized structures due to the low viscosity of bioinks and also gravitational constraints [ 11 ]. These limitations contribute to printing artifacts, mechanical instability and poor resolution. To overcome these challenges, researchers are now opting for embedded bioprinting – an innovative approach where low-viscosity bioinks are supported inside a supportive, shear thinning embedding bath that enables the fabrication of complex, biomimetic and self-supporting tissue constructs with improved resolution & fidelity [ 12 ]. To develop LUAD models that closely replicate the TME, including vascular networks & cellular heterogeneity- embedded bioprinting may be an ideal choice. This printing method also enables the incorporation of multiple bioinks and cell types, offering versatile platforms to investigate tumour biology and determine the efficacy of therapeutics in a closer context to in vivo microenvironment of humans [ 13 ]. By leveraging embedded bioprinting, physiologically relevant LUAD models can be fabricated and tuned to better mimic native tissue features, thereby advancing personalised medicine and drug screening efforts [ 14 ]. Therefore, the embedded bioprinting approach stands unique in successfully overcoming the limitations of conventional models and other 3D bioprinting techniques, providing promising pathways to enhance understanding of tumour progression and therapeutic outcomes in LUAD [ 15 ]. In this study, we fabricated microarchitectures of the human lung alveoli, digitally designed using Fusion 360. For the bioink, we used a low-viscosity formulation comprising gelatin (5% w/v) and PEGDA (15% w/v), which was extruded into a xanthan gum (1.5% w/v) support bath. Gelatin – a collagen derived polymer with intrinsic cell-binding motifs is widely preferred for bioprinting applications due to its ease of crosslinking and ability to support cell adhesion & proliferation. PEGDA was chosen for its capacity to undergo photocrosslinking, thereby imparting mechanical stability to bioprinted constructs and its well-known biocompatibility. Xanthan gum – known for its excellent tunability of rheological properties such as flow behaviour, yield stress and thixotropy was preferred as an ideal support bath for embedded bioprinting. Herein, we systematically evaluated the rheological properties of the pseudoplastic xanthan gum bath, including viscoelasticity, shear thinning, and structural recovery, to confirm its suitability for supporting extrusion-based embedded bioprinting. The resulting platform was demonstrated for precise printing of complex constructs and the development of a LUAD model with well-defined architectural features. Our findings reveal that the engineered gelatin-PEGDA bioink and shear thinning xanthan gum support bath offer a robust strategy for bioprinting of biomimetic lung microarchitectures. This strategy holds excellent promise towards developing physiologically relevant in vitro models for drug screening applications. 2. Materials and Methods 2.1. Materials Poly (Ethylene Glycol) Diacrylate (PEGDA) Mn 700 g/mol, Xanthan gum from Xanthomonas campestris , and gelatin from porcine skin, gel strength 300 Type A were purchased from Sigma Aldrich, India. Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) was purchased from TCI Chemicals, Tokyo Chemical Industry Co., Ltd, Tokyo, Japan. Trypsin EDTA, live/dead assay kit and fetal bovine serum (FBS) were procured from Thermo Fisher Scientific, India. MTS Reagent was purchased from Abcam, India. Phenol and Triton X-100 were procured from Merck, India. Dulbecco's Phosphate Buffer Saline (DPBS) and Dulbecco's Modified Eagle Medium (DMEM) were purchased from HiMedia Laboratories Private Limited, Mumbai, India. Transglutaminase (Enzyme activity 200 U/g) was procured from Ultreze Enzyme Private Limited, Surat, India. A549 (CCL-185) Lung cancer cell line was procured from National Centre for Cell Science (NCCS) Pune, India. Human dermal fibroblast (HDFa) ( PCS-201-012 ) cells were purchased from HiMedia Laboratories Private Limited, Mumbai, India. All cell lines were sourced from ATCC, a well-established, authenticated repository with no reported incidents of misidentification or contamination, and the cell lines were certified to be free of mycoplasma contamination. 2.2. Preparation of low viscosity bioink The low viscosity bioink suitable for embedded bioprinting was prepared as follows. Gelatin at a concentration of 5% (w/v) was completely dissolved in 1X PBS at 50 ˚C for 15 minutes. Subsequently, PEGDA (15% v/v) was added to the gelatin solution, which was equilibrated at room temperature and stirred further for another 15 minutes. Once both polymers were thoroughly dissolved, the solution was transferred to a suitable container. LAP (0.25% w/v) was then added under dark conditions, with continued stirring until completely dissolved [ 16 ]. 2.3. Preparation of Support Bath Medium The support bath was prepared by dissolving xanthan gum at concentrations of 0.5%, 1%, 1.5% and 2% w/v in 1X PBS and allowing the mixture to hydrate overnight at room temperature. Prior to printing, the xanthan gum slurry was centrifuged at 500 rpm to remove bubbles and then used as substrate for printing [ 17 ]. 2.4. Crosslinking of bulk and printed gel Hydrogels were prepared by manually dispensing/printing the ink mixed with 0.25% (w/v) LAP into titer well plates. Photocrosslinking was performed by illuminating 405 nm light for 15 seconds (rheology) / 1 minute (swelling behaviour analysis) / 5 minutes (stability analysis), 15 minutes (complex structures). To prepare dual crosslinked hydrogels, photocrosslinked hydrogels (as above) were incubated in 10% (w/v) transglutaminase solution for 1 hour. Based on the optimization studies on crosslinking, the duration of photocrosslinking was increased for bulk and bioprinted constructs with larger dimension (complex structures) to ensure complete crosslinking. 2.5. Rheological characterization 2.5.1. Bioink The rheological properties of the bioink and support bath were studied using an Anton Parr Rheometer MCR302 with a parallel plate setup (dia 25 mm, distance 1 mm). Storage (G’) and loss modulus (G”) of the hydrogels were determined. Hydrogels were prepared by dispensing 500 µL of the ink mixed with 0.25% (w/v) LAP into 12 well plates. Photocrosslinking was performed by illuminating 405 nm light for 15 seconds (“P”), dual crosslinked hydrogels (“P-G”), were photocrosslinked for 15 seconds, followed by incubation in 10% (w/v) transglutaminase solution for 1 hour. Amplitude sweep test was performed (0.01 to 1000% strain rate at 10 Hz) to determine the linear viscoelastic region (LVR), and frequency sweep analysis covered a range from 0.1 to 100 Hz. The strain % was set from the range of values within the linear viscoelastic region measured from the amplitude sweep analysis. Strain % of 1.91% and 2.67% was set for the photocrosslinked and dual crosslinked hydrogels, respectively. All experiments were conducted in triplicate [ 18 , 19 ]. 2.5.2. Support Bath The rheological properties of the xanthan gum support bath at different concentrations, 0.5%, 1%, 1.5%, and 2% (w/v), were analyzed. Flow curve analysis was performed to study the shear thinning behaviour of the pseudoplastic support bath by varying the shear rate from 0.01 s − 1 to 100 s − 1 . The amplitude and frequency sweep analysis were carried out under the previously described conditions. In addition, the shear recovery properties of each concentration were evaluated using oscillation thixotropy and shear thixotropic analyses. Oscillation thixotropy was analysed by measuring the hydrogel modulus over three cycles, alternating between low strain (1%) for 60 s and high strain (100%) for 60 s. Shear thixotropy analysis involved measuring viscosity at a low shear rate (1 s –1 ) for 60 s, followed by higher shear rate (100 s –1 ) for 5 s, and subsequently recovery at a low shear rate (1 s –1 ) for 120 s. All experiments were done in triplicate [ 20 ]. 2.6. Swelling Behaviour and Stability Analysis The swelling behaviour of photocrosslinked and dual crosslinked hydrogels was determined as follows. For photocrosslinked hydrogels, 500 µL of bioink was manually dispensed into a 24-well plate and photocrosslinked for 1 minute. Dual crosslinked hydrogels were prepared by incubating the photocrosslinked hydrogels (manually dispensed) in 10% (w/v) TG for 1 hour. After crosslinking, the constructs were lyophilized, and their initial dry weights were taken (W 1 ). The constructs were then rehydrated in 1 ml of 1X PBS, and the wet weight was recorded at various time points: 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h and 24 h (W 2 ). The swelling ratio was calculated using the following formula. The stability analysis for the protein-polymer hydrogels was conducted over a period of 2 weeks to evaluate their degradation profile and structural integrity during incubation in 1X PBS at 37 ˚C. The stability analysis of the bulk hydrogel was compared with that of the hollow printed construct to contrast the stability of the hydrogel formulation across different material infill (100% and 0%). In addition, rheological studies, showed that the dual crosslinked constructs exhibit greater mechanical strength compared to the only-photocrosslinked constructs. Therefore, only dual crosslinked constructs were employed for further experiments.. Bulk hydrogels (8 mm dia, 16 mm height) were prepared by dispensing the bioink into a custom made mould, followed by photocrosslinking for 5 minutes. Hollow cylindrical constructs (8 mm dia, 16 mm height) were fabricated using embedded bioprinting, followed by photocrosslinking for 5 minutes. Subsequently, the printed constructs were enzymatically crosslinked (10% w/v TG for 1 hour) and then incubated in 1X PBS at physiological conditions for 14 days. At predetermined time points, the constructs were retrieved, photographed [ 21 ] . 