Femtosecond label-free imaging empowers mesenchymal stem cells quality control

Femtosecond label-free imaging empowers mesenchymal stem cells quality control | 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 Femtosecond label-free imaging empowers mesenchymal stem cells quality control Jiawei Liu, Yang Liu, Benhan Xiong, Hu Cao, Xue Li, Chunyan Tian, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8327902/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Human Cell → Version 1 posted 4 You are reading this latest preprint version Abstract Mesenchymal stem cells (MSCs) hold significant promise in regenerative medicine, yet their clinical application is hindered by challenges such as cellular heterogeneity and quality control. This study aims to develop a rapid, non-invasive method for evaluating MSCs quality using femtosecond laser label-free imaging (FLI). We examined the proliferation, metabolic dynamics, and differentiation potential of MSCs from various tissue sources, including human dental pulp, umbilical cord, and fat, across different passages. Our findings indicate that with the passages increasing, the morphology of MSCs alters, proliferation capacity decreases, β-galactosidase activity linked to aging rises, and both osteogenic and adipogenic differentiation abilities markedly decline. FLI technology effectively captures these changes: reduced NAD(P)H/FAD ratio in the cells of higher passage suggests decreased metabolic activity, while enhanced aging-related fluorescence signals, such as lipofuscin, align with cellular senescence. During differentiation, increased fluorescence intensity of NAD(P)H and FAD signals heightened metabolic activity within the cells. Due to varying differentiation potentials among cells from different sources, NAD(P)H and FAD change patterns also differ. Difference in the optical REDOX ratio (FAD/(NAD(P)H+FAD)) among differentiation directions indicate that differentiation potential of MSCs correlates with metabolic reprogramming. Three-dimensional FLI of suspension cells further revealed that the cells of lower-passage exhibit greater spatial heterogeneity in metabolic signals, possibly reflecting more active mitochondrial function. This study confirms that FLI technology can effectively assess the proliferation activity, senescence, and differentiation potential of MSCs through non-invasive, dynamic monitoring of their metabolic status and morphological features, offering a novel approach for standardized quality assessment of MSCs preparations. FLI MSCs Metabolism Quality control Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mesenchymal stem cells (MSCs) are pluripotent stromal cells capable of differentiating into mesoderm-derived cell types. Their potential for multi-directional differentiation, along with immunomodulatory and paracrine functions, has led to significant advancements in clinical applications, particularly in regenerative medicine and the treatment of autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, cardiovascular diseases, and neurological injuries [ 1 ]. However, the lack of standardized clinical quality control for MSCs limits their broader application. The natural heterogeneity of MSCs, influenced by factors such as tissue source (e.g., bone marrow, umbilical cord, fat), donor variability, and in vitro amplification procedures, complicates the assurance of functional consistency and safety across different cell product batches. While current quality control systems address basic requirements like cell survival rate, surface markers, and sterility, they fall short in providing standardized assessments of "effectiveness." Functional experiments, such as those assessing differentiation, immunomodulatory ability, and cell cycle analysis of MSCs, can only be conducted using batch-specific inspections. Furthermore, the survival rate, biological efficacy, and homogeneity of MSCs used in preclinical practice are subject to change during preservation, transportation, resuscitation, and preparation, making it challenging to ensure consistent stem cell quality. Consequently, there is a need for a rapid, dynamic, and non-invasive method for stem cell quality evaluation. In recent years, various label-free and glassless imaging techniques, primarily based on the nonlinear optical processes of femtosecond laser pulses, have been developed and widely applied in imaging biological tissues and cells [ 2 , 3 ]. The femtosecond laser label-free imaging (FLI) microscope system is a novel technology that uses laser pulses focused through high numerical aperture objectives to interact with endogenous biomolecules and structures in biological tissues, achieving high-resolution, label-free, and section-free imaging. This system can simultaneously collect multiple nonlinear optical signals, including three-harmonic generation, two-photon fluorescence emission, second-harmonic generation, and three-photon fluorescence emission. These signals are gathered through a high-efficiency multi-channel system and processed using a specific mathematical model. After algorithmic reconstruction, the system produces two forms of results: structured composite graphs that integrate various types of information and datasets that retain information from multiple independent channels. Glycolysis and oxidative phosphorylation are crucial metabolic pathways that determine the metabolic state of cells. Glycolysis takes place in the cytoplasm, where glucose is converted into pyruvate, producing ATP and reducing NAD + to NADH. NADH then transports electrons to the mitochondria. Within the inner mitochondrial membrane, these electrons are transferred to complex I of the electron transport chain (ETC), initiating oxidative phosphorylation. FAD serves as a cofactor for succinate dehydrogenase, facilitating the conversion of succinate to fumaric acid while accepting two hydrogen atoms to form FADH2. The electrons from FADH2 enter the ubiquinone pool, proceed through complex III, and contribute to ATP synthesis. Additionally, NADH and NAD + play roles in calcium homeostasis, gene expression, oxidative stress, aging, and apoptosis. The phosphorylated and form, NADPH and NADP+, is involved in the reductive biosynthesis of fatty acids and steroids, antioxidation, and oxidative stress [ 4 ]. FAD acts as a cofactor in various flavin proteins, participating in DNA repair, nucleotide biosynthesis, fatty acid β-oxidation, amino acid decomposition, and other processes. Importantly, NAD⁺ and FADH2 lack autofluorescence, so non-invasive metabolic studies typically measure the relative fluorescence of NAD(P)H and FAD [ 5 ]. In addition to capturing endogenous fluorescence signals, FLI can also collect triple-frequency signals generated by non-uniform interfaces with refractive index variations. This capability allows for the depiction of cell membranes, nuclear membranes, lipid droplets, and other intracellular or extracellular structures, enabling high-contrast imaging of subcellular structures and providing valuable insights for cell morphology studies. This study aims to utilize FLI to monitor MSCs from various tissue sources and different passages after in vitro expansion. By comprehensively analyzing their dynamic changes in proliferation ability, osteogenic and adipogenic differentiation potential, and cell metabolism, to establish a new, rapid and non-invasive method for assessing stem cell quality. Methods Stem cell culture Human dental pulp stem cell (DPSC), umbilical cord mesenchymal stem cell (UCMSC), and adipose-derived MSC (ADSC) are gifted by Beijing SH Biotechnology. MSCs were seeded at a density of 1×10⁴ cells/cm² and cultured in Minimum Essential Medium α (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). Cells were maintained in a 37°C incubator with 5% CO2, and digested with 0.25% trypsin at day 4. Cell Cycle Analysis MSCs at passages 4, 6, 8, 10, and 12 were digested, centrifuged at 1000 r/min, and fixed overnight at 4°C with 1 ml of 70% ice-cold ethanol. After fixation, the cells were centrifuged at 1500 r/min for 5 min, and then the pellet was suspended and stained with 500 µl of PI/RNase A staining working solution at 37°C for 30 min in the dark. The stained cells were analyzed using a flow cytometer to determine the cell cycle distribution. β-Galactosidase Activity Assay β-Galactosidase Activity Assay was performed using Senescence β-Galactosidase Staining Kit (C0602, Beyotime, China) according to the manufacturer’s protocol. Briefly, MSCs at passages 4, 6, 8, 10, and 12 were fixed with β-galactosidase staining fixative for 15 min, followed by incubation with β-galactosidase staining solution overnight at 37°C in a CO₂-free environment. Cellular staining was examined under a microscope. The