Artificial ovary construction requires a biocompatible scaffold that mimics native extracellular matrix. This study developed a novel decellularization technique combining physical preconditioning (freeze-thaw cycle, multipoint needling, and 10% NaCl hypertonic solution) with shortened chemical detergent exposure (0.5% sodium dodecyl sulfate, 1% Triton X-100, and 2% sodium deoxycholate) for intact porcine ovaries. Compared to conventional chemical-only decellularization, the novel method better preserved glycosaminoglycan and collagen content, induced smaller changes in tissue elastic modulus, and supported higher granulosa cell fusion density in vitro. After subcutaneous implantation in mice, the novel scaffolds promoted greater cell infiltration and neovascularization, while triggering a lower M1-macrophage response. These results demonstrate that integrating physical steps enhances decellularization efficiency and biocompatibility, offering a promising strategy for generating functional ovarian bioscaffolds.
Structural protein alignment plays a vital role in determining the mechanical function and biological performance of engineered tissues. During tissue remodeling, alignment of fibrous components such as collagen and actin can evolve with cellular forces and geometric constraints. Previously, we developed a polarized Raman spectroscopy (PRS) technique that provides a nondestructive, label-free method to quantify protein alignment in tissues and hydrated constructs. Spectral data are analyzed using principal component analysis (PCA) to compute an amplitude alignment metric (AAM), reflecting molecular orientation. In this study, we employed PRS to quantify the spatiotemporal evolution of protein alignment in myoblast-seeded collagen constructs over a 5-day incubation period. Raman spectra were collected at seven polarization angles (0°-180°) across three defined regions-middle center (MC), middle edge (ME), and corner edge (CE)-on days 1, 2, and 5. PRS suggested a region-dependent temporal redistribution of alignment patterns, with early higher alignment at MC and later increased alignment at CE. On day 1, MC exhibited the highest alignment (AAM = 0.71 ± 0.38), while CE showed minimal organization (0.16 ± 0.23). By day 5, alignment was highest at CE (0.62 ± 0.28), while MC decreased (0.57 ± 0.36), indicating a spatial redistribution of matrix organization. There were significant main effects of region and region-by-time interaction, highlighting location- and time-dependent realignment. Within the CE region, alignment on days 2 and 5 was significantly higher than on day 1, consistent with delayed but sustained peripheral alignment. Qualitative immunofluorescence imaging provided complementary visualization of increasing fiber density and directional organization over time. Together, these findings show that PRS, with PCA-based spectral analysis, can detect early spatial and temporal changes in molecular anisotropy within myoblast-seeded collagen constructs. The observed region-dependent alignment changes are consistent with the combined influence of cellular contractility, boundary constraints, and matrix remodeling. Future studies with matched acellular controls and broader remodeling stages and construct conditions will help clarify matrix-associated contributions to PRS-derived alignment during engineered tissue maturation.
Efficient delivery of large gene-editing plasmids, such as the mCherry-CRISPR dCas9 system, into nucleus pulposus (NP) cells is a key step in generating sufficient cells for tissue-engineered intervertebral disc (IVD) constructs and other regenerative therapies for degenerative disc disease (DDD). However, the transfection and transduction of these environmentally sensitive cells remain challenging. This study aimed to identify the best protocol for delivery with minimal cytotoxicity and greatest efficiency. Transfection conditions in HEK293T cells were evaluated using Lipofectamine 3000, Lipofectamine Classic, and ViaFect at different reagent-to-DNA ratios and DNA amounts. Transfection efficiency was quantified by flow cytometry based on mCherry expression. Lentivirus was produced and concentrated by comparing PEG8000, a commercial Lenti-X concentrator, and ultracentrifugation. For NP cell transduction, polybrene and protamine sulfate were tested at multiple concentrations to maximize efficiency and viability. The optimized protocol was validated by delivering a CRISPR/dCas9 Synergistic Activation Mediator (SAM) system to activate endogenous FOXA2, and by seeding the resulting cells onto membranes to assess in vitro NP-like tissue formation. Lipofectamine 3000 at a 2:1 reagent-to-DNA ratio with 0.5 µg DNA per well yielded the highest transfection efficiency in HEK293T cells while minimizing cytotoxicity. Coprecipitation methods for lentiviral concentration, particularly the in-house PEG8000 concentrator, were better than ultracentrifugation. Protamine sulfate at 30 µg/mL yielded efficient NP cell transduction with higher viability than polybrene, as assessed by survival after antibiotic selection. Application of this protocol upregulated endogenous FOXA2 mRNA and protein expression, demonstrating functional efficacy. SAM-FOXA2 cells produced thicker tissue on membrane inserts than SAM controls, confirming compatibility of the protocol with downstream tissue engineering applications. Therefore, an optimized protocol balancing high transfection/transduction efficiency with minimized cytotoxicity was developed, supporting tissue-engineered IVD constructs and other CRISPR-based regenerative therapies for DDD.