2.7. Cytotoxicity Analysis 2.7.1. In vitro extract cytotoxicity assay The cytotoxicity of photocrosslinked, dual crosslinked scaffolds, as well as xanthan gum support bath was determined according to the procedures described in ISO standard 10993-5. Initially, the crosslinked hydrogel scaffolds were incubated in the culture medium (DMEM high glucose supplemented with 10% FBS and 1% antibiotic antimycotic) for about 24 h to prepare sterile extracts. These extracts were collected and serially diluted with fresh culture medium to obtain concentrations of 12.5% (E1), 25% (E2), 50% (E3) and 100% (E4). Liquified phenol (1.5% v/v) and culture medium served as positive and negative controls, respectively. HDFa were seeded at a density of 10,000 cells/well in a 96 well plate and cultured for 24 h. The culture medium was then replaced with respective extracts (E1, E2, E3 and E4), positive and negative control, followed by a 24 h incubation period. Post-incubation, cells were examined microscopically for morphological changes and detachment. Cell viability was qualitatively analyzed using live/dead assay and compared with TCPS and phenol treated group. The culture media was removed and incubated with 2 µL calcein AM (green) and 4 µL of ethidium homodimer − 1 (red) reagent for 10 min under dark conditions. Images were taken using laser scanning confocal microscope (Olympus FV1000, Japan). Quantitative viability assessment was conducted via MTS assay. Briefly, the culture medium was removed from each well and cells were washed twice with PBS. Serum free DMEM (90 µL) and 10 µL of MTS reagent were added to each well and incubated for 3 h. Absorbance was measured at 490 nm, and cell viability (%) was calculated using the following equation [ 19 ]. where OD T is the absorbance of test samples and OD NC is the absorbance of the negative control. 2.7.2. Direct contact cytotoxicity HDFa were seeded at a density of 50,000 cells/ well in 24 well plates for direct cytotoxicity analysis. and cultured until they reached 80–90% confluency. The culture medium was then removed, and cells were gently washed with DPBS. Photocrosslinked and dual crosslinked hydrogel scaffolds were carefully placed on top of the fibroblast monolayer. MTS assay was performed after 24 h of incubation to assess cell viability. Additionally, live/dead staining was performed at 24 h post-incubation with the constructs to evaluate the cytotoxic effects on the HDFa. Images of the hydrogel constructs placed on top of the cell monolayer were captured using an Olympus Inverted Microscope (CKX3-SLP) [ 22 ]. 2.8. Embedded Bioprinting of complex constructs Prior to embedded bioprinting, the well plates and petri dishes were filled with 1.5% (w/v) xanthan gum support bath. The bioink supplemented with LAP photoinitiator was loaded into a 3 mL cartridge and refrigerated at 4 ˚C for 10 minutes. After cooling, the cartridge was transferred to a temperature-controlled printhead maintained at 26 ˚C. Printing was performed using a 25 G needle with an inner diameter of 250 microns. Various complex structures, including a human anatomical heart model, a human brain model, a bifurcated Y-shaped tube, human coronary artery model, were printed using the Cellink BioX6 bioprinter. Extrusion pressure was varied between 50–75 kPa. For heart & brain models, infill density was set at 25%, while for other models, infill was set at 0%. 3D models were downloaded from Thingiverse ( https://www.thingiverse.com/ ) and sliced using Simplify 3D software. 2.9. Development of LUAD Model using Embedded Bioprinting Considering the versatility of embedded bioprinting in fabricating complex and physiologically relevant tissue constructs, a 3D LUAD model was developed to replicate the alveolar sac architecture of native lung tissue. The digital model was created using Autodesk Fusion 360 with dimensions of 5 mm in length and 2 mm in height. The bioinks were prepared and sterilized as follows: 15% (v/v) PEGDA was added to 5% (w/v) of gelatin solution which was dissolved at 50 ˚C. Then, 0.25% (w/v) of LAP was added to the polymer solution (at 40 ˚C) and then filter sterilized using 0.22 µm sterile filter. For support bath preparation, xanthan gum powder was exposed to UV light for 30 minutes. The UV sterilized xanthan gum was then mixed with sterile PBS until a slurry formed. All steps were carried out under sterile cell culture conditions. For embedded bioprinting, A549 lung adenocarcinoma cells were suspended in bioink at a density of 10 million cells/mL. This cell-laden bioink was embedded bioprinted within 24 well plates with 1.5% (w/v) xanthan gum support bath. Immediately after the completion of printing process, constructs were photocrosslinked for 15 seconds, and then gently removed from the support bath using a spatula, incubated in 10% TG solution for an hour at 37 ˚C. This step simultaneously allows both the removal of xanthan gum support bath and efficient enzymatic crosslinking of gelatin. Post enzymatic crosslinking, fresh media was added to the bioprinted LUAD models and the constructs were cultured for 14 days at 37 ˚C. These crosslinking processes improved the shape fidelity, mechanical stability and biomimetic properties. By leveraging the advantages of embedded bioprinting, this LUAD model aims to offer biomimetic structural features to better study tumour progression, drug efficacy and cellular interactions. 2.10. Cell Viability and Proliferation within the LUAD Model At pre-determined timepoints, cell viability of the LUAD model was evaluated using the live/dead assay kit as per the manufacturer’s protocol, as described previously [ 18 ]. The cell viability of the bioprinted LUAD Model was determined qualitatively using a Leica confocal microscope (Stellaris-Leica, Germany). The Z-stack images were captured using the tile scan feature. The proliferative ability of the A549 cells on the 3D LUAD model was evaluated using the MTS assay at various time points. Briefly, the constructs were rinsed with DPBS and transferred to a fresh well plate. Subsequently, serum-free DMEM (100 µL) and MTS reagent (20 µL) were added to each well, followed by incubation for 3 h. Absorbance was measured at 490 nm using a multi-mode reader (Infinite 200 M, Tecan, USA). Four replicates were included for each condition, and the results were expressed as mean ± SD. 2.11. Statistical analysis All results are shown as mean ± SD with n equal to the number of samples. Statistical differences in the mechanical properties of crosslinked hydrogels, cell viability for cytotoxicity assays and cell proliferation calculated using one–way ANOVA followed by the Tukey post–hoc test. All the values for statistical significance were set as p < 0.05. 3. Results and discussion 3.1. Characteristics of bioink and support bath In the present study, the developed low viscous protein-polymer bioink formulation demonstrated remarkable versatility by enabling tunability over mechanical properties, enhanced stiffness, improved biocompatibility and biodegradability. The incorporation of PEGDA contributed significantly to higher diffusion and swelling profiles, without compromising stability [ 23 , 24 ]. Gelatin, in addition, further promoted cellular proliferation and viability, yielding a hybrid formulation with the required mechanical properties and increased compatibility with tunable thermoresponsive gelation kinetics. This protein-polymer blend has two important features: mechanical integrity and biological interactions – a balance imperative for tissue engineering applications. To the best of our knowledge, this unique combination of protein & polymer bioink has not yet been explored for embedded bioprinting, highlighting its novelty. The pseudoplastic xanthan gum support bath used in this study remains well recognized due to its shear thinning behaviour. Shear forces disrupt the highly ordered polymeric network by intermittently disrupting intermolecular hydrogen bonds, resulting in a reversible decrease in viscosity. Interestingly, these hydrogen bonds reform rapidly upon removal of shear stress, allowing the bath to regain its initial viscosity rapidly, which is essential to achieve print fidelity [ 25 , 26 ]. Figure 2 illustrates various complex structures fabricated using embedded bioprinting, demonstrating the capability of the developed platform to produce biomimetic constructs. Figure 3 depicts the development and characterization of LUAD model, showcasing the promising potential of the developed bioink & fabrication methods. 3.2. Rheological properties of the bioink and support bath The primary requirement of any bioink is to possess shear thinning property, which is essential for extrusion based bioprinting. Shear thinning refers to a decrease in viscosity profile of the bioink as the shear forces increase, facilitating smooth extrusion through the printing nozzle while maintaining fidelity post-printing. The bioink formulations developed in this study exhibited excellent shearthinning behaviour with a prominent reduction in viscosity with increasing shear rate. Notably, the storage modulus (G’) of dual crosslinked hydrogels was found to be higher than that of photocrosslinked constructs, indicating that the dual crosslinking strategy improves the mechanical properties of the hydrogels Fig. 4 A, B. For the xanthan gum support bath, viscosity decreased with increasing shear rate, demonstrating classic shear thinning behaviour that was dependent on concentration (Fig. 4 E ). Frequency sweep and amplitude sweep analysis confirmed that the pseudoplastic support bath retained its gel like characteristics under the testing conditions (Fig. 4 C and D) . The relatively lower yield stress values observed for all xanthan gum concentrations show that the material underwent shape deformation during printing, which is advantageous for smooth extrusion of bioink (Fig. 4 H). Shear recovery analysis demonstrated better recovery behaviour for 1.5% and 2% (w/v) xanthan gum when compared to other concentrations, attributed to their enhanced ability to reform hydrogen bonds following shear-induced disruption (happen during printing), thereby promoting shape fidelity after printing (Fig. 4 F & G) . The fluidity is higher for lower concentrations of xanthan gum and therefore maintaining shape fidelity of the printed constructs remains a huge challenge. Although, all xanthan gum concentrations exhibit higher recovery, 1.5% (w/v) xanthan gum was selected as the support bath based on its pseudoplasticity and storage modulus values in the ideal range [ 13 ]. Rheological data showed no significant improvement in modulus at higher xanthan gum concentrations 2% (w/v). Hence, 1.5% (w/v) xanthan gum was chosen for further experiments. Xanthan gum is a widely used support bath material due to its pseudoplastic behaviour and instantaneous reversible viscosity changes under shear forces. Previous studies indicate that xanthan gum undergoes gel-to-sol phase transition at 126% strain [ 17 ]. For an ideal support material, the yield stress values should range from 1–10 Pa, with storage modulus (G’) values between 10–100 Pa [ 27 ]. By comparison, silk fibroin (SF) has been investigated as a viable support bath for breast tumour spheroid formation, exhibiting a yield stress around 7.5 Pa at low shear rates (1–5 s − 1 ) [ 13 ]. In our study, 1.5% (w/v) xanthan gum had storage modulus value within the ideal range and the yield stress slightly above the ideal value. From the preliminary studies, it was observed that 1.5% (w/v) xanthan gum support bath yielded high fidelity constructs which were easily retrievable. Hence 1.5% (w/v) xanthan gum was optimized for further experiments. 