number of positive cells was counted in five randomly selected fields of view. Osteogenic and adipogenic differentiation MSCs at passages 4, 8, and 12 were seeded into 24-well confocal glass-bottom plates (P24-1.5H-N, Cellvis, USA). When cell density reached approximately 70%, osteogenic differentiation was induced using the OriCell® Human Mesenchymal Stem Cell Osteogenic Differentiation Kit (HUXXC-90021, OriCell, China), with the medium replaced every 3 days. On day 14, Alizarin Red S staining was performed to assess mineralization. When cell density reached approximately 90%, adipogenic differentiation was induced using the OriCell® Human Mesenchymal Stem Cell Adipogenic Differentiation Kit (HUXXC-90031, OriCell, China), with the medium replaced every 4 days. On day 21, Oil Red O staining was conducted to evaluate lipid droplet formation. Femtosecond laser label-free imaging (FLI) The images were acquired by a FLI microscopy system (FI-100, Femtosecond Research Center (Guangzhou) Co., Ltd., China). The system employed sub-5 femtosecond laser pulses with a broad spectral range (900–1200 nm) at a repetition rate of 12.5MHz. These pulses were focused through a high numerical aperture objective (UAPON 40XW340, Olympus; NA1.15) to interact with endogenous biomolecules and structures for live or fixed biological tissues without labeling or sectioning. Each image was acquired at a field of view (FOV) of 360 × 360 µm² (500 × 500 pixels, 1s). For larger regions (up to 10×10 mm), a motorized stage captured serial images with 5% overlap for artifact-free mosaicking. Statistical analysis All data are expressed as the mean ± standard deviation (SD), and differences were considered statistically significant at P < 0.05. The results were analyzed using one-way ANOVA analysis followed by Fisher’s post hoc test. Results The changes of morphology, cell cycle, and senescence of different passages of MSCs We observed morphological changes of MSCs as passages increase under a light microscope, and found that MSCs of passage 4 (P4) and 6 exhibited uniform morphology, relatively small, and grew with a spindle-shaped vortex pattern. With the extension of culture time in vitro , cell bodies enlarge, cells turn to flatten and spread, and their proliferation rate decreased. P12 MSCs appeared distinctly flat or egg-shaped and were arranged disorderly (Fig. 1 A). Cell cycle provides real-time insights into the proliferation activity, health status, and environmental response of cells [ 6 ]. The results revealed that with increased passage, the proportion of MSCs in the G0/G1 phase significantly raised, while the proportion in the S phase, where genetic material is replicated in preparation for division, declined (Fig. 1 B). Consequently, more cells exited the active division cycle and entered a resting state. Additionally, the activity of senescence-associated β-galactosidase (SA-β-Gal) increased in higher-passage cells (Fig. 1 C). These results indicated that MSCs of P4-P6 are in an active division phase, while cells from higher passage are prone to replicative senescence, resulting in diminished proliferation ability and weakened therapeutic function. Metobolic changes and cell senescence of different passages of MSCs can be detected by the multi-photon fluorescence of FLI The working principle of FLI is shown as Fig. 2 A, and it enables high-resolution analysis of cell morphology by detecting fluorescence lifetime difference of endogenous fluorophores. Its results align closely with optical microscopy in depicting overall cell morphology and can distinctly illustrate changes such as increased cell body size and flattening after multiple passages. Additionally, FLI can observe subcellular structures. As the passages increase, the volume of nucleus significantly expanded (Fig. 2 B). An increased NAD(P)H/FAD ratio indicated strong intracellular reducing power and a more "reducing" metabolic environment, often associated with rapid cell growth and a high demand for biosynthetic materials, signifying vigorous metabolism. The 3PFE (Ctr1 and Ctr2 channel) modality of FLI sensitively captures signals of NAD(P)H and FAD. With passages increasing, the NAD(P)H/FAD ratio of MSCs gradually declines, correlating with decreased cell vitality and function, consistent with reduced proliferation capacity (Fig. 2 C). FLI's multi-channel separation technology allows for the distinct collection of signals from endogenous fluorophores other than NAD(P)H and FAD. The 2PFE modality of FLI (Ctr4 channel) captures auto fluorescence of lipofuscin, an oxidized lipid-protein polymer that accumulates in senescent cells [ 7 ]. Up to P30, significant senescence is observed in most ADSC cells, and the lipofuscin signals captured by the Ctr4 channel overlap with SA-β-Gal staining results (Fig. 2 D). This finding suggests that FLI can indicate cellular senescence by evaluating lipofuscin deposition without labeling. Osteogenic and Adipogenic Differentiation of MSCs The ability of differentiation is a key indicator for assessing the function of MSCs and is crucial for their potential use in regenerative medicine. MSCs migrate to damaged areas and differentiate into specific cell types, such as osteoblasts and chondrocytes, to replace or repair damaged tissues. Strong differentiation ability suggests a higher potential for tissue regeneration and repair [ 8 ]. However, cell replicative senescence, resulting from prolonged in vitro expansion, significantly alters their multidirectional differentiation potential. As the passages increases, the osteogenic and adipogenic capabilities of the three types of MSCs gradually decline. Compared to P4 cells, P12 cells showed a marked reduction in the formation, size, and alizarin red staining depth of calcium nodules, which are markers of osteogenic differentiation (Fig. 3 A). Additionally, the number of lipid droplets in higher passage cells decreased significantly after adipogenic differentiation induction, this change particularly noticeable in ADSC (Fig. 3 B). Changes in the 3PFE channel of FLI can indicate the differentiation direction and potential of MSCs MSCs undergo significant remodeling during differentiation. To evaluate metabolic changes during osteogenic and adipogenic differentiation, we analyzed the fluorescence intensity of NAD(P)H and FAD. Compared with undifferentiated cells, FAD and NAD(P)H levels in the three differentiated MSCs showed a marked increase (Fig. 4 A & 4 B). This suggests a notable enhancement in intracellular energy metabolism, with the rise in FAD indicating activation of oxidative phosphorylation (OxPhos) and a shift from glycolysis to oxidative phosphorylation. After differentiation, cells undergo metabolic reprogramming to support more complex and energy-intensive physiological functions. Different MSCs exhibit varying functions and differentiation potentials (Fig. 4 C-E). The stronger osteogenic differentiation capacity of DPSC resulted in more pronounced changes in FAD and NAD(P)H in the osteogenic direction (Fig. 4 C). ADSC, with its robust adipogenic differentiation ability, shows greater changes in FAD and NAD(P)H in the adipogenic direction compared to the osteogenic direction (Fig. 4 E). However, these differences diminish as passage increases. Thus, FLI can serve as a method to assess differentiation direction. Additionally, during osteogenic differentiation, the optical REDOX ratio decreases with continuous passage, whereas during adipogenic differentiation, it increases. FLI effectively captures changes in the differentiation potential of MSCs, which are influenced by source and passage. Spatial metabolic changes in Suspended MSCs can be detected by Femtosecond laser layer scanning technology In clinical, MSCs from a certified working bank are typically prepared as an injection solution and administered to patients. The quality of these cell preparations directly influences therapeutic outcomes. To assess how passages affect cell quality, we analyzed the metabolic changes in suspended MSCs with varying passages. FLI enables 3D layer scanning imaging of suspension cells, displaying their three-dimensional morphology through continuous optical sectioning along the Z-axis and subsequent reconstruction (Fig. 5 A-C). Our observations indicated that the NAD(P)H/FAD values in lower passages of MSCs decreased with increasing Z-axis depth. In contrast, the variation in NAD(P)H/FAD values along the Z-axis in higher passage of MSCs is smaller than in lower passage (Fig. 5 D). We hypothesize that this may be due to differences in the spatial function and quality of subcellular structures, primarily mitochondria. Lower passage MSCs likely possesses more active and spatially diverse mitochondrial networks and energy