Decellularized cartilage xenograft is an emerging alternative to autograft or allograft techniques, though its application has been limited by the toxicity that conventional chemical decellularizing agents have on tissue structure and the environment. We describe a nontoxic, scalable, and environmentally sustainable ovine cartilage decellularization protocol utilizing supercritical CO2(scCO2). Five scCO2 decellularization protocols were evaluated in various ovine cartilage dimensions, of which the multicycle scCO2 method was selected for in vivo evaluation of ovine zested cartilage, ovine minced cartilage, and human minced cartilage. Cartilage groups were enclosed in porous polylactic acid (PLA) scaffolds and implanted subcutaneously on rat dorsa along with an empty scaffold control group. Rats were euthanized after 1, 3, and 6 months for volumetric, histological, and biomechanical analysis of implants. The efficiencies of scCO2 decellularization was enhanced by multicycle process or additional reagents, as well as the smaller dimensions of processed cartilage, as confirmed by DNA quantification and nucleic staining. Preserved structural integrity was evidenced by intact cartilage structure, lacunae components, and collagenous content postprocessing on histology. Implantation in a murine model for 6 months demonstrated excellent neo-tissue ingrowth with minimal inflammatory response. Biomechanical testing revealed no significant differences in moduli or hydraulic permeability of minced and zested samples, before and after the decellularization treatment. The resulting stiffness after implantation mimicked the physical properties of other cartilaginous tissues. In conclusion, we demonstrate the effective decellularization of ovine cartilage graft with a scCO2 protocol while maintaining native tissue architecture and biomechanical properties with a minimal immunogenic response in vivo. Further development of this approach promises to augment surgeon choice of materials used for cartilage reconstruction.
Skeletal muscle tissue engineering (SMTE) is a rapidly evolving field with applications spanning regenerative medicine, disease modeling, drug screening, and biohybrid robotics. Effective SMTE requires scaffolds that reproduce the anisotropic architecture and mechanical properties of native muscle while supporting macroscale tissue formation. Decellularized tissues are strong candidates; however, existing approaches face key limitations. Whole-muscle decellularization requires complex perfusion systems and often fails to fully clear the tissue core. In contrast, minced-tissue processing completely disrupts native architecture and necessitates technically demanding reconstruction. As a result, producing long, continuous scaffolds needed to model or restore physiologically relevant muscle units remains challenging. Here, we present an intermediate strategy that enables the fabrication of long, aligned scaffolds from native muscle bundles. Bundles exceeding 10 cm in length were dissected to preserve native alignment and subjected to mild detergent-based decellularization, achieving efficient removal of cellular material while maintaining extracellular matrix structure and mechanics. The resulting scaffolds supported myogenic cell adhesion, proliferation, and differentiation, demonstrating their suitability for in vitro muscle tissue culture. This accessible approach provides a straightforward route to generate macroscale, structurally faithful skeletal muscle scaffolds, bridging the gap between whole-muscle and minced-tissue decellularization methods.