3.3. Cytotoxicity Profiles To demonstrate the compatibility of the protein polymer-bioink formulation, toxicity profiling was performed. It was found that the bioink formulations exhibited high cell viability regardless of the crosslinking strategy. According to ISO 10993-5, which considers materials cytocompatible when cell viability is 70% or higher, all groups treated with various extract concentrations exhibited cell viability above 70%, indicating excellent cytocompatibility of the bioink. ( Fig. 5 A i,ii). Live dead staining also revealed the native spindle morphology of the fibroblast, indicating the non-cytotoxic behaviour. In direct contact assay, where hydrogels were placed directly onto cell monolayers, increased cell viability was observed, further affirming the non-cytotoxicity of the printed hydrogel constructs (Fig. 5 B i,ii ). Similar to these findings, Sumana et al., developed a PEGDA-Gelatin hydrogel exhibiting Bingham fluid behaviour, characterized by restricted flow properties. Cytotoxicity evaluation using MC3T3-E1 mouse osteoblastic cells showed cell viability of more than 70% after 24 hours, indicating cytocompatibility. [ 28 ]. In addition, 0.25% (w/v) LAP was chosen as the optimal concentration as reported in the literature [ 29 ]. Lower concentrations of LAP might prolong the crosslinking duration of the printed constructs remaining in the support bath. Higher concentrations of LAP might remain in the hydrogel system in their non radicalized form, severely affecting the cell viability. A drastic decrease in the cellular viability was observed with increasing concentrations of LAP in a previous study [ 29 ]. 3.4. Swelling behaviour and stability analysis Controlled swelling behaviour is a critical attribute of hydrogel constructs, as it facilitates efficient media and nutrient permeation throughout the construct. In this study, the developed protein-polymer hydrogels exhibited a swelling ratio of 400% for photocrosslinked hydrogels and 310% for dual crosslinked constructs after 24 hours (Fig. 6 B ) . The photocrosslinked hydrogels showed a higher swelling ratio compared to the dual crosslinked hydrogels. While the higher water retention property of PEGDA could have contributed to increased swelling characteristics, in photocrosslinked constructs, the uncrosslinked gelatin, which has undergone only physical interactions with PEGDA, might have also resulted in higher swelling owing to the porosity differences. The covalent isopeptide bond formation induced by the enzymatic crosslinking between the glutamine and lysine residues of gelatin resulted in a denser polymer network with reduced swelling. This covalent crosslinking restricts polymer chain mobility, thereby decreasing the swelling ratio compared to only photocrosslinked constructs [ 19 ]. Moreover, stability assessments of both bulk and hollow printed cylindrical constructs revealed over a two weeks incubation period, highlighting enhanced structural integrity of the hydrogels (Fig. 6 A ) . Similarly, P. R. Avallone et al., reported that gelatin and PEDGA hybrid hydrogels with higher PEGDA concentration form covalent crosslinks that confine polymer chains’ mobility. The incorporation of gelatin further improves network integrity, resulting in controlled swelling behaviour and improved mechanical robustness [ 30 ]. 3.5. Cellular viability and proliferation within the printed constructs The developed protein polymer hydrogel matrix supports increased cellular proliferation, attributed to its favourable biophysical cues, alongside the presence of bioactive gelatin. Cellular proliferation within the bioprinted constructs was determined using the MTS Assay for 14 days (Fig. 7 C ) , which demonstrated a significant increase in cellular proliferation over time, indicating that the hydrogel matrix promotes cell infiltration and proliferation ( Fig. 7 A ) . Furthermore, cell viability remained prominent within the bioprinted constructs, with visible cell alignment along the printed strands, suggesting that the hydrogel matrix promotes organized cell proliferation ( Fig. 7 A &B). A similar study employing a sodium alginate-gelatin-fibrinogen based bioink to develop a bioprinted lung cancer model reported that initial cell proliferation, assessed by alamar blue assay, was slower at day 3, increased significantly after 12 days of culture, consistent with gradual cell adaptation and proliferation within the printed milieu [ 31 ]. 4. Conclusion 3D in vitro tumour models are invaluable testing platforms for preclinical drug screening and personalized medicine development. Conventional 3D bioprinting strategies have limitations in fully recapitulating the complex, vascularized tumour microenvironment. However, embedding bioprinting enables the fabrication of highly complex native tissue architecture with perfusable channels. This study successfully designed a low viscosity bioink optimized for bioprinting of native tissues and a LUAD model. The bioink exhibited good printability at optimal printing parameters - pressure between 50–60 kPa, print speed of 3 mm/s, printhead temperature of 26°C and a gauge size of 25G (0.250 mm in diameter): yielding smooth & continuous extrusion. Further, rheological characterization showed that the hydrogel exhibited greater mechanical strength and stiffness. The dual crosslinking strategy enhanced network density, resulting in increased stability and reduced swelling ability compared to only photo-crosslinked hydrogel. Constructs remained stable for a period of two weeks when observed under physiological conditions. Additionally, cytocompatibility assays with more than 70% cell viability in both indirect and direct cytotoxicity assays, confirming bioink’s biocompatibility. The bioink provided a suitable microenvironment for A549 cells demonstrated by uniform distribution and proliferation within the printed construct. Further studies will focus on incorporating stromal cells and endothelial cells to recapitulate the vascularized network present in tumours with greater biological complexity. Integration with microfluidic perfusion systems and bioreactors will enable dynamic culture conditions, resembling in vivo tumour environment through controlled media flow. Thus, embedded bioprinting with this novel bioink serves as a suitable strategy in developing vascularized 3D tumour models suitable for high throughput drug screening, offering a powerful alternative to animal models, especially in oncology research. Declarations Acknowledgement The authors wish to acknowledge the Nano Mission, Department of Science & Technology (DST) (SR/NM/TP–83/2016 (G)), and Prof. T. R. Rajagopalan, R&D Cell of SASTRA Deemed University, for financial and infrastructural support. We also acknowledge the Adhoc funding from the Indian Council of Medical Research (ICMR) (17x3/Adhoc/23/2022–ITR), the ANRF CRG (Exponential Technologies) grant (CRG/2021/007847) and the ANRF-SURE grant (SUR/2022/003181) for financial support. We also thank the TCS Foundation, Mumbai for infrastructural support through Technology Innovation Centre grant. Data availability Data will be made available on request . CRediT authorship contribution statement Aiswarya Ganapthisankarakrishnan: Writing – original draft, Validation, Methodology, Formal analysis, Data curation . Dona Shaji: Methodology, Formal analysis, Data curation . Amrutha Krishnamoorthy: Methodology, Formal analysis, Data curation . Swaminathan Sethuraman: Writing – review & editing, Funding acquisition, Formal analysis . Dhakshinamoorthy Sundaramurthi: Writing – review & editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. 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Budharaju, S. Sethuraman, and D. Sundaramurthi, “Preparation of thermoresponsive & enzymatically crosslinkable gelatin-gellan gum bioink: A protein-polysaccharide hydrogel for 3D bioprinting of complex soft tissues,” Int. J. Biol. Macromol. , 2024; vol. 295, no. p. 139563, 2025, https://doi.org/10.1016/j.ijbiomac.2025.139563. H. Budharaju, D. R. Chellappan, D. Sundaramurthi, and S. Sethuraman, “Protein–in–polysaccharide bioink for 3D bioprinting of muscle mimetic tissue constructs to treat volumetric muscle loss,” Carbohydr. Polym. , 2025; vol. 367, p. 123993, https://doi.org/10.1016/j.carbpol.2025.123993 M. A. Alioglu et al. , “Nested Biofabrication: Matryoshka‐Inspired Intra‐Embedded Bioprinting,” Small Methods , 2024; vol. 8 https://doi.org/10.1002/smtd.202301325. A. Zennifer, D. R. Chellappan, P. Chinnaswamy, A. Subramanian, D. Sundaramurthi, and S. Sethuraman, “Efficacy of 3D printed anatomically equivalent thermoplastic polyurethane guide conduits in promoting the regeneration of critical-sized peripheral nerve defects,” Biofabrication , 2024; vol. 16, no. 4, p. 045015, https://doi.org/10.1002/smtd.202301325 T. Şener Raman et al. , “A study on the material properties of novel PEGDA/gelatin hybrid hydrogels polymerized by electron beam irradiation,” Front. Chem. , 2023; vol. 10, https://doi.org/10.3389/fchem.2022.1094981. H. Yue, Y. Wang, S. Fernandes, C. Vyas, and P. Bartolo, “Bioprinting of GelMA/PEGDA Hybrid Bioinks for SH‐SY5Y Cell Encapsulation: Role of Molecular Weight and Concentration,” Macromol. Biosci. , 2025; vol. 25, no. 6, https://doi.org/10.1002/mabi.202400587 N. Noor, A. Shapira, R. Edri, I. Gal, L. Wertheim, and T. Dvir, “3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts,” 2019; Adv. Sci. , vol. 6, no. 11, https://doi.org/10.1002/advs.201900344 V. D. Trikalitis et al. , “Embedded 3D printing of dilute particle suspensions into dense complex tissue fibers using shear thinning xanthan baths,” Biofabrication , 2023; vol. 15, no. 1, p. 015014, https://doi.org/10.1088/1758-5090/aca124 G. Lai and L. Meagher, “Versatile xanthan gum-based support bath material compatible with multiple crosslinking mechanisms: rheological properties, printability, and cytocompatibility study,” Biofabrication . 