metabolism activities. Conversely, higher passage MSCs may undergo mitochondrial function homogenization or decline during in vitro expansion, leading to reduced spatial heterogeneity in their metabolic signals. In conclusion, the spatial distribution characteristics revealed by FLI-based metabolic imaging offer a rapid, non-invasive method for preclinical quality control for assessment of cell preparations. Discussion Label-free imaging technology is extensively utilized in the functional analysis of stem cells due to its ability to non-invasively monitor cell morphology and function simultaneously. Stringari C et al. employed phasor fluorescence lifetime microscopy (phasor-FLIM) to differentiate and isolate human embryonic stem cells (hESC) and their differentiating progeny by detecting differences in the fluorescence lifetime of endogenous fluorophores, such as NAD(P)H, within the cells [ 9 ]. Belinda K et al. used phasor-fluorescence lifetime imaging microscopy to spatially locate NAD(P)H in both undifferentiated stem cells and differentiated muscle cells, thereby linking stem cell metabolic activity to their differentiation fate [ 10 ]. By using two-photon fluorescence lifetime imaging microscopy, Aleksandra V et al. discovered that MSCs exhibit stronger glycolytic metabolic characteristics following osteogenic and chondrogenic differentiation, highlighting the significant role of metabolism in determining cell fate [ 11 ]. Priyanka et al. integrated label-free differential phase contrast imaging (DPC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with lipidomics analysis to investigate the functional heterogeneity of MSCs at the single-cell level [ 12 ]. In this study, we examined the proliferation, differentiation potential, and metabolic dynamics of MSCs from various tissue sources and in vitro expansion passages by utilizing FLI technology alongside traditional biological detection methods. The results confirmed that FLI technology can not only capture the light refraction signals of subcellular structures but also process the optical signals into visual images, clearly demonstrating phenomena such as cell flattening and increased nuclear volume, which cannot be observed under an ordinary optical microscope, in MCSs with passages increasing. These phenomena are associated with the dysregulation of nuclear lamina protein or increased genomic instability caused by replicative senescence. The cellular energy metabolic state is an important indicator of its functional state. Among them, NAD(P)H and FAD, as key coenzymes, are widely involved in cellular life activities, and their slight fluctuations can reflect the dynamic changes in cellular metabolic pathways. FLI has the advantages of being label - free and capable of real- time dynamic monitoring. It can perform non-invasive quantitative analysis of autofluorescent substances such as NAD(P)H and FAD, and accurately characterize their concentrations in a digital manner to reflect cellular metabolic activity. The NAD(P)H/FAD ratio can directly indicate the cellular redox state. We found that NAD(P)H/FAD ratio showed a downward trend with passages increasing, suggesting a weakened cellular reducing ability, which is related to the increased consumption or impaired regeneration of intracellular NAD(P)H. The differentiation ability of MSCs is one of their core biological functions. FLI detected a significant increase in the fluorescence signal intensities of NAD(P)H and FAD after osteogenic and adipogenic differentiation of MSCs, indicating that the differentiation process is accompanied by drastic energy changes. Moreover, the optical redox ratio(FAD/(NAD(P)H + FAD))showed regular changes with passages increasing, and the different changing trends of MSCs from different tissue sources during osteogenic and adipogenic differentiation provide a novel and quantifiable functional evaluation index for assessing the differentiation ability of MSCs. Although in Fig. 3 , we also found through traditional induced differentiation that the differentiation ability of MSCs decreased with passages increasing, and there were differences in the differentiation directions of MSCs from different tissue sources, this classic method usually requires an induction period as long as 14 to 21 days. Essentially, it is a “post - hoc verification” method and cannot achieve early and rapid “prediction” of differentiation potential. In contrast, FLI technology demonstrates its unique advantages: the quantifiable optical redox parameters can be quickly obtained by FLI, enabling immediate assessment of the differentiation potential of MSCs. Traditional microscopy techniques face multiple challenges when imaging suspended cells. The random movement of cells easily leads to blurred imaging. In addition, two-dimensional imaging can only capture planar information, while the imaging method of fixed cells destroys their physiological activity. In the clinical, stem cell products are usually cell suspensions, and the stability and activity of cells are important quality factors for application. Currently, although mass spectrometry- based spatial omics technology can provide unbiased information at the molecular level, its complex sample preparation and time-consuming data analysis hinder its ability to fulfill the demands of real-time clinical detection. Therefore, we employed FLI to conduct Z-axis layer scanning of suspended MSCs. Our findings revealed that the variation range of NAD(P)H/FAD ratio in the Z-axis of high-passage MSCs was significantly smaller than that observed in low-passage, functionally active cells. This suggests that cell quality is correlated with the spatial energy changes within the cells. FLI offers a rapid, non-invasive, and high-throughput method for the direct detection of suspended cells, highlighting its potential applications in clinical treatment. However, this study has several limitations. The current cell culture system is primarily based on laboratory-scale conditions, resulting in a limited sample size. To enhance the robustness and representativeness of our dataset, it is essential to incorporate additional samples. While we observed changes in NAD(P)H and FAD fluorescence intensity using 3PFE, and the detection of lipofuscin signals through 2PFE of FLI further supported its utility in assessing cellular senescence, the potential application of SHG—another critical component of FLI—in the quality control of MSCs remains underexplored. Presently, SHG imaging has been utilized in tissues such as the lung and skin for the visualization and quantitative analysis of highly ordered structures, including collagen fibers [ 13 , 14 ]. Although MSCs do not generate robust SHG signals, the collagen matrix secreted during their osteogenic differentiation, as well as the extracellular matrix formed through interactions with specific biomaterials, can serve as targets for SHG detection. Consequently, we aim to further investigate the application of FLI for the multidimensional and non-invasive assessment of MSCs functional states and the alterations in their microenvironment. As deep learning networks rapidly evolve, FLI can be combined with AI to analyze the fine changes in cells that the human eye cannot detect. By training on extensive scanned image data, AI can learn to extract subtle morphological features related to cell activity, proliferation ability, differentiation tendency, and aging state. This analysis can directly output quality scores or classification results (such as "excellent" or "subexcellent"), establishing a prediction model for stem cells function and creating opportunities for direct assess the quality of MSCs. Research indicated that the efficacy of stem cell therapy wasn't directly proportional to the injection dose; instead, there existed an optimal dose window [ 15 ]. Thus, ensuring the high quality and consistency of cell products across batches at a specific dose is crucial for maximizing therapeutic outcomes. A core challenge in MSCs preparation is the considerable variation between cell products from different production platforms and even among batches within the same platform. Unfortunately, this study has not systematically verified the therapeutic efficacy differences among various batches of MSCs products in animal models or clinical trials. Future research must urgently integrate FLI and functional experiments to develop an in vitro potency evaluation index system capable of predicting in vivo efficacy, thereby advancing the creation of clinical precision quality control standards. Conclusions This study employed femtosecond imaging technology to examine the proliferation, senescence, and metabolic states during in vitro amplification of MSCs from various sources. Our findings suggest that FLI could effectively capture real-time changes in MSCs. We anticipate its application as a rapid, dynamic, and non-invasive method for assessing the quality of stem cells in both laboratory research and clinical trials. Declarations Authors’ Contributions Jiawei Liu, Yang Liu: Study design; execution, acquisition of data, analysis and interpretation; Drafting and revising the article; Benhan Xiong: Execution, acquisition of data, analysis and interpretation; Hu Cao, Xue Li: Execution, acquisition of data; Chunyan Tian: Conception; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; Hua Wang: Conception, study design, revising and critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Data availability All data generated and analyzed during this study are included in this manuscript. Funding Not applicable. Conflict of interests/Competing Interests The authors have no relevant financial or nonfinancial interests to disclose. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Acknowledgements We are grateful to Beijing SH Biotechnology for gifting us DPSC, UCMSC and ADSC. References Zhou T, Yuan Z, Weng J, et al. Challenges and advances in clinical applications of mesenchymal stromal cells. J Hematol Oncol. 2021;14(1):24. https://doi.org/10.1186/s13045-021-01037-x. König TT, Goedeke J, Muensterer OJ. Multiphoton microscopy in surgical oncology- a systematic review and guide for clinical translatability. Surg Oncol. 2019;31:119-131. https://doi.org:/10.1016/j.suronc.2019.10.011. Boppart SA, You S, Li L, Chen J, Tu H. Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond. APL Photonics. 2019;4(10):100901. https://doi.org/10.1063/1.5098349. Ying W. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal. 2008;10(2):179-206. https://doi.org/10.1089/ars.2007.1672. Kolenc OI, Quinn KP. Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD. Antioxid Redox Signal. 2019;30(6):875-889. https://doi.org/10.1089/ars.2017.7451. Huang J, Li Q, Yuan X, Liu Q, Zhang W, Li P. Intrauterine infusion of clinically graded human umbilical cord-derived mesenchymal stem cells for the treatment of poor healing after uterine injury: a phase I clinical trial. Stem Cell Res Ther. 2022;13(1):85. https://doi.org/10.1186/s13287-022-02756-9. Satija NK, Singh VK, Verma YK, et al. Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine. J Cell Mol Med. 2009;13(11-12):4385-402. https://doi.org/10.1111/j.1582-4934.2009.00857.x. Song SB, Shim W, Hwang ES. Lipofuscin Granule Accumulation Requires Autophagy Activation. Mol Cells. 2023;46(8):486-495. https://doi.org/10.14348/molcells.2023.0019. Stringari C, Sierra R, Donovan PJ, Gratton E. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy. J Biomed Opt. 2012;17(4):046012. https://doi.org/10.1117/1.JBO.17.4.046012. Wright BK, Andrews LM, Markham J, et al.. NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. Biophys J. 2012;103(1):L7-9. https://doi.org/10.1016/j.bpj.2012.05.038. Meleshina AV, Dudenkova VV, Bystrova AS, Kuznetsova DS, Shirmanova MV, Zagaynova EV. Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation. Stem Cell Res Ther. 2017;8(1):15. https://doi.org/10.1186/s13287-017-0484-7. Priyadarshani P, Van Grouw A, Liversage AR, et al. Investigation of MSC potency metrics via integration of imaging modalities with lipidomic characterization. Cell Rep. 2024;43(8):114579. https://doi.org/10.1016/j.celrep.2024.114579. Wang W, Liu P, Zhu W, et al. Skin organoid transplantation promotes tissue repair with scarless in frostbite. Protein Cell. 2025;16(4):240-259. https://doi.org/10.1093/procel/pwae055. Xiao Z, Ding L, Yu Y, et al. Tanreqing injection inhibits stemness and enhances sensitivity of non-small cell lung cancer models to gefitinib through ROS/STAT3 signaling pathway. J Cancer. 2024;15(13):4259-4274. https://doi.org/10.7150/jca.94438. Kabat M, Bobkov I, Kumar S, Grumet M. Trends in mesenchymal stem cell clinical trials 2004-2018: Is efficacy optimal in a narrow dose range? Stem Cells Transl Med. 2020;9(1):17-27. https://doi.org/10.1002/sctm.19-0202. Cite Share Download PDF Status: Published Journal Publication published 10 Mar, 2026 Read the published version in Human Cell → Version 1 posted Reviewers agreed at journal 09 Jan, 2026 Reviewers invited by journal 18 Dec, 2025 Editor assigned by journal 17 Dec, 2025 First submitted to journal 13 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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. 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13:11:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8327902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8327902/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-026-01366-4","type":"published","date":"2026-03-10T15:59:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":99307876,"identity":"8e499dd2-a370-489c-a73d-e4676aa74508","added_by":"auto","created_at":"2025-12-31 16:06:58","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9741,"visible":true,"origin":"","legend":"","description":"","filename":"huceHUCED2500713.xml","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/92a14f73a239864ed83a882e.xml"},{"id":99307351,"identity":"b613a2f6-d6db-48df-8acc-425ce172850e","added_by":"auto","created_at":"2025-12-31 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17:18:44","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":69316,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/75183a11dab4276c52ab1a4d.html"},{"id":98820822,"identity":"f7449878-5170-41de-a40f-ba7d48f71f3d","added_by":"auto","created_at":"2025-12-22 17:18:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":504237,"visible":true,"origin":"","legend":"\u003cp\u003eBiological change of MSCs from different sources and different passages.\u003cstrong\u003e \u003c/strong\u003e(A) Representative images of the morphology of DPSC, UCMSC, and ADSC at different passages. Scale bar = 200 μm. (B) Cell cycle changes in DPSC, UCMSC and ADSC at different passages. n = 3. Representative images (C) and statistical analysis (D) of β-galactosidase staining in DPSC, UCMSC, and ADSC at different passages. Scale bar = 200 μm. n = 5. Data are presented as the mean ± SD. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/421afc0587deb58db169db5e.png"},{"id":98820823,"identity":"073d4962-0d95-443b-a76e-49f534d80764","added_by":"auto","created_at":"2025-12-22 17:18:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":681116,"visible":true,"origin":"","legend":"\u003cp\u003eFLI captures metobolic changes and cell senescence of MCSs. (A) Working principles of FLI imaging, data acquisition and analysis. (B) Multimodal fitting graph synthesized by FLI. (Yellow: Ctr0; Pink: Ctr1+Ctr2). Scale bar = 100 μm. (C) The quantification results of Ctr1 (NAD(P)H)/ Ctr2 (FAD) collected by FLI. n = 10. (D) Comparison results of Ctr4 pseudo-color images collected by FLI with β-Gal staining. Scale bar = 50 μm. Data are presented as the mean ± SD\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/ffbcecd529e018cf43f239e6.png"},{"id":98820827,"identity":"14e4686f-7b32-4dd5-8ebc-a303d8a72c13","added_by":"auto","created_at":"2025-12-22 17:18:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":960771,"visible":true,"origin":"","legend":"\u003cp\u003eThe osteogenic and adipogenic differentiation of MSCs.\u003cstrong\u003e \u003c/strong\u003e(A) Representative images of Alizarin Red S staining for osteogenic differentiation of MSCs. (B) Representative images of Oil Red O staining for adipogenic differentiation of MSCs. Scale bar = 100 μm\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/7a106d594dadd5e0cbc34335.png"},{"id":99307559,"identity":"a17bde03-4248-4a1b-8ce8-21fe52320266","added_by":"auto","created_at":"2025-12-31 16:06:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":538790,"visible":true,"origin":"","legend":"\u003cp\u003eFLI of differentiated MSCs.\u003cstrong\u003e \u003c/strong\u003e(A) Multimodal fitting diagram of osteogenic differentiated MSCs (Yellow: Ctr0; Pink: Ctr1+Ctr2). Scale bar = 100 μm. (B) Multimodal fitting diagram of adipogenic differentiated MSCs (Yellow: Ctr0; Pink: Ctr1+Ctr2). Scale bar = 100 μm. (C) Statistical results of NAD(P)H, FAD and optical REDOX ratio (FAD / (NAD(P)H) + FAD) before and after differentiation of DPSC (C), UCMSC (D) and ADSC (E). Data are presented as the mean ± SD. n = 10. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/4e6fbc4a294db438ca1db208.png"},{"id":99307897,"identity":"f70621a8-1205-4edc-9dfe-eb04ea6ef2ef","added_by":"auto","created_at":"2025-12-31 16:07:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179466,"visible":true,"origin":"","legend":"\u003cp\u003eFLI of MSCs suspension.