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Aim: Adipose tissue-derived microvascular fragments (ad-MVF) are vascularization units for regenerative medicine. We investigated whether University of Wisconsin (UW) solution is suitable for their xeno-free storage. Materials & methods: Murine ad-MVF were cultivated for 24 h in 4°C or 20°C UW solution and 20°C endothelial cell growth medium (control). The ad-MVF were seeded onto collagen–glycosaminoglycan scaffolds, which were analyzed in dorsal skinfold chambers by intravital fluorescence microscopy and histology. Results: All implants exhibited microvascular networks on day 14 with the highest functional microvessel density in controls. Ad-MVF cultivation in UW solution at 4°C resulted in an improved scaffold vascularization compared with cultivation at 20°C. Conclusion: UW solution is suitable for the hypothermic storage of ad-MVF.

In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the treatment of diseases has increased significantly. The cell quantities required for such therapeutic approaches, between 1012 and 1013, have thus far been predominantly produced using commercially available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning), which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bioreactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems, while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems, adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also referred to as microcarriers (MCs).This chapter describes the steps which need to be realized to guarantee successful hMSC expansion within a stirred single-use bioreactor (Eppendorf's BioBLU® 0.3c) operated using MCs under serum- and xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were performed under comparable process conditions.

Collagen type I, commonly derived from xenogenic sources, is extensively used as a biomaterial for tissue engineering applications. However, the use of xenogenic collagen is typically associated with species specific variation in mechanical, structural, and biological properties that are known to influence cellular response and remodeling. In addition, immunological complications and risks of disease transmission are also major concerns. The goal of this study is to characterize a new xeno-free human skin-derived collagen and assess its applicability as a bioink for cell-laden 3 D bioprinting. Four different concentrations of human collagen (i.e., 0.5 mg/mL, 1 mg/mL, 3 mg/mL and 6 mg/mL) were employed for the synthesis of collagen hydrogels. In addition, bovine collagen was used as a xenogenic control. Results from SDS-PAGE analysis showed the presence of α1, α2, and β chains, confirming that the integrity of type I human collagen is maintained post isolation. Polymerization rate and compressive modulus increased significantly with increase in the concentration of human collagen. When comparing two different sources of collagen, the polymerization rate of xenogenic collagen was significantly faster (p −1, indicating a dense and supraorganized fibrillar structure in human collagen hydrogels. Conversely, Amide I band intensity for xenogenic collagen was comparable to that of Amide II and Amide III bands. Further, the use of 6 mg/mL human collagen as a bioink yielded 3 D printed constructs with high shape fidelity and cell viability. On the other hand, xenogenic collagen failed to yield stable 3 D printed constructs. Together, the results from this study provides an impetus for using human-derived collagen as a viable alternative to xenogenic sources for 3 D bioprinting of clinically relevant scaffolds for tissue engineering applications.

Human pluripotent stem cells (hPSCs) can be expanded and differentiatedin vitrointo almost any adult tissue cell type, and thus have great potential as a source for cell therapies with biomedical application. In this study, a fully-defined polymer synthetic substrate is identified for hPSC culture in completely defined, xeno-free conditions. This system can overcome the cost, scalability and reproducibility limitations of current hPSC culture strategies, and facilitate large-scale production. A high-throughput, multi-generational polymer microarray platform approach was used to test over 600 unique polymers and rapidly assess hPSC-polymer interactions in combination with the fully defined xeno-free medium, Essential 8TM(E8). This study identifies as novel nanoscale phase separated blend of poly(tricyclodecane-dimethanol diacrylate) and poly(butyl acrylate) (2:1 v/v), which supports long-term expansion of hPSCs and can be readily coated onto standard cultureware. Analysis of cell-polymer interface interactions through mass spectrometry and integrin blocking studies provides novel mechanistic insight into the role of the E8 proteins in promoting integrin-mediated hPSC attachment and maintaining hPSC signaling, including ability to undergo multi-lineage differentiation. This study therefore identifies a novel substrate for long-term serial passaging of hPSCs in serum-free, commercial chemically-defined E8, which provides a promising and economic hPSC expansion platform for clinical-scale application.

Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.

We describe an innovative method of culturing a three-dimensional cell mass (3DCM) of human adipose-derived stem cells (hASCs) in the xeno-free (XF) condition for the treatment of severe ischemic diseases. The majority of the mice injected with XF-3DCMs exhibited limb salvaging and displayed similar blood perfusion compared to normal limbs.

Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 &times, 105&ndash, 6 &times, 105 cells/mL within 5 days with no media exchange, maintaining the final cell population doubling level (PDL) at 16&ndash, 20. Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs.

In this study, we compared three commercially available and two widely used CPAs for their ability of cryopreserving PBMCs. Similar survival (81.0%) and recovery rate (73.7%) were observed among cells using these five CPAs. However, all the cryopreserved PBMCs exhibited a significantly lower survival rate when compared with the fresh samples (94.3%). We further evaluated effector cell subpopulation and tumoricidal activity of PBMC-derived cytokine-induced killing (CIK) cells and natural killing (NK) cells. Similar and high survival (CIK: 88.6%; NK: 87.5%) and recovery (CIK: 99.5%; NK: 99.7%) rates were detected in CIK and NK cells prepared from cryopreserved PBMCs using the five CPAs. The CD3+CD56+ effector percentage (27.3%) of cryopreserved PBMC-derived CIK cells using the five different CPAs and their tumoricidal activities on melanoma CHL-1 cells (45.7%) and bladder cancer cell line T-24 (44.7%) were similar but significantly lower than those of the fresh PBMC-derived controls (effector: 30.7%; CHL-1: 84.2%; T-24: 82.2%). Cryopreserved PBMC-derived NK cells also exhibited similar tumoricidal activities (CHL-1: 73.8%; T-24: 71.9%) but was significantly lower than that of the fresh control group. We were not able to identify a specific CPA that performed superior than others in PBMC cryopreservation.

Background Mesenchymal stem cells (MSC) are promising in cell therapy especially for their paracrine action and immunomodulatory and anti-inflammatory functions. However, for clinical application, these cells need to expand in vitro to achieve enough number for therapy. Most of the studies for MSC expansion use xenogeneic products. In order to provide a safe therapy, using animal protein-free products have been considered. In this study it was evaluated the immunomodulatory potential of umbilical cord MSC (UC-MSC) using xeno-free expansion conditions compared with UC-MSC expansion using animal derived components. Methods Six samples of UC-MSC were isolated and expanded until passage 3 (P3), under xeno-free conditions and with animal derived components. Immunomodulation was evaluated by lymphocyte proliferation assay where MSC suppress peripheral blood mononuclear cell (PBMC) proliferation. Results UC-MSC cultured under the xeno-free conditions, were able to inhibit 37.69% of T lymphocyte proliferation, in the ratio 1:2 (PBMC:MSC), 65.33% in 1:5 and 78.04% in 1:10. In the protocol using animal derived components, the MSC inhibitory capacity was 74.33% in the ratio 1:2, 74.60% in 1:5 and 79.61% in 1:10. The inhibitory capacity of UC-MSC in the ratio 1:2, was statistically significantly lower in xeno-free conditions than in the protocol using animal derived components (p=0.004). In ratios 1:5 and 1:10, the inhibitory capacity was similar, with no statistical difference (p=0.238 and p=0.792, respectively). Conclusion The inhibitory capacity of UC-MSC in the ratio 1:2 was insufficient in the xeno-free conditions, because UC-MSC inhibit less than 50% of T cells, that is under the criteria established for clinical use, which must be ≥ 50%. The T lymphocyte proliferation assay showed that a greater number of MSC in xeno-free conditions is necessary for an effective immunosuppression. The study showed that UC-MSC culture under xeno-free conditions reduced the immunomodulatory potential of these cells.

With cell therapies advancing towards the patients’ bedside, there is an increased need for robust and up-scalable protocols to generate clinical grade stem cell-derived products.The protocol we present in the study provides a xeno-free, chemically defined, scalable and robust retinal pigment epithelial cell monolayer differentiation without the need for manual isolation that uses either human embryonic stem cells or human induced pluripotent stem cells as a starting material.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryo-preserved ASCs could be applied for regenerative medicine.

