Xeno-free Medium Research Articles

Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage

Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine.

Limbal epithelial stem cell sheets from young donors have better regenerative potential

To investigate the stemness of limbal epithelial stem cell sheets in relation to the donor’s age. Human limbal explants from cadaveric donors were set on human amniotic membrane scaffolds with the xeno-free medium. We evaluated limbal epithelial sheet size, expression of stem/progenitor cell markers, and colony formation efficiency from donors of different age groups (age ≤ 45, age 45–65, and age > 65). Expression of the proliferation marker Ki67, stem/progenitor cell markers p63α and ABCG2, cornea specific marker PANCK, and differentiation marker CK12 were evaluated. To determine the effect of donor age on the storage period of limbal explant sheets, the limbal explant outgrowth sheets were stored in 4 °C for 2 days and analyzed for JC-1, p63α, and PANCK with FACS on each day. From days 6 to 12, the outgrowth area of the limbal epithelial stem cell sheet was significantly larger in the age ≤ 45 groups (296 ± 54.7 mm2, day 9) compared to the other two age groups [age 45–65 group (278 ± 62.6 mm2), age > 65 group (257 ± 44.0 mm2), day 9] (p < 0.01). In terms of stemness, outgrowth cells from aged donors (age > 65) showed lower expression of stem/progenitor cell markers p63α and ABCG2 and decreased CFE compared to the other two groups. There were significantly more p63α+ cells in outgrowth cells in the age ≤ 45 group (18.2 ± 3.6%) compared to the age > 65 group (14.1 ± 4.6%; p < 0.01). Limbal explant outgrowth sheet on the age ≤ 45 group (32.7 ± 7.5%) had higher percentages of cells resisting staining by JC-1 compared with sheets under the age > 65 groups (25.7 ± 7.1%, p < 0.01) (JC-1low). Cells from the age ≤ 45 group showed a higher clonogenic capacity than those from the other two age groups (45 < Age ≤ 65 CFE ratio = 0.7 ± 0.16, p < 0.01; 65 < Age CFE ratio = 0.3 ± 0.06, p < 0.01, vs. Age ≤ 45). In the age > 65 group, positive cells of p63α on D0, 1, and 2 were significantly lower compared to those in the age ≤ 45 group on the storage period (p < 0.01, respectively). Our results imply that donors younger than 65 years of age are a better source of limbal epithelial stem cell sheet generation with high regeneration potential.

Sourcing of human peripheral blood-derived myeloid angiogenic cells under xeno-free conditions for the treatment of critical limb ischemia

BackgroundCritical limb ischemia (CLI) is the most severe form of peripheral artery disease and exhibits a high risk of lower extremity amputations. As even the most promising experimental approaches based on mesenchymal stem cells (MSCs) demonstrated only moderate therapeutic effects, we hypothesized that other cell types with intrinsic roles in angiogenesis may exhibit a stronger therapeutic potential. We have previously established a protocol to source human peripheral blood-derived angiogenic cells (BDACs). These cells promoted revascularization and took perivascular location at sites of angiogenesis, thus resembling hematopoietic pericytes, which were only described in vivo so far. We thus hypothesized that BDACs might have a superior ability to promote revascularization and rescue the affected limb in CLI.MethodsAs standard BDAC sourcing techniques involve the use of animal-derived serum, we sought to establish a xeno- and/or serum-free protocol. Next, BDACs or MSCs were injected intramuscularly following the ligation of the iliac artery in a murine model. Their ability to enhance revascularization, impair necrosis and modulate inflammatory processes in the affected limb was investigated. Lastly, the secretomes of both cell types were compared to find potential indications for the observed differences in angiogenic potential.ResultsFrom the various commercial media tested, one xeno-free medium enabled the derivation of cells that resembled functional BDACs in comparable numbers. When applied to a murine model of CLI, both cell types enhanced limb reperfusion and reduced necrosis, with BDACs being twice as effective as MSCs. This was also reflected in histological evaluation, where BDAC-treated animals exhibited the least muscle tissue degeneration. The BDAC secretome was enriched in a larger number of proteins with pro-angiogenic and anti-inflammatory properties, suggesting that the combination of those factors may be responsible for the superior therapeutic effect.ConclusionsFunctional BDACs can be sourced under xeno-free conditions paving the way for their safe clinical application. Since BDACs are derived from an easily accessible and renewable tissue, can be sourced in clinically relevant numbers and time frame and exceeded traditional MSCs in their therapeutic potential, they may represent an advantageous cell type for the treatment of CLI and other ischemic diseases.