\u003cstrong\u003e \u003c/strong\u003e(A) Monolayer scanning image of suspension cells. Scale bar = 100 μm. (B) Schematic diagram of layer-by-layer scanning of suspension cells along the Z-axis (step size: 1 µm). (C) 3D pseudo-color image of a single suspension cell generated by applying an algorithm to delineate the cell in each layer. (Yellow \u0026amp; Blue: Ctr0; Pink: Ctr1+Ctr2). Scale bar = 10 μm. (D) Metabolic changes of suspended DPSC, UCMSC and ADSC along the Z-axis. Data are presented as the mean ± SD. n = 5. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/4f8d9dc30f3cf069a42129d8.png"},{"id":104739490,"identity":"ed256230-a838-4e57-89d2-948a24d5f3bd","added_by":"auto","created_at":"2026-03-16 16:07:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3593205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8327902/v1/1a5e3548-ca2d-4553-8b8c-a26d53eb6582.pdf"}],"financialInterests":"","formattedTitle":"Femtosecond label-free imaging empowers mesenchymal stem cells quality control","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMesenchymal stem cells (MSCs) are pluripotent stromal cells capable of differentiating into mesoderm-derived cell types. Their potential for multi-directional differentiation, along with immunomodulatory and paracrine functions, has led to significant advancements in clinical applications, particularly in regenerative medicine and the treatment of autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, cardiovascular diseases, and neurological injuries [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the lack of standardized clinical quality control for MSCs limits their broader application. The natural heterogeneity of MSCs, influenced by factors such as tissue source (e.g., bone marrow, umbilical cord, fat), donor variability, and \u003cem\u003ein vitro\u003c/em\u003e amplification procedures, complicates the assurance of functional consistency and safety across different cell product batches. While current quality control systems address basic requirements like cell survival rate, surface markers, and sterility, they fall short in providing standardized assessments of \"effectiveness.\" Functional experiments, such as those assessing differentiation, immunomodulatory ability, and cell cycle analysis of MSCs, can only be conducted using batch-specific inspections. Furthermore, the survival rate, biological efficacy, and homogeneity of MSCs used in preclinical practice are subject to change during preservation, transportation, resuscitation, and preparation, making it challenging to ensure consistent stem cell quality. Consequently, there is a need for a rapid, dynamic, and non-invasive method for stem cell quality evaluation.\u003c/p\u003e \u003cp\u003eIn recent years, various label-free and glassless imaging techniques, primarily based on the nonlinear optical processes of femtosecond laser pulses, have been developed and widely applied in imaging biological tissues and cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The femtosecond laser label-free imaging (FLI) microscope system is a novel technology that uses laser pulses focused through high numerical aperture objectives to interact with endogenous biomolecules and structures in biological tissues, achieving high-resolution, label-free, and section-free imaging. This system can simultaneously collect multiple nonlinear optical signals, including three-harmonic generation, two-photon fluorescence emission, second-harmonic generation, and three-photon fluorescence emission. These signals are gathered through a high-efficiency multi-channel system and processed using a specific mathematical model. After algorithmic reconstruction, the system produces two forms of results: structured composite graphs that integrate various types of information and datasets that retain information from multiple independent channels.\u003c/p\u003e \u003cp\u003eGlycolysis and oxidative phosphorylation are crucial metabolic pathways that determine the metabolic state of cells. Glycolysis takes place in the cytoplasm, where glucose is converted into pyruvate, producing ATP and reducing NAD\u0026thinsp;+\u0026thinsp;to NADH. NADH then transports electrons to the mitochondria. Within the inner mitochondrial membrane, these electrons are transferred to complex I of the electron transport chain (ETC), initiating oxidative phosphorylation. FAD serves as a cofactor for succinate dehydrogenase, facilitating the conversion of succinate to fumaric acid while accepting two hydrogen atoms to form FADH2. The electrons from FADH2 enter the ubiquinone pool, proceed through complex III, and contribute to ATP synthesis. Additionally, NADH and NAD\u0026thinsp;+\u0026thinsp;play roles in calcium homeostasis, gene expression, oxidative stress, aging, and apoptosis. The phosphorylated and form, NADPH and NADP+, is involved in the reductive biosynthesis of fatty acids and steroids, antioxidation, and oxidative stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. FAD acts as a cofactor in various flavin proteins, participating in DNA repair, nucleotide biosynthesis, fatty acid β-oxidation, amino acid decomposition, and other processes. Importantly, NAD⁺ and FADH2 lack autofluorescence, so non-invasive metabolic studies typically measure the relative fluorescence of NAD(P)H and FAD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to capturing endogenous fluorescence signals, FLI can also collect triple-frequency signals generated by non-uniform interfaces with refractive index variations. This capability allows for the depiction of cell membranes, nuclear membranes, lipid droplets, and other intracellular or extracellular structures, enabling high-contrast imaging of subcellular structures and providing valuable insights for cell morphology studies.\u003c/p\u003e \u003cp\u003eThis study aims to utilize FLI to monitor MSCs from various tissue sources and different passages after \u003cem\u003ein vitro\u003c/em\u003e expansion. By comprehensively analyzing their dynamic changes in proliferation ability, osteogenic and adipogenic differentiation potential, and cell metabolism, to establish a new, rapid and non-invasive method for assessing stem cell quality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStem cell culture\u003c/h2\u003e \u003cp\u003eHuman dental pulp stem cell (DPSC), umbilical cord mesenchymal stem cell (UCMSC), and adipose-derived MSC (ADSC) are gifted by Beijing SH Biotechnology. MSCs were seeded at a density of 1\u0026times;10⁴ cells/cm\u0026sup2; and cultured in Minimum Essential Medium α (α-MEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). Cells were maintained in a 37\u0026deg;C incubator with 5% CO2, and digested with 0.25% trypsin at day 4.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Cycle Analysis\u003c/h3\u003e\n\u003cp\u003eMSCs at passages 4, 6, 8, 10, and 12 were digested, centrifuged at 1000 r/min, and fixed overnight at 4\u0026deg;C with 1 ml of 70% ice-cold ethanol. After fixation, the cells were centrifuged at 1500 r/min for 5 min, and then the pellet was suspended and stained with 500 \u0026micro;l of PI/RNase A staining working solution at 37\u0026deg;C for 30 min in the dark. The stained cells were analyzed using a flow cytometer to determine the cell cycle distribution.\u003c/p\u003e\n\u003ch3\u003eβ-Galactosidase Activity Assay\u003c/h3\u003e\n\u003cp\u003eβ-Galactosidase Activity Assay was performed using Senescence β-Galactosidase Staining Kit (C0602, Beyotime, China) according to the manufacturer\u0026rsquo;s protocol. Briefly, MSCs at passages 4, 6, 8, 10, and 12 were fixed with β-galactosidase staining fixative for 15 min, followed by incubation with β-galactosidase staining solution overnight at 37\u0026deg;C in a CO₂-free environment. Cellular staining was examined under a microscope. The number of positive cells was counted in five randomly selected fields of view.\u003c/p\u003e\n\u003ch3\u003eOsteogenic and adipogenic differentiation\u003c/h3\u003e\n\u003cp\u003eMSCs at passages 4, 8, and 12 were seeded into 24-well confocal glass-bottom plates (P24-1.5H-N, Cellvis, USA). When cell density reached approximately 70%, osteogenic differentiation was induced using the OriCell\u0026reg; Human Mesenchymal Stem Cell Osteogenic Differentiation Kit (HUXXC-90021, OriCell, China), with the medium replaced every 3 days. On day 14, Alizarin Red S staining was performed to assess mineralization.\u003c/p\u003e \u003cp\u003eWhen cell density reached approximately 90%, adipogenic differentiation was induced using the OriCell\u0026reg; Human Mesenchymal Stem Cell Adipogenic Differentiation Kit (HUXXC-90031, OriCell, China), with the medium replaced every 4 days. On day 21, Oil Red O staining was conducted to evaluate lipid droplet formation.