2024; vol. 16, no. 3, p. 035005, https://doi.org/10.1088/1758-5090/ad39a8 S. Posritong, R. Flores Chavez, T.-M. G. Chu, and A. Bruzzaniti, “A Pyk2 inhibitor incorporated into a PEGDA-gelatin hydrogel promotes osteoblast activity and mineral deposition,” Biomed. Mater. , 2019; vol. 14, no. 2, p. 025015, https://doi.org/10.1088/1748-605X/aafffa P. A. Jansen et al. , “Spectrophotometric determination of LAP photoinitiator radicalization in ophthalmic applications,” Biomed. Mater. , 2026; vol. 21, no. 2, p. 025011, , doi: 10.1088/1748-605X/ae4a62. P. R. Avallone, N. Russo, N. Gargiulo, N. Grizzuti, and S. Costanzo, “Design and Characterization of Hybrid Gelatin/PEGDA Hydrogels with Tunable Viscoelastic Properties,” Biomacromolecules . 2025; vol. 26, no. 8, pp. 5450–5460, https://doi.org/10.1021/acs.biomac.5c01048 S. Zou, J. Ye, Y. Wei, and J. Xu, “Characterization of 3D-Bioprinted In Vitro Lung Cancer Models Using RNA-Sequencing Techniques,” Bioengineering . 2023; vol. 10, no. 6, p. 667 , https://doi.org/10.3390/bioengineering10060667 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 09 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 28 Apr, 2026 Editor assigned by journal 26 Apr, 2026 Submission checks completed at journal 26 Apr, 2026 First submitted to journal 24 Apr, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9513495","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635630209,"identity":"5dbf2fe5-465c-406d-9dd4-2f1bf97d0e34","order_by":0,"name":"Aiswarya Ganapathisankarakrishnan","email":"","orcid":"","institution":"SASTRA Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Aiswarya","middleName":"","lastName":"Ganapathisankarakrishnan","suffix":""},{"id":635630210,"identity":"34416fa5-7993-49f0-b827-b5dc7f9ac02e","order_by":1,"name":"Dona Shaji","email":"","orcid":"","institution":"SASTRA Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Dona","middleName":"","lastName":"Shaji","suffix":""},{"id":635630211,"identity":"32f96c79-15ee-4a4e-ad37-f24147b9a26e","order_by":2,"name":"Amrutha Krishnamoorthy","email":"","orcid":"","institution":"SASTRA Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Amrutha","middleName":"","lastName":"Krishnamoorthy","suffix":""},{"id":635630212,"identity":"90155dfb-9bc1-41bd-a4de-fe7bf68b6307","order_by":3,"name":"Swaminathan Sethuraman","email":"","orcid":"","institution":"SASTRA Deemed University","correspondingAuthor":false,"prefix":"","firstName":"Swaminathan","middleName":"","lastName":"Sethuraman","suffix":""},{"id":635630213,"identity":"6de0d97b-da66-4c0b-afcf-65ce8e7e9e85","order_by":4,"name":"Dhakshinamoorthy Sundaramurthi","email":"data:image/png;base64,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","orcid":"","institution":"SASTRA Deemed University","correspondingAuthor":true,"prefix":"","firstName":"Dhakshinamoorthy","middleName":"","lastName":"Sundaramurthi","suffix":""}],"badges":[],"createdAt":"2026-04-24 07:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9513495/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9513495/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108944247,"identity":"12fd2502-d254-491d-91b8-d6b0fcc725e3","added_by":"auto","created_at":"2026-05-11 05:57:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the workflow of embedded bioprinting, including printing inside the support bath, photocrosslinking, followed by enzymatic crosslinking and finally construct retrieval from support bath\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/f123b30e6ff933080b6c23e9.jpeg"},{"id":108944248,"identity":"46d7d242-6a01-431c-bf9f-584982f572b0","added_by":"auto","created_at":"2026-05-11 05:57:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1388608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrinting of complex structures using embedded bioprinting approach. Structures include the [A] human anatomical heart, [B] human coronary artery, [C] human ear, [D] a bifurcated Y shaped tube and [E] human anatomical brain. CAD models are included as insets in the top right corners of the respective images.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/393619a0ec08bcafc6a3b57a.jpeg"},{"id":108944232,"identity":"6356fe30-9931-4ea6-b2a2-f49ffa9e8e41","added_by":"auto","created_at":"2026-05-11 05:57:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1181475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment of LUAD model. [A] Schematic representation of alveolar pulmonary network, [B] CAD model mimicking the alveolar compartment, [C] Top view of the CAD model, [D] Side view of the CAD model, [E] SEM images of the printed LUAD model tilted at 35˚, [F] Side view of the printed LUAD model. Insets in panels [E] and [F] show corresponding stereomicrographs.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/9a97b6cc902a729599e01615.jpeg"},{"id":108944245,"identity":"7951e7eb-6702-45ba-bdbe-93b0a2f8011c","added_by":"auto","created_at":"2026-05-11 05:57:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":998739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRheological characterization of crosslinked hydrogels and xanthan gum support bath. [A] \u0026amp; [B] show amplitude and frequency sweep analyses of photocrosslinked \u0026amp; dual crosslinked hydrogel respectively, [C] Amplitude \u0026amp; [D] frequency sweep results for varying concentrations of the xanthan gum support bath, [E] depicts flow curve analysis results, [F] Oscillation thixotropy, [G] Shear thixotropy and [H] Yield Stress of various xanthan gum support bath concentrations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/26029726f6d2f865f7470ccf.jpeg"},{"id":108944234,"identity":"2c0824b7-5795-420c-8703-933cfa21679a","added_by":"auto","created_at":"2026-05-11 05:57:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1859779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[A] Extract cytotoxicity analysis of the crosslinked hydrogels. (i) Live-dead images of HDFa cells treated with different dilutions of hydrogel extracts (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 2); Photo denotes only Photocrosslinked hydrogels, Dual denotes both photocrosslinked and enzymatically crosslinked hydrogels, XG denotes Xanthan Gum. (ii) Quantitative analysis of HDFa cell viability using MTS assay (*\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 4), [B] (i) Live-dead and phase contrast images of HDFa cells in direct contact with crosslinked hydrogels, demonstrating cell viability \u0026amp; morphology (scale bar : 100 μm) (n = 2). G denotes crosslinked hydrogels, C denotes HDFa cells. (ii) Quantitative analysis of viability of HDFa cells in direct contact with crosslinked hydrogels using MTS assay (*\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026lt; 0.05, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e = 4)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/2cdf1718d73d5946dab0c3a1.png"},{"id":108944190,"identity":"9f020ba2-cf75-484e-a6b4-285d1dbd785d","added_by":"auto","created_at":"2026-05-11 05:57:21","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[A] Photographs showing stability of bulk and printed hydrogel constructs over 14 days, revealing no visible deformation, [B] Swelling behaviour of photo and dual crosslinked hydrogels measured upto 24 hours, showing controlled swelling profiles.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/22704e581fb41bc16affde1f.jpeg"},{"id":108944233,"identity":"f78d32fe-9bea-4cd8-a44a-148b39d7d867","added_by":"auto","created_at":"2026-05-11 05:57:47","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":369356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[A] Live/dead images of bioprinted LUAD model on Day 7. (i) Top view (scale bar: 2 mm). (ii) Lateral view (scale bar: 2 mm). (iii) Lateral view of the sectioned region of LUAD model - 1 mm, [B] Depth Profile analysis of LUAD Day 7. (i) Top view 2 mm. (ii) Lateral view of the sectioned region of LUAD model (scale bar: 200 µm), [C] Quantitative cell proliferation assessment for bioprinted LUAD model using MTS assay.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/2d13f5e7c81297b292c95f80.jpeg"},{"id":108944447,"identity":"c7222bdf-b3f7-4ced-befe-a041797f2870","added_by":"auto","created_at":"2026-05-11 05:58:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6333037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9513495/v1/7ac9bb7d-2d2c-4397-ba6a-31c5114022b5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Low Viscosity PEGDA - Gelatin Bioink for Embedded Bioprinting of Complex Structures and Lung Adenocarcinoma (LUAD) Model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTissue \u0026amp; disease models emerge as promising non-animal platforms to investigate disease progression and test therapeutic drugs. Precisely recapitulating complex human physiological systems with \u003cem\u003ein vitro\u003c/em\u003e models require biomimetic cellular organization and structural complexity due to the dependency of tissue functionality on morphological and geometric resemblance to native tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Conventional cancer tissue models, such as 2D monolayer cultures and basic tumour spheroids or organoids, fail to replicate the three-dimensional tumour microenvironment imperative to determine disease progression \u0026amp; drug responses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In recent times, there has been a significant shift from exclusive reliance on chemotherapy to modern therapies, such as immunotherapies and targeted therapies, which contributed to a considerable reduction in mortality rates. However, the successful clinical outcomes of these novel interventions mostly depend on precise prognosis and pathological evaluation. Unfortunately, the poor predictive accuracy of the available preclinical models remains a major bottleneck, often leading to increased attrition rates for new therapeutic drug candidates during clinical trials [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although animal models may offer better insights, alternatively, they fail to fully replicate the human-specific responses due to species level genetic \u0026amp; physiological differences. Considering the above, there is a huge clinical demand to establish next-generation \u003cem\u003ein vitro\u003c/em\u003e cancer models that have the potential to offer precise predictions on therapeutic outcome and enable mechanistic understanding of tumour development in a clinically relevant context comparable to humans [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOver the last decade, LUAD happened to be the most common subtype of lung cancer, amounting for about 40% of all cases across the globe. Typically associated with chronic smoking, incidence of LUAD has been notably rising among regular smokers and also among non-smokers underscoring its complexity in aetiology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite considerable breakthroughs in clinical treatment strategies (targeted immunotherapies and genetically targeted therapies), LUAD continues to be a challenging global health burden. Additionally, the five year survival rate generally remains between 5% and 20% (influenced by healthcare access, geographic location and individual patient variability), highlighting the dire need for enhanced therapeutic outcomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Existing conventional models (2D monolayer cultures, tumour spheroids, and \u003cem\u003eex vivo\u003c/em\u003e tissue cultures) for LUAD poorly reproduce the tumour microenvironment (TME), though offer valuable insights to some extent. The TME comprises a complex vascular network around the cancer tissue, immune cells \u0026amp; components, fibroblasts, extracellular matrix components and other factors (soluble growth factors, signalling molecules like chemokines, cytokines) that are crucial in tumour progression and therapeutic response. Nevertheless, these conventional models have failed to capture cell-cell \u0026amp; cell-matrix interactions, spatial and mechanical heterogeneity that are hallmarks of tumours, reducing the predictive efficacy for drug responses and tumour behaviour [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn order to better mimic the TME, advanced fabrication methods have paved new ways for creating physiologically relevant LUAD models by enabling precise spatial localization of multiple cell types and matrix components [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Still, traditional extrusion-based 3D bioprinting techniques experience several limitations, particularly in fabricating complex, overhanging vascularized structures due to the low viscosity of bioinks and also gravitational constraints [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These limitations contribute to printing artifacts, mechanical instability and poor resolution. To overcome these challenges, researchers are now opting for embedded bioprinting \u0026ndash; an innovative approach where low-viscosity bioinks are supported inside a supportive, shear thinning embedding bath that enables the fabrication of complex, biomimetic and self-supporting tissue constructs with improved resolution \u0026amp; fidelity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To develop LUAD models that closely replicate the TME, including vascular networks \u0026amp; cellular heterogeneity- embedded bioprinting may be an ideal choice. This printing method also enables the incorporation of multiple bioinks and cell types, offering versatile platforms to investigate tumour biology and determine the efficacy of therapeutics in a closer context to \u003cem\u003ein vivo\u003c/em\u003e microenvironment of humans [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. By leveraging embedded bioprinting, physiologically relevant LUAD models can be fabricated and tuned to better mimic native tissue features, thereby advancing personalised medicine and drug screening efforts [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the embedded bioprinting approach stands unique in successfully overcoming the limitations of conventional models and other 3D bioprinting techniques, providing promising pathways to enhance understanding of tumour progression and therapeutic outcomes in LUAD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we fabricated microarchitectures of the human lung alveoli, digitally designed using Fusion 360. For the bioink, we used a low-viscosity formulation comprising gelatin (5% w/v) and PEGDA (15% w/v), which was extruded into a xanthan gum (1.5% w/v) support bath. Gelatin \u0026ndash; a collagen derived polymer with intrinsic cell-binding motifs is widely preferred for bioprinting applications due to its ease of crosslinking and ability to support cell adhesion \u0026amp; proliferation. PEGDA was chosen for its capacity to undergo photocrosslinking, thereby imparting mechanical stability to bioprinted constructs and its well-known biocompatibility. Xanthan gum \u0026ndash; known for its excellent tunability of rheological properties such as flow behaviour, yield stress and thixotropy was preferred as an ideal support bath for embedded bioprinting. Herein, we systematically evaluated the rheological properties of the pseudoplastic xanthan gum bath, including viscoelasticity, shear thinning, and structural recovery, to confirm its suitability for supporting extrusion-based embedded bioprinting. The resulting platform was demonstrated for precise printing of complex constructs and the development of a LUAD model with well-defined architectural features. Our findings reveal that the engineered gelatin-PEGDA bioink and shear thinning xanthan gum support bath offer a robust strategy for bioprinting of biomimetic lung microarchitectures. This strategy holds excellent promise towards developing physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e models for drug screening applications.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003ePoly (Ethylene Glycol) Diacrylate (PEGDA) Mn 700 g/mol, Xanthan gum from \u003cem\u003eXanthomonas campestris\u003c/em\u003e, and gelatin from porcine skin, gel strength 300 Type A were purchased from Sigma Aldrich, India. Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) was purchased from TCI Chemicals, Tokyo Chemical Industry Co., Ltd, Tokyo, Japan. Trypsin EDTA, live/dead assay kit and fetal bovine serum (FBS) were procured from Thermo Fisher Scientific, India. MTS Reagent was purchased from Abcam, India. Phenol and Triton X-100 were procured from Merck, India. Dulbecco\u0026apos;s Phosphate Buffer Saline (DPBS) and Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) were purchased from HiMedia Laboratories Private Limited, Mumbai, India. Transglutaminase (Enzyme activity 200 U/g) was procured from Ultreze Enzyme Private Limited, Surat, India. A549 \u003cstrong\u003e(CCL-185)\u003c/strong\u003e Lung cancer cell line was procured from National Centre for Cell Science (NCCS) Pune, India. Human dermal fibroblast (HDFa) (\u003cstrong\u003ePCS-201-012\u003c/strong\u003e) cells were purchased from HiMedia Laboratories Private Limited, Mumbai, India. All cell lines were sourced from ATCC, a well-established, authenticated repository with no reported incidents of misidentification or contamination, and the cell lines were certified to be free of mycoplasma contamination.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Preparation of low viscosity bioink\u003c/h2\u003e\n \u003cp\u003eThe low viscosity bioink suitable for embedded bioprinting was prepared as follows. Gelatin at a concentration of 5% (w/v) was completely dissolved in 1X PBS at 50 ˚C for 15 minutes. Subsequently, PEGDA (15% v/v) was added to the gelatin solution, which was equilibrated at room temperature and stirred further for another 15 minutes. Once both polymers were thoroughly dissolved, the solution was transferred to a suitable container. LAP (0.25% w/v) was then added under dark conditions, with continued stirring until completely dissolved [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Preparation of Support Bath Medium\u003c/h2\u003e\n \u003cp\u003eThe support bath was prepared by dissolving xanthan gum at concentrations of 0.5%, 1%, 1.5% and 2% w/v in 1X PBS and allowing the mixture to hydrate overnight at room temperature. Prior to printing, the xanthan gum slurry was centrifuged at 500 rpm to remove bubbles and then used as substrate for printing [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Crosslinking of bulk and printed gel\u003c/h2\u003e\n \u003cp\u003eHydrogels were prepared by manually dispensing/printing the ink mixed with 0.25% (w/v) LAP into titer well plates. Photocrosslinking was performed by illuminating 405 nm light for 15 seconds (rheology) / 1 minute (swelling behaviour analysis) / 5 minutes (stability analysis), 15 minutes (complex structures). To prepare dual crosslinked hydrogels, photocrosslinked hydrogels (as above) were incubated in 10% (w/v) transglutaminase solution for 1 hour. Based on the optimization studies on crosslinking, the duration of photocrosslinking was increased for bulk and bioprinted constructs with larger dimension (complex structures) to ensure complete crosslinking.