Xeno-free and chemically-defined poly(ethylene glycol) (PEG)-based hydrogels are increasingly used for in vitro culture and differentiation of induced pluripotent stem cells (iPSCs). These synthetic matrices provide highly tunable gelation kinetics and adaptable material properties that are crucial for guiding stem cell fate processes. In this work, we integrated sequential norbornene-click chemistries for forming synthetic and dynamically tunable PEG-peptide hydrogels to promote human iPSCs culture and differentiation. Specifically, iPSCs were photo-encapsulated in xeno-free thiol-norbornene hydrogels crosslinked by multi-arm PEG-norbornene (PEG-NB) and protease-labile peptide crosslinkers. These matrices were used to evaluate growth of iPSCs under the influence of matrix degradability, adhesion ligands, cell density, and ROCK inhibition. We further employed tetrazine-norbornene (Tz-NB) inverse electron demand Diels-Alder (iEDDA) reaction to dynamically stiffen these cell-laden hydrogels. Fast reactive Tz and its more stable derivative methyltetrazine (mTz) were tethered to multi-arm PEG, yielding mono-functionalized PEG-Tz and PEG-mTz, as well as dual-functionalized PEG-Tz/mTz that readily reacted with PEG-NB to form additional crosslinks in the cell-laden hydrogels. The versatility of this stiffening approach was demonstrated with different Tz-modified macromers (e.g., hyaluronic acid-Tz and heparin-Tz) or by intermittent incubation of PEG-Tz for temporally regulated stiffening. Finally, the Tz-NB-mediated dynamic stiffening was explored for 4D culture and definitive endoderm differentiation of human iPSCs. Overall, this dynamic hydrogel platform affords exquisite controls of hydrogel crosslinking for serving as a xeno-free and dynamic stem cell niche.

This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

Background & Aim Extracellular vesicles (EVs) are increasingly being investigated as both drug delivery vehicles and as a cell-free therapy. EVs from mesenchymal stem/stromal cells (MSC-EVs), for example, have been shown to recapitulate many of the therapeutic effects of the cells themselves. A critical barrier in the development of MSC-EVs as a commercial therapy, however, is generating the large amount of EVs that will be required per dose. In a recent survey, the majority of those working with EVs were working with less than 100mL of sample (ISEV 2016). In this study, two strategies were investigated to maximize EV yield. First, timing parameters for culturing human bone-marrow derived MSCs and collecting MSC-EVs were evaluated to increase EV yield. Second, conditioning the cells was investigated by adding an EV activator during the EV collection period. Methods, Results & Conclusion MSCs were first cultured in an expansion media (RoosterNourishTM, RoosterBio) in a fed-batch system. The cultures were then switched to a collection media (RoosterCollectTM-EV, RoosterBio) with or without an EV activator (EV BoostTM, RoosterBio). After two days, the conditioned media was harvested. The EVs in the collected conditioned media were analyzed for particle concentration and particle size distributions by nanoparticle tracking analysis, protein expression, RNA expression, and wound healing capability. Particle concentration (particles per L of media) was used as a measure of EV yield. Particle concentration increased with increased culture time in expansion media, with confluent cultures generating the most EVs. The EV activator at a low concentration increased EV yield about 2-fold compared to without the activator after 24hrs. The EV yield from the low EV activator culture at 24hrs was still about 45% greater than the yield of the culture without the activator after 48hrs. After 6hrs of collection, the high EV activator concentration increased EV yield to about the same level as without the activator after 48hrs of collection. Optimizing protocols for EV yield will become increasingly important as MSC-EVs move towards the clinic and lot sizes of clinically-relevant doses are required. Pre-conditioning of cells or conditioning cells during EV collection offers one possibility to further increase EV yield and decrease process time.

This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block-DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells' morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost-effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno-free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3-D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 104 and 1.4 ± 0.3 × 104 cells/cm2 . UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5-23.0 × 104 cells/cm2 ) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno-free expansion processes represents an important step toward a GMP compliant large-scale production platform for MSC-based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358-1367, 2017.

Currently, stem cell research faces a major bottleneck related to the low efficiency of methods to produce large quantities of human embryonic stem cells (ESC) for use in clinical trials. Most culture media currently employed for human ESC cultivation contain animal compounds, and cells are grown in static flasks. Besides the immediate contamination with nonhuman compounds, cell expansion in flasks tends to be laborious and nonefficient. Here we cultured human ESC in stirred microcarrier (MC) systems using an animal/human-component-free medium, to overcome both issues. The method developed to culture cells on suspended beads combined the use of polymeric MCs in stirred vessels with an optimized culture medium free of supplements of animal and human origin. This approach generated approximately 160 million cells within 6 days, which were shown to remain pluripotent. The process developed herein provides a step forward toward therapy due to the economic advantages in the production of human ESC and to their consequent low immunogenic potential.

The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source of human fibroblasts is essential for long-term xeno-free hESC maintenance.

The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno-free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner-flasks. A maximum 4.0 ± 0.8-fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno-free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration-based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier-based hiPSC expansion, leading to a more cost-effective and efficient bioprocess.

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

AIM: To optimize a xeno-free cryopreservation protocol for primary human hepatocytes. METHODS: The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7. RESULTS: The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r2 = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples. CONCLUSION: The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.