\u003c/p\u003e\n\u003ch3\u003eFemtosecond laser label-free imaging (FLI)\u003c/h3\u003e\n\u003cp\u003eThe images were acquired by a FLI microscopy system (FI-100, Femtosecond Research Center (Guangzhou) Co., Ltd., China). The system employed sub-5 femtosecond laser pulses with a broad spectral range (900\u0026ndash;1200 nm) at a repetition rate of 12.5MHz. These pulses were focused through a high numerical aperture objective (UAPON 40XW340, Olympus; NA1.15) to interact with endogenous biomolecules and structures for live or fixed biological tissues without labeling or sectioning. Each image was acquired at a field of view (FOV) of 360 \u0026times; 360 \u0026micro;m\u0026sup2; (500 \u0026times; 500 pixels, 1s). For larger regions (up to 10\u0026times;10 mm), a motorized stage captured serial images with 5% overlap for artifact-free mosaicking.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results were analyzed using one-way ANOVA analysis followed by Fisher\u0026rsquo;s post hoc test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe changes of morphology, cell cycle, and senescence of different passages of MSCs\u003c/h2\u003e \u003cp\u003eWe observed morphological changes of MSCs as passages increase under a light microscope, and found that MSCs of passage 4 (P4) and 6 exhibited uniform morphology, relatively small, and grew with a spindle-shaped vortex pattern. With the extension of culture time \u003cem\u003ein vitro\u003c/em\u003e, cell bodies enlarge, cells turn to flatten and spread, and their proliferation rate decreased. P12 MSCs appeared distinctly flat or egg-shaped and were arranged disorderly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Cell cycle provides real-time insights into the proliferation activity, health status, and environmental response of cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The results revealed that with increased passage, the proportion of MSCs in the G0/G1 phase significantly raised, while the proportion in the S phase, where genetic material is replicated in preparation for division, declined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consequently, more cells exited the active division cycle and entered a resting state. Additionally, the activity of senescence-associated β-galactosidase (SA-β-Gal) increased in higher-passage cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results indicated that MSCs of P4-P6 are in an active division phase, while cells from higher passage are prone to replicative senescence, resulting in diminished proliferation ability and weakened therapeutic function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMetobolic changes and cell senescence of different passages of MSCs can be detected by the multi-photon fluorescence of FLI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe working principle of FLI is shown as Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, and it enables high-resolution analysis of cell morphology by detecting fluorescence lifetime difference of endogenous fluorophores. Its results align closely with optical microscopy in depicting overall cell morphology and can distinctly illustrate changes such as increased cell body size and flattening after multiple passages. Additionally, FLI can observe subcellular structures. As the passages increase, the volume of nucleus significantly expanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). An increased NAD(P)H/FAD ratio indicated strong intracellular reducing power and a more \"reducing\" metabolic environment, often associated with rapid cell growth and a high demand for biosynthetic materials, signifying vigorous metabolism. The 3PFE (Ctr1 and Ctr2 channel) modality of FLI sensitively captures signals of NAD(P)H and FAD. With passages increasing, the NAD(P)H/FAD ratio of MSCs gradually declines, correlating with decreased cell vitality and function, consistent with reduced proliferation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). FLI's multi-channel separation technology allows for the distinct collection of signals from endogenous fluorophores other than NAD(P)H and FAD. The 2PFE modality of FLI (Ctr4 channel) captures auto fluorescence of lipofuscin, an oxidized lipid-protein polymer that accumulates in senescent cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Up to P30, significant senescence is observed in most ADSC cells, and the lipofuscin signals captured by the Ctr4 channel overlap with SA-β-Gal staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This finding suggests that FLI can indicate cellular senescence by evaluating lipofuscin deposition without labeling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eOsteogenic and Adipogenic Differentiation of MSCs\u003c/h2\u003e \u003cp\u003eThe ability of differentiation is a key indicator for assessing the function of MSCs and is crucial for their potential use in regenerative medicine. MSCs migrate to damaged areas and differentiate into specific cell types, such as osteoblasts and chondrocytes, to replace or repair damaged tissues. Strong differentiation ability suggests a higher potential for tissue regeneration and repair [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, cell replicative senescence, resulting from prolonged \u003cem\u003ein vitro\u003c/em\u003e expansion, significantly alters their multidirectional differentiation potential. As the passages increases, the osteogenic and adipogenic capabilities of the three types of MSCs gradually decline. Compared to P4 cells, P12 cells showed a marked reduction in the formation, size, and alizarin red staining depth of calcium nodules, which are markers of osteogenic differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, the number of lipid droplets in higher passage cells decreased significantly after adipogenic differentiation induction, this change particularly noticeable in ADSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChanges in the 3PFE channel of FLI can indicate the differentiation direction and potential of MSCs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMSCs undergo significant remodeling during differentiation. To evaluate metabolic changes during osteogenic and adipogenic differentiation, we analyzed the fluorescence intensity of NAD(P)H and FAD. Compared with undifferentiated cells, FAD and NAD(P)H levels in the three differentiated MSCs showed a marked increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This suggests a notable enhancement in intracellular energy metabolism, with the rise in FAD indicating activation of oxidative phosphorylation (OxPhos) and a shift from glycolysis to oxidative phosphorylation. After differentiation, cells undergo metabolic reprogramming to support more complex and energy-intensive physiological functions. Different MSCs exhibit varying functions and differentiation potentials (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E). The stronger osteogenic differentiation capacity of DPSC resulted in more pronounced changes in FAD and NAD(P)H in the osteogenic direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). ADSC, with its robust adipogenic differentiation ability, shows greater changes in FAD and NAD(P)H in the adipogenic direction compared to the osteogenic direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). However, these differences diminish as passage increases. Thus, FLI can serve as a method to assess differentiation direction. Additionally, during osteogenic differentiation, the optical REDOX ratio decreases with continuous passage, whereas during adipogenic differentiation, it increases. FLI effectively captures changes in the differentiation potential of MSCs, which are influenced by source and passage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSpatial metabolic changes in Suspended MSCs can be detected by Femtosecond laser layer scanning technology\u003c/h2\u003e \u003cp\u003eIn clinical, MSCs from a certified working bank are typically prepared as an injection solution and administered to patients. The quality of these cell preparations directly influences therapeutic outcomes. To assess how passages affect cell quality, we analyzed the metabolic changes in suspended MSCs with varying passages. FLI enables 3D layer scanning imaging of suspension cells, displaying their three-dimensional morphology through continuous optical sectioning along the Z-axis and subsequent reconstruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Our observations indicated that the NAD(P)H/FAD values in lower passages of MSCs decreased with increasing Z-axis depth. In contrast, the variation in NAD(P)H/FAD values along the Z-axis in higher passage of MSCs is smaller than in lower passage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). We