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Rheological characterization\u003c/h2\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.1. Bioink\u003c/h2\u003e\n \u003cp\u003eThe rheological properties of the bioink and support bath were studied using an Anton Parr Rheometer MCR302 with a parallel plate setup (dia 25 mm, distance 1 mm). Storage (G\u0026rsquo;) and loss modulus (G\u0026rdquo;) of the hydrogels were determined. Hydrogels were prepared by dispensing 500 \u0026micro;L of the ink mixed with 0.25% (w/v) LAP into 12 well plates. Photocrosslinking was performed by illuminating 405 nm light for 15 seconds (\u0026ldquo;P\u0026rdquo;), dual crosslinked hydrogels (\u0026ldquo;P-G\u0026rdquo;), were photocrosslinked for 15 seconds, followed by incubation in 10% (w/v) transglutaminase solution for 1 hour. Amplitude sweep test was performed (0.01 to 1000% strain rate at 10 Hz) to determine the linear viscoelastic region (LVR), and frequency sweep analysis covered a range from 0.1 to 100 Hz. The strain % was set from the range of values within the linear viscoelastic region measured from the amplitude sweep analysis. Strain % of 1.91% and 2.67% was set for the photocrosslinked and dual crosslinked hydrogels, respectively. All experiments were conducted in triplicate [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.2. Support Bath\u003c/h2\u003e\n \u003cp\u003eThe rheological properties of the xanthan gum support bath at different concentrations, 0.5%, 1%, 1.5%, and 2% (w/v), were analyzed. Flow curve analysis was performed to study the shear thinning behaviour of the pseudoplastic support bath by varying the shear rate from 0.01 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The amplitude and frequency sweep analysis were carried out under the previously described conditions. In addition, the shear recovery properties of each concentration were evaluated using oscillation thixotropy and shear thixotropic analyses. Oscillation thixotropy was analysed by measuring the hydrogel modulus over three cycles, alternating between low strain (1%) for 60 s and high strain (100%) for 60 s. Shear thixotropy analysis involved measuring viscosity at a low shear rate (1 s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) for 60 s, followed by higher shear rate (100 s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) for 5 s, and subsequently recovery at a low shear rate (1 s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) for 120 s. All experiments were done in triplicate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Swelling Behaviour and Stability Analysis\u003c/h2\u003e\n \u003cp\u003eThe swelling behaviour of photocrosslinked and dual crosslinked hydrogels was determined as follows. For photocrosslinked hydrogels, 500 \u0026micro;L of bioink was manually dispensed into a 24-well plate and photocrosslinked for 1 minute. Dual crosslinked hydrogels were prepared by incubating the photocrosslinked hydrogels (manually dispensed) in 10% (w/v) TG for 1 hour. After crosslinking, the constructs were lyophilized, and their initial dry weights were taken (W\u003csub\u003e1\u003c/sub\u003e). The constructs were then rehydrated in 1 ml of 1X PBS, and the wet weight was recorded at various time points: 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h and 24 h (W\u003csub\u003e2\u003c/sub\u003e). The swelling ratio was calculated using the following formula.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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mzh8+DD++c9/YmtrSz/RrqysPNDlkjzPw6lTp5DL5XD69GlMT093fH8PipwHuypabG5uAgB+/vOf20kdpBr6Xqp433vvPbRarW2tnCHXY9R3Iv8/TrW4rO5xL684VfW0eywuLsL3/cA1eeTIETiOg2q12reVggDg6aefBiKGW0lzVjKZ7FgtR9Y+DSuUSF4r+fNeM/DgiXbJMuzp+ZtvvgEi1tGTJ6833ngDW1tbgXFP0k62uroKz/MCHYWErOO3tLQUWgUS9nl22rlz51CtVu3ND5yM8bJLXLVaLfRmMpnVPM1mM7J0JuQ8FIvFjn2l1sBur44ipeJeVU22SqWiOwmZna+kGsp+wPr8888Dv9vk/++GUjoNp1qthvn5efzlL38JXJOJREJ3djt27FjHPfOgpFIpff3aneM++eQTAMDJkycD22HUFn744YeB7c1mE57nIZlMhubne4Ld/bbfpNu04ziB8XzLy8t6CEPYXIjm8IxuYz7DhqIIc/xULpdT+XxeLSws6L81hzx0m5sxamiJUv8bnmGPVTS7bdvDMMxxjyYZqhL1ymQyHUNIwkg387DvTVlDRAqFgiqXyyqfz+uhLWHHY47jzGQyKp/PB85pt+OV9wwb5+k4zraOSUQNVerGdd3I6yQZMil8tzGgMq1Z1PsR9WKO5w4bdmXeS/aQvV58Y5L2qLxVyPA1x5gYXu7LqOtfWflho9EIjBEPO569YuDBs1wu6/lr5YKQjDSXy3U9uXIRhGWuEpR7zaNYKBT0+0imZ//fjY0NnSnLPvaYQHmZAcl8XzOQhL2f/D95aJA08yI1b6qolxMy36TJ/rxhAV+1v7+wsY3mA4cdQM2xuq6xBFG34zX/ttd52A4J/FHj02zyQBJ1U8t1JGN+5f23uz9RHL7vd71X7HS513qR+zbqFfWwt7GxEVhezswLujHzD8R4sN/NBh48afskYw67COWJEF0y9lHhtpdR6hV4ZfB5r6d3KUVL5mE/NAi/x/Jm9D37ATHsQUx1qYFRIUEk6j2IBoXBc0hJtXG3jH47M+yMAjOIxam+vR9SK7CdoE3BKTmjgp5Z4gk7j1JFzmuehsGOdBii3v7zn//Ymzo0m004jhMYbzWKJiYmcPfuXQDAvn37Ojo8PGie52Hfvn0AgLt370ZOpk3/I8PNAOChhx4KpAlzFrGwGaakt7PrutvqUEbUTwyeQ2p6ehqu66JYLGJxcTEw+1GtVkM2m0W1Wr2nVUD2okQigfX1dZw8eRLvvvtu3wKo53l49913cfLkSayvr/O736ZEIqF7c4YNa6hUKmi1WnqilLDhR9JLvt9jHom2g8FziK2srCCfz+Ozzz5DMpnUg+RfeOEFjI+Pw/f9vdsN/B5ls1msr6/37Xvp9/vvZb/5zW+AiKFFV69eRTKZxO9///vIfWTYlFmKJdopY+r74RVERH1Vq9Xw+OOPA/h+qkDT1NQUDh48iCNHjuCRRx6J3AcA1tfXA9uJdgJLnkQ0EKlUSs/WZE5KIqsg7d+/H4lEQk/nFrbP0aNH9TaincTgSUQDEzad2+3bt+E4jp41TPa5c+eO3keWHGSVLQ0LBk8iGhiZ59Rs07x06ZIOmDDWt11bW9PbLl68iEwmww5aNDQYPIloYGQe6lKppOdpXV1dDUwebs5VjfaQrFKp1HOCcc/zMDk5OZTzVNPew+BJRAMzMTGh2zRv3bqlh6hIwDT3abVaqNfrPatsJWi+/PLLHcsZEvULgycRDZSM5bxx4wauXr0K13U7JpqQatzbt2/j0qVLkVW2zWYT58+fx/nz57G8vGwnE/UNgycRDdT+/fuBdrXs6upqoL1TSLvn9evXUSwWI6tsE4kEVlZWMD09HTlzEVE/MHgS0UDJ1IbVahXValWv8WqSatylpSWgS5Ut0U5h8CSigUokEnBdFwDgOE7oPLVm22hUlS3RTmLwJKKBe+mllwCjbTOMpEVV2RLtJE7PR0R7QqVSwYEDB1Aul0NLs0QPEkueRLQnfPPNNwCAb7/91k4ieuBY8iSiXe348eNYXV1FtVoF2u2oTz31FJ588km8+eab9u5EDwSDJxERUUystiUiIoqJwZOIiCgmBk8iIqKYGDyJiIhiYvAkIiKKicGTiIgoJgZPIiKimBg8iYiIYmLwJCIiionBk4iIKCYGTyIiopgYPImIiGJi8CQiIoqJwZOIiCgmBk8iIqKYGDyJiIhiYvAkIiKKicGTiIgopv8H8JAejpOVX64AAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n \u003cp\u003eThe stability analysis for the protein-polymer hydrogels was conducted over a period of 2 weeks to evaluate their degradation profile and structural integrity during incubation in 1X PBS at 37 ˚C. The stability analysis of the bulk hydrogel was compared with that of the hollow printed construct to contrast the stability of the hydrogel formulation across different material infill (100% and 0%). In addition, rheological studies, showed that the dual crosslinked constructs exhibit greater mechanical strength compared to the only-photocrosslinked constructs. Therefore, only dual crosslinked constructs were employed for further experiments.. Bulk hydrogels (8 mm dia, 16 mm height) were prepared by dispensing the bioink into a custom made mould, followed by photocrosslinking for 5 minutes. Hollow cylindrical constructs (8 mm dia, 16 mm height) were fabricated using embedded bioprinting, followed by photocrosslinking for 5 minutes. Subsequently, the printed constructs were enzymatically crosslinked (10% w/v TG for 1 hour) and then incubated in 1X PBS at physiological conditions for 14 days. At predetermined time points, the constructs were retrieved, photographed [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Cytotoxicity Analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.1. \u003cem\u003eIn vitro\u003c/em\u003e extract cytotoxicity assay\u003c/h2\u003e\n \u003cp\u003eThe cytotoxicity of photocrosslinked, dual crosslinked scaffolds, as well as xanthan gum support bath was determined according to the procedures described in ISO standard 10993-5. Initially, the crosslinked hydrogel scaffolds were incubated in the culture medium (DMEM high glucose supplemented with 10% FBS and 1% antibiotic antimycotic) for about 24 h to prepare sterile extracts. These extracts were collected and serially diluted with fresh culture medium to obtain concentrations of 12.5% (E1), 25% (E2), 50% (E3) and 100% (E4). Liquified phenol (1.5% v/v) and culture medium served as positive and negative controls, respectively. HDFa were seeded at a density of 10,000 cells/well in a 96 well plate and cultured for 24 h. The culture medium was then replaced with respective extracts (E1, E2, E3 and E4), positive and negative control, followed by a 24 h incubation period. Post-incubation, cells were examined microscopically for morphological changes and detachment. Cell viability was qualitatively analyzed using live/dead assay and compared with TCPS and phenol treated group. The culture media was removed and incubated with 2 \u0026micro;L calcein AM (green) and 4 \u0026micro;L of ethidium homodimer\u0026thinsp;\u0026minus;\u0026thinsp;1 (red) reagent for 10 min under dark conditions. Images were taken using laser scanning confocal microscope (Olympus FV1000, Japan). Quantitative viability assessment was conducted via MTS assay. Briefly, the culture medium was removed from each well and cells were washed twice with PBS. Serum free DMEM (90 \u0026micro;L) and 10 \u0026micro;L of MTS reagent were added to each well and incubated for 3 h. Absorbance was measured at 490 nm, and cell viability (%) was calculated using the following equation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003ewhere OD\u003csub\u003eT\u003c/sub\u003e is the absorbance of test samples and OD\u003csub\u003eNC\u003c/sub\u003e is the absorbance of the negative control.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e2.7.2. Direct contact cytotoxicity\u003c/h2\u003e\n \u003cp\u003eHDFa were seeded at a density of 50,000 cells/ well in 24 well plates for direct cytotoxicity analysis. and cultured until they reached 80\u0026ndash;90% confluency. The culture medium was then removed, and cells were gently washed with DPBS. Photocrosslinked and dual crosslinked hydrogel scaffolds were carefully placed on top of the fibroblast monolayer. MTS assay was performed after 24 h of incubation to assess cell viability. Additionally, live/dead staining was performed at 24 h post-incubation with the constructs to evaluate the cytotoxic effects on the HDFa. Images of the hydrogel constructs placed on top of the cell monolayer were captured using an Olympus Inverted Microscope (CKX3-SLP) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Embedded Bioprinting of complex constructs\u003c/h2\u003e\n \u003cp\u003ePrior to embedded bioprinting, the well plates and petri dishes were filled with 1.5% (w/v) xanthan gum support bath. The bioink supplemented with LAP photoinitiator was loaded into a 3 mL cartridge and refrigerated at 4 ˚C for 10 minutes. After cooling, the cartridge was transferred to a temperature-controlled printhead maintained at 26 ˚C. Printing was performed using a 25 G needle with an inner diameter of 250 microns. Various complex structures, including a human anatomical heart model, a human brain model, a bifurcated Y-shaped tube, human coronary artery model, were printed using the Cellink BioX6 bioprinter. Extrusion pressure was varied between 50\u0026ndash;75 kPa. For heart \u0026amp; brain models, infill density was set at 25%, while for other models, infill was set at 0%. 3D models were downloaded from Thingiverse (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.thingiverse.com/\u003c/span\u003e\u003c/span\u003e) and sliced using Simplify 3D software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Development of LUAD Model using Embedded Bioprinting\u003c/h2\u003e\n \u003cp\u003eConsidering the versatility of embedded bioprinting in fabricating complex and physiologically relevant tissue constructs, a 3D LUAD model was developed to replicate the alveolar sac architecture of native lung tissue. The digital model was created using Autodesk Fusion 360 with dimensions of 5 mm in length and 2 mm in height. The bioinks were prepared and sterilized as follows: 15% (v/v) PEGDA was added to 5% (w/v) of gelatin solution which was dissolved at 50 ˚C. Then, 0.25% (w/v) of LAP was added to the polymer solution (at 40 ˚C) and then filter sterilized using 0.22 \u0026micro;m sterile filter. For support bath preparation, xanthan gum powder was exposed to UV light for 30 minutes. The UV sterilized xanthan gum was then mixed with sterile PBS until a slurry formed. All steps were carried out under sterile cell culture conditions.