The absence of induced pluripotent stem cell (iPSC) lines derived from Emirati patients with developmental disease hampers region-specific disease modeling and therapeutic research. Herein, we describe the creation of an iPSC line from peripheral blood mononuclear cells obtained from a 21-year-old Emirati female patient with ventricular septal defect (VSD) using Sendai virus-mediated delivery of reprogramming factors. The resulting line, UAEUi001-A, exhibited typical colony morphology, was mycoplasma negative, successfully generated embryoid bodies), and demonstrated strong alkaline phosphatase activity. These iPSCs were further characterized for pluripotency potential and their differentiation potential into the three germ layers under in vitro culture conditions through immunostaining using stage-specific markers. To the best our knowledge, this is the first reported generation of an iPSC line from an Emirati patient with VSD. Overall, this iPSC line may serve as a valuable model for establishing an Emirati-specific iPSC repository, supporting disease modeling and drug discovery relevant to the Emirati population.Impact StatementThis study establishes the first induced pluripotent stem cell (iPSC) line derived from an Emirati patient with ventricular septal defect, addressing a critical gap in region-specific disease models. It provides a valuable platform for understanding developmental cardiac disorders in underrepresented populations and supports the development of precision medicine and targeted therapeutic strategies relevant to the Emirati population.
Several recent advances in microphysiological systems or organ-on-chip technology have demonstrated its potential for replacing traditional in vitro and animal models in the coming years. Despite the physiological relevance and cost-effectiveness of organ chips, there are several hurdles that must be overcome for widespread adoption for biological studies. Many manufacturing and scalability challenges have been overcome by a transition from polydimethylsiloxane to thermoplastics. However, challenges have arisen in these sealed, brittle systems related to end-point tissue analyses, harvest, and high-resolution imaging, which is particularly difficult for multilayer organ chips. Here, we present low-cost organ chips that are fluidically sealed but demountable, fabricated using a cut-and-assemble method without the need for cleanroom technologies. We have validated the capabilities of this method by demonstrating the culture of human aortic smooth muscle cells and induced pluripotent stem cell-derived neural cells, encapsulated in gelatin methacryloyl (GelMA) hydrogel on chip, for up to 27 days. The three-dimensional (3D) culture layer of the organ chip was removed, and high-resolution images were obtained following immunostaining. Furthermore, these organ chips facilitate rapid redesign and manufacture for alternative tissue and/or interface systems. To the best of our knowledge, this is the first innervated organ chip with multiple removable cell culture layers, as well as the first humanized nerve-artery model that includes a 3D hydrogel culture. In future work, these unique features of our platform can be utilized for investigating the crosstalk mechanisms between different cell types in coculture.Impact StatementWe present here a new method for fabricating low-cost demountable organ-on-a-chip platforms. This method leverages our recent cut-and-assemble method for layered three-dimensional organ chips comprised of gas impermeable thermoplastics.
Semantic segmentation was performed on 177 large histological images of re-endothelialized mouse lung vasculatures. Specifically, patch-based semantic segmentation algorithms were used to classify pixels corresponding to two classes: organ tissue, which includes lung and heart tissue; and ruptured and/or dilated vessels, which are abnormal vessels formed during re-endothelialization. Semantic segmentation is a potential means to automate the end-to-end analysis of these images, circumventing denoising and enhancement operations to visualize tissue and bypassing the manual diagnosis of ruptured and/or dilated vessels. To increase data quantity, images were compressed to sizes 1024 × 1024, 768 × 768, and 512 × 512 and then divided into nonoverlapping 256 × 256 patches. To benchmark the performance of the patch-based models, a vanilla model trained on complete images compressed to size 256 × 256 was also evaluated. The U-Net and LinkNet architectures were used to train and test each model using a data augmentation and transfer learning approach, and their results were ensembled. The loss of image context in the 3 × 3 and 4 × 4 patch-based models negatively impacted performance, generating many false positive predictions for target classes, whereas the low-image quantity of the vanilla model hindered performance. The 2 × 2 ensemble patch-based model returned the best performance, classifying organ tissue with a precision, recall, and intersection over union (IOU) of 88.0% ± 5.7%, 84.7% ± 9.2%, and 76.3% ± 10.9%, respectively, and classifying ruptured/dilated vessels with a precision, recall, and IOU of 78.4% ± 5.2%, 60.2% ± 11.4%, and 51.0% ± 8.4%, respectively.Impact StatementTo evaluate re-endothelization quality, the absence of ruptured and dilated vessels must be confirmed in the resultant re-endothelialized lung scaffold through subjective, work-intensive, and time-consuming per-image manual analysis. In this study, we apply computer vision to detect the ruptured and/or dilated vessels from re-endothelialized histology images via end-to-end semantic segmentation. We also investigate the viability of a patch-based semantic segmentation approach to detect ruptured and/or dilated vessels and generate high-resolution masks. Through this work, the potential of computer vision to automate and standardize the characterization of recellularized lung histology is demonstrated.
