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INTRODUCTION AND OBJECTIVE:In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS), however, animal components introduce the risk o...

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

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.

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.

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.

Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods; however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f; a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.

Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.

Human extended pluripotent stem (EPS) cells have shown great applicative potentials in basic and translational research. One prerequisite for the wide applications of EPS cells is the development of a xeno-free culture system for maintain these cells in vitro. Here we report a protocol for culturing and generating human EPS cells using a chemically defined and xeno-free culture system.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of Endometrial Mesenchymal Stromal Cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, the surgical curettage and the Vacuum Aspiration Biopsy Random Assay, and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell lines were isolated after a mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential and multilineage differentiation. The expression of mesenchymal and stemness markers was tested by FACS analysis and Real-Time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed to efficiently isolate and expand E-MSCs using a xeno-free method preserving their mesenchymal and stemness phenotype, proliferative potential and multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting Vabra endometrial sampling as alternative to surgical curettage.

Human induced pluripotent stem cells hold the promise to revolutionize medicine in the near future. For application in medicine, human induced pluripotent stem cells must be manufactured under culture conditions that enable consistent and robust production of safe and therapeutically functional products. This chapter provides a summary of the protocols and methods exploited to culture human pluripotent stem cells to date, with an emphasis on the various xeno-free systems that enable production of cells for clinical applications. Although promising results have been generated in vitro and in preclinical animal models, so far very few human induced pluripotent stem cell–derived products have entered the clinical phase. The development and implementation of state-of-the-art culture technologies, in adherence with international manufacturing guidelines, are critical to produce clinical-grade cells for routine medical applications.

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.

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar 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.

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.

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.

Mesenchymal stem cells (MSCs) have been used in therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. To foster commercialization and implementation of stem cells treatments, researchers have recently derived MSCs from human induced pluripotent stem cells (iMSCs). For therapeutic applications, human iMSCs must be produced in xeno-free culture conditions and following procedures that are compatible with the principles of Good Manufacturing Practice.

BACKGROUND: Human induced pluripotent stem cells (hiPSC) are expected to become powerful tools for disease modelling, for the discovery and testing of new drugs and, ultimately, for regenerative therapies. The success of these applications depends on the development of scalable bioprocesses capable of generating large numbers of hiPSC and derivatives. RESULTS: In this work, the novel vertical‐wheel single‐use bioreactors were used for the first time for the expansion of hiPSC under xeno‐free conditions. Cultures were performed on microcarriers in two different scales of vessels (100 and 500 mL with 80 and 300 mL working volumes, respectively), leading to maximum cell densities up to 1.21 ± 0.02 × 10⁶ cells mL⁻¹ and volumetric productivities of 2.01 ± 0.04 × 10⁵ cells mL⁻¹ day⁻¹. The pluripotency as well as a normal karyotype were maintained after cell expansion. Consistency of the processes was confirmed with a different hiPSC line, which is an important aspect for a personalized medicine approach. CONCLUSION: The results here described demonstrate the feasibility of scalable production of hiPSC in a microcarrier‐based system using vertical‐wheel bioreactors. The protocols developed in this study provide a Good Manufacturing Practices (GMP)‐compliant system for hiPSC manufacturing which may be an important step towards the successful implementation of hiPSC‐based products. © 2018 Society of Chemical Industry

Background and Objectives Stem cells from human exfoliated deciduous teeth (SHED) are a promising clinical resource for various tissue defects, including lumbar spondylosis, neural compression, and cleft palate. Use of media containing animal-derived serum carries potential risk of infectious diseases and unwanted immunogenicity. To increase the potential utility of SHED for clinical application, SHED was adapted to xeno-free conditions. Methods Define xeno-free culture media were compared with the conventional serum containing media in the culture of SHED. Cultured SHED in different media were further characterized through proliferative capacities, cellular phenotype, and differentiation potential. Results Selected xeno-free media were capable of supporting the growth of SHED. MSCGM-CD Bulletkit medium greatly increased the number and proliferate capacity of colony-forming unit-fibroblast than SHED cultured in other media. In addition, the characteristic surface markers expression and multipotent differentiation potential of SHED in the MSCGM-CD Bulletkit medium were comparable to those observed with serum-containing medium. Conclusions The xeno-free medium described herein has the potential to be further used for the safe expansion and to determine efficient way to produce clinical grade dental stem cells for therapeutic applications.