\u003c/p\u003e\n \u003cp\u003eFor embedded bioprinting, A549 lung adenocarcinoma cells were suspended in bioink at a density of 10\u0026nbsp;million cells/mL. This cell-laden bioink was embedded bioprinted within 24 well plates with 1.5% (w/v) xanthan gum support bath. Immediately after the completion of printing process, constructs were photocrosslinked for 15 seconds, and then gently removed from the support bath using a spatula, incubated in 10% TG solution for an hour at 37 ˚C. This step simultaneously allows both the removal of xanthan gum support bath and efficient enzymatic crosslinking of gelatin. Post enzymatic crosslinking, fresh media was added to the bioprinted LUAD models and the constructs were cultured for 14 days at 37 ˚C. These crosslinking processes improved the shape fidelity, mechanical stability and biomimetic properties. By leveraging the advantages of embedded bioprinting, this LUAD model aims to offer biomimetic structural features to better study tumour progression, drug efficacy and cellular interactions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. Cell Viability and Proliferation within the LUAD Model\u003c/h2\u003e\n \u003cp\u003eAt pre-determined timepoints, cell viability of the LUAD model was evaluated using the live/dead assay kit as per the manufacturer\u0026rsquo;s protocol, as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The cell viability of the bioprinted LUAD Model was determined qualitatively using a Leica confocal microscope (Stellaris-Leica, Germany). The Z-stack images were captured using the tile scan feature.\u003c/p\u003e\n \u003cp\u003eThe proliferative ability of the A549 cells on the 3D LUAD model was evaluated using the MTS assay at various time points. Briefly, the constructs were rinsed with DPBS and transferred to a fresh well plate. Subsequently, serum-free DMEM (100 \u0026micro;L) and MTS reagent (20 \u0026micro;L) were added to each well, followed by incubation for 3 h. Absorbance was measured at 490 nm using a multi-mode reader (Infinite 200 M, Tecan, USA). Four replicates were included for each condition, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll results are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD with n equal to the number of samples. Statistical differences in the mechanical properties of crosslinked hydrogels, cell viability for cytotoxicity assays and cell proliferation calculated using one\u0026ndash;way ANOVA followed by the Tukey post\u0026ndash;hoc test. All the values for statistical significance were set as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characteristics of bioink and support bath\u003c/h2\u003e \u003cp\u003eIn the present study, the developed low viscous protein-polymer bioink formulation demonstrated remarkable versatility by enabling tunability over mechanical properties, enhanced stiffness, improved biocompatibility and biodegradability. The incorporation of PEGDA contributed significantly to higher diffusion and swelling profiles, without compromising stability [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Gelatin, in addition, further promoted cellular proliferation and viability, yielding a hybrid formulation with the required mechanical properties and increased compatibility with tunable thermoresponsive gelation kinetics. This protein-polymer blend has two important features: mechanical integrity and biological interactions \u0026ndash; a balance imperative for tissue engineering applications. To the best of our knowledge, this unique combination of protein \u0026amp; polymer bioink has not yet been explored for embedded bioprinting, highlighting its novelty. The pseudoplastic xanthan gum support bath used in this study remains well recognized due to its shear thinning behaviour. Shear forces disrupt the highly ordered polymeric network by intermittently disrupting intermolecular hydrogen bonds, resulting in a reversible decrease in viscosity. Interestingly, these hydrogen bonds reform rapidly upon removal of shear stress, allowing the bath to regain its initial viscosity rapidly, which is essential to achieve print fidelity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates various complex structures fabricated using embedded bioprinting, demonstrating the capability of the developed platform to produce biomimetic constructs. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicts the development and characterization of LUAD model, showcasing the promising potential of the developed bioink \u0026amp; fabrication methods.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Rheological properties of the bioink and support bath\u003c/h2\u003e \u003cp\u003eThe primary requirement of any bioink is to possess shear thinning property, which is essential for extrusion based bioprinting. Shear thinning refers to a decrease in viscosity profile of the bioink as the shear forces increase, facilitating smooth extrusion through the printing nozzle while maintaining fidelity post-printing. The bioink formulations developed in this study exhibited excellent shearthinning behaviour with a prominent reduction in viscosity with increasing shear rate. Notably, the storage modulus (G\u0026rsquo;) of dual crosslinked hydrogels was found to be higher than that of photocrosslinked constructs, indicating that the dual crosslinking strategy improves the mechanical properties of the hydrogels Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B.\u003c/p\u003e \u003cp\u003eFor the xanthan gum support bath, viscosity decreased with increasing shear rate, demonstrating classic shear thinning behaviour that was dependent on concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e Frequency sweep and amplitude sweep analysis confirmed that the pseudoplastic support bath retained its gel like characteristics under the testing conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003eand D)\u003c/b\u003e. The relatively lower yield stress values observed for all xanthan gum concentrations show that the material underwent shape deformation during printing, which is advantageous for smooth extrusion of bioink (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Shear recovery analysis demonstrated better recovery behaviour for 1.5% and 2% (w/v) xanthan gum when compared to other concentrations, attributed to their enhanced ability to reform hydrogen bonds following shear-induced disruption (happen during printing), thereby promoting shape fidelity after printing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF \u003cb\u003e\u0026amp; G)\u003c/b\u003e. The fluidity is higher for lower concentrations of xanthan gum and therefore maintaining shape fidelity of the printed constructs remains a huge challenge. Although, all xanthan gum concentrations exhibit higher recovery, 1.5% (w/v) xanthan gum was selected as the support bath based on its pseudoplasticity and storage modulus values in the ideal range [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Rheological data showed no significant improvement in modulus at higher xanthan gum concentrations 2% (w/v). Hence, 1.5% (w/v) xanthan gum was chosen for further experiments. Xanthan gum is a widely used support bath material due to its pseudoplastic behaviour and instantaneous reversible viscosity changes under shear forces. Previous studies indicate that xanthan gum undergoes gel-to-sol phase transition at 126% strain [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For an ideal support material, the yield stress values should range from 1\u0026ndash;10 Pa, with storage modulus (G\u0026rsquo;) values between 10\u0026ndash;100 Pa [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. By comparison, silk fibroin (SF) has been investigated as a viable support bath for breast tumour spheroid formation, exhibiting a yield stress around 7.5 Pa at low shear rates (1\u0026ndash;5 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, 1.5% (w/v) xanthan gum had storage modulus value within the ideal range and the yield stress slightly above the ideal value. From the preliminary studies, it was observed that 1.5% (w/v) xanthan gum support bath yielded high fidelity constructs which were easily retrievable. Hence 1.5% (w/v) xanthan gum was optimized for further experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cytotoxicity Profiles\u003c/h2\u003e \u003cp\u003eTo demonstrate the compatibility of the protein polymer-bioink formulation, toxicity profiling was performed. It was found that the bioink formulations exhibited high cell viability regardless of the crosslinking strategy. According to ISO 10993-5, which considers materials cytocompatible when cell viability is 70% or higher, all groups treated with various extract concentrations exhibited cell viability above 70%, indicating excellent cytocompatibility of the bioink. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003ei,ii).\u003c/b\u003e Live dead staining also revealed the native spindle morphology of the fibroblast, indicating the non-cytotoxic behaviour. In direct contact assay, where hydrogels were placed directly onto cell monolayers, increased cell viability was observed, further affirming the non-cytotoxicity of the printed hydrogel constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u003cb\u003ei,ii\u003c/b\u003e). Similar to these findings, Sumana et al., developed a PEGDA-Gelatin hydrogel exhibiting Bingham fluid behaviour, characterized by restricted flow properties. Cytotoxicity evaluation using MC3T3-E1 mouse osteoblastic cells showed cell viability of more than 70% after 24 hours, indicating cytocompatibility. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, 0.25% (w/v) LAP was chosen as the optimal concentration as reported in the literature [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Lower concentrations of LAP might prolong the crosslinking duration of the printed constructs remaining in the support bath. Higher concentrations of LAP might remain in the hydrogel system in their non radicalized form, severely affecting the cell viability. A drastic decrease in the cellular viability was observed with increasing concentrations of LAP in a previous study [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Swelling behaviour and stability analysis\u003c/h2\u003e \u003cp\u003eControlled swelling behaviour is a critical attribute of hydrogel constructs, as it facilitates efficient media and nutrient permeation throughout the construct. In this study, the developed protein-polymer hydrogels exhibited a swelling ratio of 400% for photocrosslinked hydrogels and 310% for dual crosslinked constructs after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The photocrosslinked hydrogels showed a higher swelling ratio compared to the dual crosslinked hydrogels. While the higher water retention property of PEGDA could have contributed to increased swelling characteristics, in photocrosslinked constructs, the uncrosslinked gelatin, which has undergone only physical interactions with PEGDA, might have also resulted in higher swelling owing to the porosity differences. The covalent isopeptide bond formation induced by the enzymatic crosslinking between the glutamine and lysine residues of gelatin resulted in a denser polymer network with reduced swelling. This covalent crosslinking restricts polymer chain mobility, thereby decreasing the swelling ratio compared to only photocrosslinked constructs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, stability assessments of both bulk and hollow printed cylindrical constructs revealed over a two weeks incubation period, highlighting enhanced structural integrity of the hydrogels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Similarly, P. R. Avallone et al., reported that gelatin and PEDGA hybrid hydrogels with higher PEGDA concentration form covalent crosslinks that confine polymer chains\u0026rsquo; mobility. The incorporation of gelatin further improves network integrity, resulting in controlled swelling behaviour and improved mechanical robustness [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Cellular viability and proliferation within the printed constructs\u003c/h2\u003e \u003cp\u003eThe developed protein polymer hydrogel matrix supports increased cellular proliferation, attributed to its favourable biophysical cues, alongside the presence of bioactive gelatin. Cellular proliferation within the bioprinted constructs was determined using the MTS Assay for 14 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, which demonstrated a significant increase in cellular proliferation over time, indicating that the hydrogel matrix promotes cell infiltration and proliferation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Furthermore, cell viability remained prominent within the bioprinted constructs, with visible cell alignment along the printed strands, suggesting that the hydrogel matrix promotes organized cell proliferation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e\u0026amp;B).\u003c/b\u003e A similar study employing a sodium alginate-gelatin-fibrinogen based bioink to develop a bioprinted lung cancer model reported that initial cell proliferation, assessed by alamar blue assay, was slower at day 3, increased significantly after 12 days of culture, consistent with gradual cell adaptation and proliferation within the printed \u003cem\u003emilieu\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e3D \u003cem\u003ein vitro\u003c/em\u003e tumour models are invaluable testing platforms for preclinical drug screening and personalized medicine development. Conventional 3D bioprinting strategies have limitations in fully recapitulating the complex, vascularized tumour microenvironment. However, embedding bioprinting enables the fabrication of highly complex native tissue architecture with perfusable channels. This study successfully designed a low viscosity bioink optimized for bioprinting of native tissues and a LUAD model. The bioink exhibited good printability at optimal printing parameters - pressure between 50\u0026ndash;60 kPa, print speed of 3 mm/s, printhead temperature of 26\u0026deg;C and a gauge size of 25G (0.250 mm in diameter): yielding smooth \u0026amp; continuous extrusion. Further, rheological characterization showed that the hydrogel exhibited greater mechanical strength and stiffness. The dual crosslinking strategy enhanced network density, resulting in increased stability and reduced swelling ability compared to only photo-crosslinked hydrogel. Constructs remained stable for a period of two weeks when observed under physiological conditions. Additionally, cytocompatibility assays with more than 70% cell viability in both indirect and direct cytotoxicity assays, confirming bioink\u0026rsquo;s biocompatibility. The bioink provided a suitable microenvironment for A549 cells demonstrated by uniform distribution and proliferation within the printed construct. Further studies will focus on incorporating stromal cells and endothelial cells to recapitulate the vascularized network present in tumours with greater biological complexity. Integration with microfluidic perfusion systems and bioreactors will enable dynamic culture conditions, resembling \u003cem\u003ein vivo\u003c/em\u003e tumour environment through controlled media flow. Thus, embedded bioprinting with this novel bioink serves as a suitable strategy in developing vascularized 3D tumour models suitable for high throughput drug screening, offering a powerful alternative to animal models, especially in oncology research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to acknowledge the Nano Mission, Department of Science \u0026amp; Technology (DST) (SR/NM/TP\u0026ndash;83/2016 (G)), and Prof. T. R. Rajagopalan, R\u0026amp;D Cell of SASTRA Deemed University, for financial and infrastructural support. We also acknowledge the Adhoc funding from the Indian Council of Medical Research (ICMR) (17x3/Adhoc/23/2022\u0026ndash;ITR), the ANRF CRG (Exponential Technologies) grant (CRG/2021/007847) and the ANRF-SURE grant (SUR/2022/003181) for financial support. We also thank the TCS Foundation, Mumbai for infrastructural support through Technology Innovation Centre grant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAiswarya Ganapthisankarakrishnan:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; original draft, Validation, Methodology, Formal analysis, Data curation\u003cstrong\u003e. Dona Shaji: \u0026nbsp;\u003c/strong\u003eMethodology, Formal analysis, Data curation\u003cstrong\u003e. Amrutha Krishnamoorthy:\u0026nbsp;\u003c/strong\u003eMethodology, Formal analysis, Data curation\u003cstrong\u003e. Swaminathan Sethuraman:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Funding acquisition, Formal analysis\u003cstrong\u003e. Dhakshinamoorthy Sundaramurthi:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eY. 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Xu, \u0026ldquo;Characterization of 3D-Bioprinted In Vitro Lung Cancer Models Using RNA-Sequencing Techniques,\u0026rdquo; \u003cem\u003eBioengineering\u003c/em\u003e. 2023; vol. 10, no. 6, p. 667 , https://doi.org/10.3390/bioengineering10060667\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Embedded bioprinting, LUAD Model, low viscosity, support bath, shear thinning, PEGDA","lastPublishedDoi":"10.21203/rs.3.rs-9513495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9513495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer (NSCLC), accounting for 40% of all lung cancer cases. The development of 3D \u003cem\u003ein vitro\u003c/em\u003e cancer models offers better replication of the native tumour microenvironment, facilitating patient-specific drug screening and therapeutic evaluation. However, conventional extrusion printing approaches are limited in fabricating functional vascularized tissue models for transplantation, drug screening and disease modelling, owing to restrictions caused by gravity \u0026amp; structural complexities. In this study, we employ embedded bioprinting to fabricate self-supporting biomimetic LUAD with intricate geometries. A low-viscosity bioink comprising polyethylene glycol diacrylate (PEGDA) \u0026amp; gelatin was extruded into a xanthan gum support bath, which exhibited pseudoplastic and shear thinning behaviour to overcome the gravitational and overhang limitations of standard extrusion printing. Gelatin offers biomimetic cell-binding motifs, while PEGDA enhances mechanical stability and tunable tissue stiffness. Rheological analysis confirmed shear thinning and recovery properties of the support bath system. \u003cem\u003eIn vitro\u003c/em\u003e assessments further demonstrated the cytocompatibility of both the printed construct and support bath. This approach highlights the feasibility of engineering physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e LUAD models, presenting a promising alternative to animal models for preclinical screening of therapeutics and personalised medicine applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Development of a Low Viscosity PEGDA - Gelatin Bioink for Embedded Bioprinting of Complex Structures and Lung Adenocarcinoma (LUAD) Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 05:55:09","doi":"10.21203/rs.3.rs-9513495/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-09T11:25:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21902554966631363768159561974350370702","date":"2026-05-06T15:15:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325266885305273227519701519887663048119","date":"2026-04-29T01:26:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T12:59:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-27T02:48:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-27T02:48:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"In vitro models","date":"2026-04-24T06:54:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"in-vitro-models","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [In vitro models](https://link.springer.com/journal/44164)","snPcode":"44164","submissionUrl":"https://submission.springernature.com/new-submission/44164/3","title":"In vitro models","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"23b26ea6-915a-4f86-b4a3-d9112f54f497","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-09T11:25:54+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"21902554966631363768159561974350370702","date":"2026-05-06T15:15:37+00:00","index":18,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T05:55:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 05:55:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9513495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9513495","identity":"rs-9513495","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}