hypothesize that this may be due to differences in the spatial function and quality of subcellular structures, primarily mitochondria. Lower passage MSCs likely possesses more active and spatially diverse mitochondrial networks and energy metabolism activities. Conversely, higher passage MSCs may undergo mitochondrial function homogenization or decline during \u003cem\u003ein vitro\u003c/em\u003e expansion, leading to reduced spatial heterogeneity in their metabolic signals. In conclusion, the spatial distribution characteristics revealed by FLI-based metabolic imaging offer a rapid, non-invasive method for preclinical quality control for assessment of cell preparations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLabel-free imaging technology is extensively utilized in the functional analysis of stem cells due to its ability to non-invasively monitor cell morphology and function simultaneously. Stringari C et al. employed phasor fluorescence lifetime microscopy (phasor-FLIM) to differentiate and isolate human embryonic stem cells (hESC) and their differentiating progeny by detecting differences in the fluorescence lifetime of endogenous fluorophores, such as NAD(P)H, within the cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Belinda K et al. used phasor-fluorescence lifetime imaging microscopy to spatially locate NAD(P)H in both undifferentiated stem cells and differentiated muscle cells, thereby linking stem cell metabolic activity to their differentiation fate [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. By using two-photon fluorescence lifetime imaging microscopy, Aleksandra V et al. discovered that MSCs exhibit stronger glycolytic metabolic characteristics following osteogenic and chondrogenic differentiation, highlighting the significant role of metabolism in determining cell fate [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Priyanka et al. integrated label-free differential phase contrast imaging (DPC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with lipidomics analysis to investigate the functional heterogeneity of MSCs at the single-cell level [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we examined the proliferation, differentiation potential, and metabolic dynamics of MSCs from various tissue sources and \u003cem\u003ein vitro\u003c/em\u003e expansion passages by utilizing FLI technology alongside traditional biological detection methods. The results confirmed that FLI technology can not only capture the light refraction signals of subcellular structures but also process the optical signals into visual images, clearly demonstrating phenomena such as cell flattening and increased nuclear volume, which cannot be observed under an ordinary optical microscope, in MCSs with passages increasing. These phenomena are associated with the dysregulation of nuclear lamina protein or increased genomic instability caused by replicative senescence.\u003c/p\u003e \u003cp\u003eThe cellular energy metabolic state is an important indicator of its functional state. Among them, NAD(P)H and FAD, as key coenzymes, are widely involved in cellular life activities, and their slight fluctuations can reflect the dynamic changes in cellular metabolic pathways. FLI has the advantages of being label - free and capable of real- time dynamic monitoring. It can perform non-invasive quantitative analysis of autofluorescent substances such as NAD(P)H and FAD, and accurately characterize their concentrations in a digital manner to reflect cellular metabolic activity. The NAD(P)H/FAD ratio can directly indicate the cellular redox state. We found that NAD(P)H/FAD ratio showed a downward trend with passages increasing, suggesting a weakened cellular reducing ability, which is related to the increased consumption or impaired regeneration of intracellular NAD(P)H.\u003c/p\u003e \u003cp\u003eThe differentiation ability of MSCs is one of their core biological functions. FLI detected a significant increase in the fluorescence signal intensities of NAD(P)H and FAD after osteogenic and adipogenic differentiation of MSCs, indicating that the differentiation process is accompanied by drastic energy changes. Moreover, the optical redox ratio(FAD/(NAD(P)H\u0026thinsp;+\u0026thinsp;FAD))showed regular changes with passages increasing, and the different changing trends of MSCs from different tissue sources during osteogenic and adipogenic differentiation provide a novel and quantifiable functional evaluation index for assessing the differentiation ability of MSCs. Although in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we also found through traditional induced differentiation that the differentiation ability of MSCs decreased with passages increasing, and there were differences in the differentiation directions of MSCs from different tissue sources, this classic method usually requires an induction period as long as 14 to 21 days. Essentially, it is a \u0026ldquo;post - hoc verification\u0026rdquo; method and cannot achieve early and rapid \u0026ldquo;prediction\u0026rdquo; of differentiation potential. In contrast, FLI technology demonstrates its unique advantages: the quantifiable optical redox parameters can be quickly obtained by FLI, enabling immediate assessment of the differentiation potential of MSCs.\u003c/p\u003e \u003cp\u003eTraditional microscopy techniques face multiple challenges when imaging suspended cells. The random movement of cells easily leads to blurred imaging. In addition, two-dimensional imaging can only capture planar information, while the imaging method of fixed cells destroys their physiological activity. In the clinical, stem cell products are usually cell suspensions, and the stability and activity of cells are important quality factors for application. Currently, although mass spectrometry- based spatial omics technology can provide unbiased information at the molecular level, its complex sample preparation and time-consuming data analysis hinder its ability to fulfill the demands of real-time clinical detection. Therefore, we employed FLI to conduct Z-axis layer scanning of suspended MSCs. Our findings revealed that the variation range of NAD(P)H/FAD ratio in the Z-axis of high-passage MSCs was significantly smaller than that observed in low-passage, functionally active cells. This suggests that cell quality is correlated with the spatial energy changes within the cells. FLI offers a rapid, non-invasive, and high-throughput method for the direct detection of suspended cells, highlighting its potential applications in clinical treatment.\u003c/p\u003e \u003cp\u003eHowever, this study has several limitations. The current cell culture system is primarily based on laboratory-scale conditions, resulting in a limited sample size. To enhance the robustness and representativeness of our dataset, it is essential to incorporate additional samples. While we observed changes in NAD(P)H and FAD fluorescence intensity using 3PFE, and the detection of lipofuscin signals through 2PFE of FLI further supported its utility in assessing cellular senescence, the potential application of SHG\u0026mdash;another critical component of FLI\u0026mdash;in the quality control of MSCs remains underexplored. Presently, SHG imaging has been utilized in tissues such as the lung and skin for the visualization and quantitative analysis of highly ordered structures, including collagen fibers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although MSCs do not generate robust SHG signals, the collagen matrix secreted during their osteogenic differentiation, as well as the extracellular matrix formed through interactions with specific biomaterials, can serve as targets for SHG detection. Consequently, we aim to further investigate the application of FLI for the multidimensional and non-invasive assessment of MSCs functional states and the alterations in their microenvironment. As deep learning networks rapidly evolve, FLI can be combined with AI to analyze the fine changes in cells that the human eye cannot detect. By training on extensive scanned image data, AI can learn to extract subtle morphological features related to cell activity, proliferation ability, differentiation tendency, and aging state. This analysis can directly output quality scores or classification results (such as \"excellent\" or \"subexcellent\"), establishing a prediction model for stem cells function and creating opportunities for direct assess the quality of MSCs. Research indicated that the efficacy of stem cell therapy wasn't directly proportional to the injection dose; instead, there existed an optimal dose window [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, ensuring the high quality and consistency of cell products across batches at a specific dose is crucial for maximizing therapeutic outcomes. A core challenge in MSCs preparation is the considerable variation between cell products from different production platforms and even among batches within the same platform. Unfortunately, this study has not systematically verified the therapeutic efficacy differences among various batches of MSCs products in animal models or clinical trials. Future research must urgently integrate FLI and functional experiments to develop an \u003cem\u003ein vitro\u003c/em\u003e potency evaluation index system capable of predicting \u003cem\u003ein vivo\u003c/em\u003e efficacy, thereby advancing the creation of clinical precision quality control standards.