Improved biological performance of human cartilage-derived progenitors in platelet lysate xenofree media in comparison to fetal bovine serum media

Primary articular cartilage-derived cells are among the preferred contenders for cell-based therapy approaches for cartilage repair. Limited access to primary human cartilage tissue necessitates the process of in vitro cell expansion to obtain sufficient cells for therapeutic purposes. Therapeutic outcomes of such cell-based approaches become highly dependent on the quality of the in vitro culture-expanded cells. The objective of this study was to determine the differential biological effects of human platelet lysate (hPL) xeno-free defined media vs FBS containing traditional media on primary human cartilage-derived cells. Our goal in pursuing this work was to identify a preferred xenofree media alternative, that can be used as a platform for expansion of cells intended for clinical applications. Primary cartilage-derived cells obtained from five patients were simultaneously cultured in two expansion media's: (1) traditional (DMEM+10%FBS+1%P/S) and (2) defined xenofree (Nutristem® complete media+0.5%hPL). Connective tissue progenitors (CTPs) were assayed by standard colony forming unit assay, morphology, proliferation in early and late passages, expression of MSC associated cell-surface markers (CD73, CD90 and CD105) and trilineage differentiation (adipogenesis, osteogenesis and chondrogenesis) were considered for comparison of biological performance. Early biological performance of primary cartilage-derived cells was significantly improved in Nutristem® expansion media in comparison to traditional expansion media with respect to (1) Colony forming efficiency tended to be higher (p = 0.058) and (2) CTPs formed larger colonies with respect to total cells per colony and colony area (p < 0.01). In the culture expanded cell population, Nutristem® expansion media was superior to traditional expansion media with respect to: (1) overall proliferation rate through passages 1–4 (p = 0.027), (2) total cells harvested at end of passage 4 (p = 0.028) and (3) total positive stain area of CD73 (p = 0.006), CD90 (p = 0.001) and CD105 (p = 0.049). Nutristem®-hPL expanded cells when differentiated in respective xenofree serum-free defined MSCgo™ differentiated media's, also showed significant improvement in adipogenic, osteogenic and chondrogenic marker expression. Overall, we convincingly demonstrated that a low concentration of hPL in combination with defined xenofree media is an effective and economic growth supplement to culture expand primary cartilage-derived cells. It can be manufactured under cGMP conditions to improve clinical-grade cell products’ quality for therapeutic applications.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media

Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)—an undefined and variable mixture of biomolecules—in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.

Pulsed Electric Fields for Valorization of Platelets with No Therapeutic Value towards a High Biomedical Potential Product—A Proof of Concept

Nowadays, the standard media used in clinical-scale mesenchymal stem cell (MSC) production to supply hundreds of clinical trials uses animal-derived components as supplements, which raises several health concerns. Consequently, the development of xeno-free media supplements has emerged. In the current study, the effect of pulse electric field (PEF) application to platelet concentrates (PC) with no therapeutic value for producing platelet releasates (PR) able to sustain the ability of bone marrow-MSCs (BM-MSCs) to self-renew and differentiate was tested. It was demonstrated that PEF application to PC induces platelet activation and growth factor (GF) release, namely PDGF, FGF, IGF, and TGF-β. The highest GF release was observed for TGF-β, achieving similar levels to those attained in platelet lysates (PL). BM-MSCs expanded in the presence of PR obtained by the application of PEF (7 pulses of 10 and 12.5 kV/cm) to PC (PR PEF) retained the characteristic MSC cell-surface markers, and the ability to proliferate and differentiate into osteogenic, adipogenic, and chondrogenic lineages. In this study, evidence is provided that PR PEF represents a suitable alternative to fetal bovine serum (FBS) for use in MSC production.

Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells

BackgroundHuman-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.MethodsIn this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down’s syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2.ResultsWe found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers.ConclusionsOur xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.

Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells.

BackgroundCell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium.MethodsIn this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step.ResultsWe showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR−, CD34−, CD45− phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies.ConclusionsThe use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.

Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

Posology and Serum-/Xeno-Free Engineered Adipose Stromal Cells Cell Sheets

Well-characterized adipose stem cells and chemically defined culture media are important factors that control the production of the cell sheet, used in translational medicine. In this study, we have developed and engineered multilayer adipose stem cell cell sheets (ASCCSs) using chemically defined/serum-free culture media: undifferentiated or differentiated into osteoblasts and chondrocytes. In addition, using the cell sheet transmittance, we estimated the number of cells per cell sheet. Undifferentiated ASCCSs were engineered in 10 days, using serum-free/xeno-free culture media. They were CD29+, CD73+, CD90+, CD105+, HLA-A+, and HLA-DR-. ASCCSs differentiated into chondrocytes and osteoblasts were also engineered using chemically defined and animal-free culture media, in only 14 days. The addition of an ROCK inhibitor improved the chondrocyte cell sheet engineering. The decrease in the cell sheet transmittance rate was higher for the osteoblast cell sheets due to the intracellular Ca2+ accumulation. The estimation of cell number per cell sheet was carried out with the transmittance, which will provide important information for cell sheet posology. In conclusion, three types of ASCCSs were engineered using serum-free, xeno-free culture media, expressing their specific markers. Their transmittance measurement allowed estimating the number of cells per cell sheet, with a non-invasive methodology.

Influence of media and cellular origin on growth factors secreted by mesenchymal stem cells

Objective: Investigating influences of the culture media and cellular origin on the expression of several growth factors carried by exosomes secreted from umbilical cord-derived mesenchymal stem cells (UCMSC) and bone marrow-derived mesenchymal stem cells (BMMSC).&#x0D; Methods: 03 UCMSC samples was cultured in StemMACs and PowerStem, 03 BMMSC samples was cultured in StemMAC sand ACF Plus. At the passage 2, conditioned media were collected for exosome isolation. Exosomes were then investigated for the expression of growth factors, including: vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2(FGF-2), hepatocellular growth factor (HGF) and platelet-derived growth factor BB(PDGF-BB).&#x0D; Results: Both UCMSC and BMMSC at secreted exosomes carrying growth factors with different amount. UCMSC cultured in PowerStem could secret exosomes with the largest amount of growth factors. Besides, UCMSC and BMMSC cultured in StemMACs secreted exosomes carrying fewer amounts of growth factors. Secretion of growth factors through exosomes depends on the cell origin where both cell types were cultured in StemMACs, only UCMSC secretedFGF-2 and PDGF-BB but BMMSC did not.&#x0D; Conclusion: Both UCMSC and BMMSC cultured in commercial serum- and xeno-free media could secret exosomes into cell culture media. These exosomes carried growth factors, including VEGF-A, FGF-2, HGF and PDGF-BB, with different expression levels.

Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions

BackgroundAlthough umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified.MethodsMononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system.ResultsThe results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth.ConclusionsUCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Influence of media and cellular origin on growth factors secreted by mesenchymal stem cells

Objective: Investigating influences of the culture media and cellular origin on the expression of several growth factors carried by exosomes secreted from umbilical cord-derived mesenchymal stem cells (UCMSC) and bone marrow-derived mesenchymal stem cells (BMMSC).&#x0D; Methods: 03 UCMSC samples was cultured in StemMACs and PowerStem, 03 BMMSC samples was cultured in StemMAC sand ACF Plus. At the passage 2, conditioned media were collected for exosome isolation. Exosomes were then investigated for the expression of growth factors, including: vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2(FGF-2), hepatocellular growth factor (HGF) and platelet-derived growth factor BB(PDGF-BB).&#x0D; Results: Both UCMSC and BMMSC at secreted exosomes carrying growth factors with different amount. UCMSC cultured in PowerStem could secret exosomes with the largest amount of growth factors. Besides, UCMSC and BMMSC cultured in StemMACs secreted exosomes carrying fewer amounts of growth factors. Secretion of growth factors through exosomes depends on the cell origin where both cell types were cultured in StemMACs, only UCMSC secretedFGF-2 and PDGF-BB but BMMSC did not.&#x0D; Conclusion: Both UCMSC and BMMSC cultured in commercial serum- and xeno-free media could secret exosomes into cell culture media. These exosomes carried growth factors, including VEGF-A, FGF-2, HGF and PDGF-BB, with different expression levels.

A critical appraisal of humanized alternatives to fetal bovine serum for clinical applications of umbilical cord derived mesenchymal stromal cells.