Three-dimensional (3D) culture systems have emerged as powerful tools to model tumor biology and bridge the gap between conventional two-dimensional (2D) assays and in vivo studies. Here, we evaluate a poly(ethylene glycol)-based hydrogel as a simplified yet functionally relevant platform for modeling premalignant lung adenocarcinoma using the A549 cell line. Cells cultured in hydrogels exhibited transcriptional profiles that more closely resembled xenograft tumors than conventional 2D monolayers. Pathway-level analysis and regression-based benchmarking revealed restoration of critical hallmark programs, including proliferation, immune signaling, developmental pathways, and stress response cascades. While the 3D model does not fully recapitulate the complexity of the tumor microenvironment, its chemically defined, tunable, and reproducible design offers an accessible, physiologically informative model of lung adenocarcinoma that restores key transcriptional and functional features lost in conventional culture systems.
This research aimed to find the optimal sterilization process for ovarian decellularized extracellular matrix (OV-dECM). OV-dECM was prepared. Four different sterilization processes, such as ultraviolet ray, 1 mg/L CuCl2 + 0.5% H2O2, 70% ethanol, and 0.1% peracetic acid (PAA), were evaluated. The sterilization efficiency and the in vitro and in vivo biocompatibility were assessed. Hematoxylin and eosin, 4',6-diamidino-2-phenylindole staining, and DNA quantitation indicated the successful production of OV-dECM. 0.1% PAA and 70% ethanol achieved better sterilization. The sterilized OV-dECM in the 70% ethanol and 0.1% PAA groups could integrate with human umbilical cord mesenchymal stem cell (between them: 7 days: p = 0.187; 10 days: p = 0.293; both p > 0.05) and keep the cell viability (CCK-8, cell counting kit-8) and cell morphology (F-actin staining). The sterilized OV-dECM in both 70% ethanol and 0.1% PAA groups had good CD31 (a neovascularization maker) fusion indexes (between them: p = 0.288; p > 0.05), while α-smooth muscle actin (another neovascularization maker) in the 0.1% PAA group was higher than that in the 70% ethanol group (between them: p = 0.012; p < 0.05). The assessment of CD68, CD86, and CD206 (macrophage phenotype makers) demonstrated that the 0.1% PAA group had better chance to promote the M1-M2 transformation of macrophage than 70% ethanol after the implantation of OV-dECM in mice. In conclusion, 0.1% PAA is the optimal sterilization process for OV-dECM with better sterilization capacity and biocompatibility.Impact StatementThis study identifies 0.1% peracetic acid as the optimal sterilization method for ovarian decellularized extracellular matrix (OV-dECM), resolving a critical barrier to artificial ovary (AO) development. It ensures OV-dECM sterility while preserving biocompatibility, promoting macrophage M1-to-M2 polarization to reduce inflammation. This advances AO translational potential, offering hope for fertility preservation in patients with cancer/leukemia and guiding standardized OV-dECM sterilization in regenerative medicine.
Metastatic dissemination in triple-negative breast cancer (TNBC) arises from extensive intratumoral heterogeneity, yet the clonal and transcriptional programs that underlie invasive behavior remain poorly defined. The field lacks experimental systems capable of simultaneously tracking the clonal identity and transcriptional state of invasive subpopulations. Here, we apply a clonal lineage-tracing platform coupled with single-cell RNA sequencing to resolve how subpopulations contribute to invasion in a model of TNBC. We demonstrate that invasion is driven by a subset of recurrent clones, indicating the presence of pre-existing subpopulations intrinsically primed for migratory behavior. Transcriptomic profiling further reveals two transcriptomically distinct invasive cell states that remain stable across independent selections. Targeted perturbations suppress these dominant invasive programs but enable invasion by alternative rare clones, demonstrating compensatory clonal dynamics in response to selective pressure. Together these findings show that invasive potential arises from stable clonal programs and that heterogenous tumors maintain invasive capacity through clonal replacement. Clonally resolved single-cell profiling provides a framework for dissecting invasive heterogeneity and highlights the need for therapeutic strategies that account for clone-specific behaviors and the dynamic restructuring of tumor subpopulations.