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study employed femtosecond imaging technology to examine the proliferation, senescence, and metabolic states during \u003cem\u003ein vitro\u003c/em\u003e amplification of MSCs from various sources. Our findings suggest that FLI could effectively capture real-time changes in MSCs. We anticipate its application as a rapid, dynamic, and non-invasive method for assessing the quality of stem cells in both laboratory research and clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiawei Liu, Yang Liu: Study design; execution, acquisition of data, analysis and interpretation; Drafting and revising the article; Benhan Xiong: Execution, acquisition of data, analysis and interpretation; Hu Cao, Xue Li: Execution, acquisition of data; Chunyan Tian: Conception; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; Hua Wang: Conception, study design, revising and critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests/Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or nonfinancial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Beijing SH Biotechnology for gifting us DPSC, UCMSC and ADSC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eZhou T, Yuan Z, Weng J, et al. Challenges and advances in clinical applications of mesenchymal stromal cells.\u003cem\u003e\u0026nbsp;\u003c/em\u003eJ Hematol Oncol. 2021;14(1):24. https://doi.org/10.1186/s13045-021-01037-x.\u003c/li\u003e\n \u003cli\u003eK\u0026ouml;nig TT, Goedeke J, Muensterer OJ. Multiphoton microscopy in surgical oncology- a systematic review and guide for clinical translatability. Surg Oncol. 2019;31:119-131. https://doi.org:/10.1016/j.suronc.2019.10.011.\u003c/li\u003e\n \u003cli\u003eBoppart SA, You S, Li L, Chen J, Tu H. Simultaneous label-free autofluorescence-multiharmonic microscopy and beyond. APL Photonics. 2019;4(10):100901. https://doi.org/10.1063/1.5098349.\u003c/li\u003e\n \u003cli\u003eYing W. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal. 2008;10(2):179-206. https://doi.org/10.1089/ars.2007.1672.\u003c/li\u003e\n \u003cli\u003eKolenc OI, Quinn KP. Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD. Antioxid Redox Signal. 2019;30(6):875-889. https://doi.org/10.1089/ars.2017.7451.\u003c/li\u003e\n \u003cli\u003eHuang J, Li Q, Yuan X, Liu Q, Zhang W, Li P. Intrauterine infusion of clinically graded human umbilical cord-derived mesenchymal stem cells for the treatment of poor healing after uterine injury: a phase I clinical trial. Stem Cell Res Ther. 2022;13(1):85. https://doi.org/10.1186/s13287-022-02756-9.\u003c/li\u003e\n \u003cli\u003eSatija NK, Singh VK, Verma YK, et al. Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine. J Cell Mol Med. 2009;13(11-12):4385-402. https://doi.org/10.1111/j.1582-4934.2009.00857.x.\u003c/li\u003e\n \u003cli\u003eSong SB, Shim W, Hwang ES. Lipofuscin Granule Accumulation Requires Autophagy Activation. Mol Cells. 2023;46(8):486-495. https://doi.org/10.14348/molcells.2023.0019.\u003c/li\u003e\n \u003cli\u003eStringari C, Sierra R, Donovan PJ, Gratton E. Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy. J Biomed Opt. 2012;17(4):046012. https://doi.org/10.1117/1.JBO.17.4.046012.\u003c/li\u003e\n \u003cli\u003eWright BK, Andrews LM, Markham J, et al.. NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. Biophys J. 2012;103(1):L7-9. https://doi.org/10.1016/j.bpj.2012.05.038.\u003c/li\u003e\n \u003cli\u003eMeleshina AV, Dudenkova VV, Bystrova AS, Kuznetsova DS, Shirmanova MV, Zagaynova EV. Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation. Stem Cell Res Ther. 2017;8(1):15. https://doi.org/10.1186/s13287-017-0484-7.\u003c/li\u003e\n \u003cli\u003ePriyadarshani P, Van Grouw A, Liversage AR, et al. Investigation of MSC potency metrics via integration of imaging modalities with lipidomic characterization. Cell Rep. 2024;43(8):114579. https://doi.org/10.1016/j.celrep.2024.114579.\u003c/li\u003e\n \u003cli\u003eWang W, Liu P, Zhu W, et al. Skin organoid transplantation promotes tissue repair with scarless in frostbite. Protein Cell. 2025;16(4):240-259. https://doi.org/10.1093/procel/pwae055.\u003c/li\u003e\n \u003cli\u003eXiao Z, Ding L, Yu Y, et al. Tanreqing injection inhibits stemness and enhances sensitivity of non-small cell lung cancer models to gefitinib through ROS/STAT3 signaling pathway. J Cancer. 2024;15(13):4259-4274. https://doi.org/10.7150/jca.94438.\u003c/li\u003e\n \u003cli\u003eKabat M, Bobkov I, Kumar S, Grumet M. Trends in mesenchymal stem cell clinical trials 2004-2018: Is efficacy optimal in a narrow dose range? Stem Cells Transl Med. 2020;9(1):17-27. https://doi.org/10.1002/sctm.19-0202.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FLI, MSCs, Metabolism, Quality control","lastPublishedDoi":"10.21203/rs.3.rs-8327902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8327902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mesenchymal stem cells (MSCs) hold significant promise in regenerative medicine, yet their clinical application is hindered by challenges such as cellular heterogeneity and quality control. This study aims to develop a rapid, non-invasive method for evaluating MSCs quality using femtosecond laser label-free imaging (FLI). We examined the proliferation, metabolic dynamics, and differentiation potential of MSCs from various tissue sources, including human dental pulp, umbilical cord, and fat, across different passages. Our findings indicate that with the passages increasing, the morphology of MSCs alters, proliferation capacity decreases, β-galactosidase activity linked to aging rises, and both osteogenic and adipogenic differentiation abilities markedly decline. FLI technology effectively captures these changes: reduced NAD(P)H/FAD ratio in the cells of higher passage suggests decreased metabolic activity, while enhanced aging-related fluorescence signals, such as lipofuscin, align with cellular senescence. During differentiation, increased fluorescence intensity of NAD(P)H and FAD signals heightened metabolic activity within the cells. Due to varying differentiation potentials among cells from different sources, NAD(P)H and FAD change patterns also differ. Difference in the optical REDOX ratio (FAD/(NAD(P)H+FAD)) among differentiation directions indicate that differentiation potential of MSCs correlates with metabolic reprogramming. Three-dimensional FLI of suspension cells further revealed that the cells of lower-passage exhibit greater spatial heterogeneity in metabolic signals, possibly reflecting more active mitochondrial function. This study confirms that FLI technology can effectively assess the proliferation activity, senescence, and differentiation potential of MSCs through non-invasive, dynamic monitoring of their metabolic status and morphological features, offering a novel approach for standardized quality assessment of MSCs preparations.","manuscriptTitle":"Femtosecond label-free imaging empowers mesenchymal stem cells quality control","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 17:18:39","doi":"10.21203/rs.3.rs-8327902/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-01-09T11:03:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-18T13:17:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T09:18:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-12-13T08:30:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"72ccd8af-7525-4398-949e-c43eabc7087a","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:03:18+00:00","versionOfRecord":{"articleIdentity":"rs-8327902","link":"https://doi.org/10.1007/s13577-026-01366-4","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2026-03-10 15:59:32","publishedOnDateReadable":"March 10th, 2026"},"versionCreatedAt":"2025-12-22 17:18:39","video":"","vorDoi":"10.1007/s13577-026-01366-4","vorDoiUrl":"https://doi.org/10.1007/s13577-026-01366-4","workflowStages":[]},"version":"v1","identity":"rs-8327902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8327902","identity":"rs-8327902","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}