The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB+ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations. The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed. Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signatureof unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44+, CD73+, CD105+, CD90+, and CD166+ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1+ (~ 21-10%) and TNAP+ (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05). Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD39)1 Sep 2021PD39-10 A XENO-FREE CULTURE METHOD FOR THE IN VITRO EXPANSION OF HUMAN SPERMATOGONIAL STEM CELLS Meghan Robinson, Ryan Flannigan, and Luke Witherspoon Meghan RobinsonMeghan Robinson More articles by this author , Ryan FlanniganRyan Flannigan More articles by this author , and Luke WitherspoonLuke Witherspoon More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002049.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract 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 of contaminating with pathogens, making them unsuitable for medical use. This study set out to develop xeno-free culture conditions for the expansion of human SSCs. METHODS: SSCs were derived from human induced pluripotent stem cells (hiPSCs) and tested in various xeno-free media conditions in combination with growth factors Prostaglandin D2 (PDG2) and Insulin-Like Growth Factor 1 (IGF1). Primary SSCs then underwent 3 passages in the best condition, were cryopreserved and thawed, and then analysed for changes in gene and protein expression by real time polymerase chain reaction (RT-qPCR) and immunocytochemistry. RESULTS: 10 ng/mL PDG2 with 10 ng/mL IGF1 was found to replace FBS and BSA without loss of viability or growth. ROCK inhibitor Y-27632 was determined to be necessary for viability upon thawing. Compared to established conditions, the SSCs shared identical protein expression profiles for the SSC markers Glial Cell-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRA1), G-Protein Coupled Receptor 125 (GPR125), Thy-1 Cell Surface Antigen (CD90), and Stage-Specific Embryonic Antigen 4 (SSEA4) (Figure 1A-B). RT-qPCR analyses revealed no significant variation in gene expression of undifferentiated germ cell markers and Inhibitor Of Differentiation 4 (ID4), and Fibroblast Growth Factor Receptor 3 (FGFR3). The greatest change seen was an increase in Deleted In Azoospermia Like (DAZL), a regulator of both SSC proliferation and differentiation (23-fold) (Figure 1C). CONCLUSIONS: This study identified a combination of growth factors capable of replacing FBS and BSA components in established SSC expansion cell culture. This xeno-free defined formulation allows standardized SSC culture and removes the risk of animal pathogens. Source of Funding: VCHRI, CUASF, CIHR, UBC © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e671-e672 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Meghan Robinson More articles by this author Ryan Flannigan More articles by this author Luke Witherspoon More articles by this author Expand All Advertisement Loading ...

Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor.

PurposeCorneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a well-known proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs).MethodsWe isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 μM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC).Results There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS.ConclusionHPL offers a xenofree and affordable medium supplement for CEC expansion that can be used in clinical applications.

Microcarrier Screening and Evaluation for Dynamic Expansion of Human Periosteum-Derived Progenitor Cells in a Xenogeneic Free Medium.

An increasing need toward a more efficient expansion of adherent progenitor cell types arises with the advancements of cell therapy. The use of a dynamic expansion instead of a static planar expansion could be one way to tackle the challenges of expanding adherent cells at a large scale. Microcarriers are often reported as a biomaterial for culturing cells in suspension. However, the type of microcarrier has an effect on the cell expansion. In order to find an efficient expansion process for a specific adherent progenitor cell type, it is important to investigate the effect of the type of microcarrier on the cell expansion. Human periosteum-derived progenitor cells are extensively used in skeletal tissue engineering for the regeneration of bone defects. Therefore, we evaluated the use of different microcarriers on human periosteum-derived progenitor cells. In order to assess the potency, identity and viability of these cells after being cultured in the spinner flasks, this study performed several in vitro and in vivo analyses. The novelty of this work lies in the combination of screening different microcarriers for human periosteum-derived progenitor cells with in vivo assessments of the cells’ potency using the microcarrier that was selected as the most promising one. The results showed that expanding human periosteum-derived progenitor cells in spinner flasks using xeno-free medium and Star-Plus microcarriers, does not affect the potency, identity or viability of the cells. The potency of the cells was assured with an in vivo evaluation, where bone formation was achieved. In summary, this expansion method has the potential to be used for large scale cell expansion with clinical relevance.

A novel serum- and xeno-free basal medium supplemented with human platelet lysate proves effective alternative for culturing and expanding human immune cells

A novel serum- and xeno-free basal medium supplemented with human platelet lysate proves effective alternative for culturing and expanding human immune cells

Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status.

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.