Background In chronic wounds, healing process gets halted in inflammatory phase owing to lack of growth factors (GF). The concept of exogenous delivery of such GF as platelet rich plasma (PRP)/fibrin (PRF) could be logical strategy for their management. We evaluated efficacy of single dose of non-activated PRP and self- polymerized platelet rich fibrin (PRF) in subjects with chronic wounds. Methods 35 Subjects with wounds of different depths and etiologies were administered with PRP or PRP + PRF and assessed with Bates-Jensen Wound Assessment Tool (BWAT). Results Majority of subjects in PRP group responded well and entered into regenerative zone. In PRP + PRF group, 36% subjects benefitted from this treatment. Conclusion Our study demonstrates that single dose of non-activated PRP is an efficacious treatment regime for chronic wounds. Further, it eliminates need for multiple doses due to continual release of growth factor over a long period of time, thus considerably reducing effective cost of procedure.

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.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiringin vitroexpansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

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.

Reprogramming somatic cells to generate induced pluripotent stem cells (iPSCs) has presented the biomedical community with a powerful platform to develop new models for human disease. To fully realize the promise of this technology in cell therapy and regenerative medicine, creating iPSCs under current Good Manufacture Practice (cGMP) conditions is paramount. Some reports have described efforts in this regard, resulting in iPSC lines that are cGMP compliant. The technology developed at Allele Biotechnology for footprint-free, feeder-free, and xeno-free reprogramming using only mRNA is very suitable for creating iPSC lines through an established cGMP process. This technology has resulted in a licensing agreement between Allele Biotechnology and Ocata (formerly ACT, now a wholly owned division of Astellas) for clinical applications. All reagents and vessels are certified as cGMP-produced, all equipment and software are certifiable, and all procedures are carried out in Industry ISO 7 or Class 10,000-grade cleanrooms. In this revised version of the unit, we describe the core improvements to implement steps toward cGMP-compliant generation of iPSCs. Recreating a process close to cGMP production in academic research will make these findings more applicable to translational research. © 2016 by John Wiley & Sons, Inc. Keywords: induced pluripotent stem cells; iPSC; mRNA reprogramming; feeder-free; xeno-free; cGMP

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.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines. Keywords: Transplantation therapy, Xeno-free, Clinical-grade hESC lines, Disease-affected hESC lines, Biosafety testing, QC testing, Certificate of analysis

In the original article, Fig. 1A was by mistakenly duplicated. The corrected image is provided in this correction article.

Aim: Endothelial cells (ECs), isolated from peripheral blood (PB), bone marrow (BM) and cord blood (CB), are limited in numbers and expansion has had limited success. We used a novel serum-free medium (EndoGo) to evaluate effects on ex vivo expansion of CB-derived ECs. Materials & methods: Flow cytometry and matrigel were used to determine expansion of ECs and for determination of the EC progenitor cell. Results: EndoGo™-containing cultures demonstrated superior expansion and stimulated proliferation of two distinct subpopulations, CD34+CD31+ and CD34-CD31+, which exhibited different morphology, phenotype and function. EndoGo also expanded the CB endothelial progenitor cells from freshly isolated CB. Conclusion: These findings demonstrate the potential of EndoGo to expand CB ECs, which could generate increased numbers of ECs for therapeutic applications., Lay abstract Endothelial cells are important for the development of blood and lymph vessels, needed for proper blood and lymph system function. Damage to these vessels can occur with various conditions and diseases. Many tissues cannot regenerate, requiring therapy. Cell therapy can be an important tool in accelerating the repair and recovery process. Development of a cell expansion protocol unique to endothelial cells, initiated from a widely available source as in cord blood, is important to achieve this process. We demonstrate a unique medium that can promote proliferation of endothelial cells that could be further developed for clinical settings.

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.

Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.

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.

Due to their immunomodulatory and chemotactic properties, hMSC are being explored to treat immune-related diseases. For their use in human therapies, it is necessary to culture hMSC in xeno-free conditions. In this study, the impact that a xeno-free medium based on a human plasma derivate has on these properties was analysed. Bone marrow-derived hMSC preserved their immunosuppressive and immunostimulatory properties, as observed with in vitro assays with hMSC cocultured with mixed leukocyte reactions or with mitogen-stimulated leukocytes. Moreover, hMSC expanded in xeno-free medium were recruited by macrophages in both migration and invasion assays, which indicates that the cells maintained their chemotactic properties. These data suggest that xeno-free expanded hMSC preserved their immunomodulatory and chemotactic properties, indicating that the described xeno-free medium composition is a potential candidate to culture and expand hMSC for human cell therapies.