There is renewed interest in three-dimensional in vitro bioengineered models that replicate key aspects of the in vivo environment for the study of cellular behavior, with one key aspect being cell interactions with matrix interfaces. Here, we developed a dual-stiffness hydrogel-encapsulated glioblastoma (GBM) spheroid model to investigate GBM spreading along a stiffness interface. GBM is an aggressive brain cancer with a patient prognosis of 12-18 months, which is known to spread to distant brain regions by following stiffness interfaces. Our model consisted of a soft, 5% w/v, polyethylene glycol (PEG) hydrogel to mimic the native brain tissue and a stiff, 10% w/v, PEG hydrogel to replicate the stiffer GBM microenvironment. To ensure spheroids fall along the boundary, we adjusted the gelation time of the gel by varying the pH of the gel precursor solution. Encapsulated spheroids were assessed for infiltration and viability for up to 7 days. Spheroids exhibited high viability in all hydrogels. Spheroids showed a higher infiltration index in the soft hydrogel, and migration across the stiffness interface occurred only from the soft to the stiff hydrogel in the dual-stiffness gels. The developed model has a simple, robust design for studying GBM behavior in vitro, a high degree of imageability, requires no specialized equipment to prepare, and is compatible with a multiwell plate format for easy handling and analysis.
Conventional drug screening models face a series of challenges in guiding individualized cancer treatment, including time-consuming processes, limited drug coverage, and insufficient accuracy in efficacy evaluation. This study aims to establish a convenient, rapid, and reliable drug screening protocol for evaluating individualized efficacy of chemotherapy and immunotherapy. We established an ex vivo mini-tumor culture platform by culturing tumor fragments in an air-liquid interface system, which was capable of sustaining tumor growth for at least 2 weeks and maintaining immune cell infiltration for over 1 week. Using this mini-tumor culture platform, we can evaluate the individualized therapeutic responses of different tumors to chemotherapy and immunotherapy, including gemcitabine, 5-fluorouracil, cisplatin, αPD-1 and αPD-L1. Furthermore, using this drug evaluation platform, we revealed distinct therapeutic responses to immunotherapy between immune-cold tumors and immune-hot tumors, and demonstrated the important role of the immunologic adjuvant resiquimod (R848) in enhancing immunotherapy efficacy. This mini-tumor culture protocol provides a feasible implementation approach for ex vivo personalized drug testing.
Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surrounding brain microenvironment and drug resistance. Multicellular spheroids are an increasingly common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogeneous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild-type (WT) and temozolomide (TMZ)-resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 1,000 to 10,000 cells in size were embedded into a methacrylamide-functionalized gelatin hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid-size-dependent effects on TMZ efficacy for both TMZ-resistant and WT cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing, while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.
In this study, we demonstrate an automated approach to efficiently and reproducibly manufacture perforated poly(ε-caprolactone) (PCL) solution electrospun tubular meshes designed for critically-sized bone defect repair. The workflow improves reproducibility and reduces fabrication time by 67% (8.7 vs. 2.7 h per 10 meshes). By directly electrospinning PCL onto a rotating cylindrical mandrel, seam-related discontinuities are eliminated, and subsequent use of an automated soldering iron system enables precise 1 mm perforations that promote vascular ingrowth during bone healing. Despite the decrease in mass of the new design compared with the original design (18.24 ± 1.5 mg for old vs. 11.48 ± 1.2 mg for new design), mechanical testing revealed similar resistance to lateral compression compared with semimanually assembled meshes. This is important to prevent collapse during surgical placement and injection of osteoinductive treatments. Further, eliminating surgical glue improves the manufacturing simplicity and scaffold reproducibility. Following implantation with bone morphogenic protein-2 loaded alginate, the new design performed similarly to the original: in vivo microcomputed tomography confirmed bone formation that significantly increased (p ≤ 0.05) over 8-weeks in an established rat femoral defect model. This study provides a novel production method of tubular scaffolds with variable dimensions and flexible perforation patterns and demonstrates improvements in fabrication efficiencies and reproducibility.
Patients with breast or prostate cancer have a high chance of developing bone metastasis, which is associated with many skeletal-related events. The development of novel bone metastasis treatments is lagging behind due to the lack of reliable models. We aimed to develop a humanized bone metastasis model comprising vital human bone discs and human metastatic cancer cells (bone metastasis discs), which were subsequently cultured ex vivo or subcutaneously implanted into nude mice. Ex vivo culture experiments confirmed that cells within the bone metastasis discs remained metabolically active, while the presence of metastatic cancer cells could be monitored using bioluminescence. Although histological analyses confirmed the presence of relevant bone cells in the human bone tissue, no apparent formation of metastatic lesions was detected over the 2-week ex vivo culture period. In contrast, subcutaneously implanted bone metastasis discs demonstrated clear metastatic lesion formation, with osteolytic characteristics, that progressed from 3 to 6 weeks after implantation for both breast and prostate cancer bone metastasis discs. Histologically, healthy bone tissue with bone marrow compartments as well as anastomosis was observed. Cisplatin treatment of ex vivo cultured bone metastasis discs significantly decreased the bioluminescent signal from (prostate) cancer cells, while no effects of cisplatin treatment were observed for in vivo implanted bone metastasis discs. Our data provide a proof of concept for an ex vivo/in vivo bone metastasis model with vital human bone and human metastatic cancer cells but require further fine-tuning to improve robustness, relevance, and quantification methods. Future research could potentially use these models for the evaluation of novel bone metastasis treatments, accelerating their potential clinical application.
Evaluating the complex, three-dimensional (3D) architecture of de novo angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for complete visualization of the microcirculatory system is needed. We present an integrated workflow for high-resolution 3D visualization of neovascularization within arteriovenous (AV) loop-based tissue constructs in a rat model. An intravascular perfusion with a cationic near-infrared fluorescent dye, MHI148-polyethylenimine, was used to 3D label the patent vasculature. Following perfusion-fixation and explantation, the construct was rendered optically transparent using an ethyl cinnamate-based clearing protocol. The fluorescent signal was then imaged using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation. Our workflow successfully achieved high-contrast, 3D visualization of the microvascular network, allowing for whole-mount and segmental analysis of the vascular tree. At day 7, imaging delineated solely the AV loop axis while by day 28, a dense and complex, interconnected capillary plexus from the central axis demonstrated a progressive neovascularization. Downstream processing compatibility was confirmed through successful rehydration and 3D nuclear counterstaining. This workflow offers a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs, overcoming key limitations of existing techniques.
Adipose tissue is an abundant and clinically accessible source of stromal cells. Stromal vascular fraction (SVF) and nanofat have been widely investigated for their regenerative potential; however, commercial systems vary considerably in yield, viability, and regulatory oversight. Most devices report fresh results only, with limited validation following cryopreservation. Mesenchymal stromal cells derived from adipose tissue have also attracted attention due to their accessibility, immunomodulatory effects, and multipotent differentiation capacity. Uvence has developed a proprietary workflow for adipose tissue processing that integrates washing, cryopreservation, thawing, and emulsification within a Human Tissue Authority-regulated laboratory. The process includes Good Manufacturing Practices (GMP) Annex 1-aligned environmental monitoring and independent quality control (QC) testing. Critically, this workflow validates postthaw cell viability, addressing a gap in current SVF/nanofat approaches. Three cryopreserved donor samples demonstrated a mean postthaw viability of ∼91% (range 90.5-92%), consistently exceeding the International Federation for Adipose Therapeutics and Science (IFATS)/ International Society for Cell and Gene Therapy (ISCT) 70% threshold. Benchmarking against global systems showed Uvence postthaw viability to be equivalent to or higher than fresh outcomes reported for enzymatic platforms (Celution, 85-91%; InGeneron, 86%) and mechanical platforms (Lipocube, Tulip, ∼96%). Unlike competitor devices, Uvence has validated freeze-thaw performance, providing a stable and compliant platform. This study also presents in vitro culture and characterization of stromal cells expanded from Uvence nanofat-derived SVF samples, including flow cytometry, morphology, and trilineage differentiation. Flow cytometry confirmed high expression of CD73, CD90, and CD105, with minimal expression of CD34/CD45, consistent with the ISCT criteria. While these findings are limited to research characterization and do not constitute approval for therapeutic use, they demonstrate that the Uvence workflow delivers a quality-focused approach to adipose tissue processing.
