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Standardized xeno- and serum-free culture platform enables large-scale expansion of high-quality mesenchymal stem/stromal cells from perinatal and adult tissue sources
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamCollege of Health Science, VinUniversity, Hanoi, Vietnam
Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, VietnamVinmec HiTech Center, Vinmec Healthcare System, Hanoi, Vietnam
Mesenchymal stem/stromal cells (MSCs) are of interest for the treatment of graft-versus-host disease, autoimmune diseases, osteoarthritis and neurological and cardiovascular diseases. Increasing numbers of clinical trials emphasize the need for standardized manufacturing of these cells. However, many challenges related to diverse isolation and expansion protocols and differences in cell tissue sources exist. As a result, the cell products used in numerous trials vary greatly in characteristics and potency.
Methods
The authors have established a standardized culture platform using xeno- and serum-free commercial media for expansion of MSCs derived from umbilical cord (UC), bone marrow and adipose-derived (AD) and examined their functional characteristics.
Results
MSCs from the tested sources stably expanded in vitro and retained their biomarker expression and normal karyotype at early and later passages and after cryopreservation. MSCs were capable of colony formation and successfully differentiated into osteogenic, adipogenic and chondrogenic lineages. Pilot expansion of UC-MSCs and AD-MSCs to clinical scale revealed that the cells met the required quality standard for therapeutic applications.
Conclusions
The authors’ data suggest that xeno- and serum-free culture conditions are suitable for large-scale expansion and enable comparative study of MSCs of different origins. This is of importance for therapeutic purposes, especially because of the numerous variations in pre-clinical and clinical protocols for MSC-based products.
The unique bioactivities of mesenchymal stem/stromal cells (MSCs), such as immunomodulation, anti-inflammation, promotion of tissue regeneration and angiogenesis, make them valuable candidates for regenerative medicine applications. MSC products have been approved in different markets to treat children with refractory and/or acute graft-versus-host disease and Crohn-related enterocutaneous fistular disease. Potential clinical applications of MSCs are expanding quickly. According to https://clinicaltrials.gov (filtered as previously described [
]), as of 2019, a total of 921 clinical trials have been conducted for MSCs, making MSCs the second most popular cell source for therapy following hematopoietic stem cells. In addition to graft-versus-host disease and other immune-related disorder applications, MSC therapies have shown promising benefits in neurological and cardiovascular diseases, orthopedic complications and tissue repair/wound healing.
Bone marrow (BM), umbilical cord (UC) and adipose tissue (AD) are the most frequently used sources of MSCs, accounting for more than 90% of registered trials [
]. Other sources include placenta, dental pulp, oral mucosa, amniotic fluid and menstrual blood. MSCs isolated from different sources, including BM, AD, UC, placenta and tonsil tissue, exhibit distinguishable gene expression patterns [
Unique molecular signatures influencing the biological function and fate of post-natal stem cells isolated from different sources: gene expression of mesenchymal stem cells from various sources.
Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.
]. In line with the molecular signatures, MSCs from different origins show tissue-specific diversity. MSCs isolated from perinatal tissues proliferate faster than those from adult tissue [
]. Moreover, MSCs are distinguished in many biological processes, such as secretion of cytokines and growth factors, immunomodulatory and anti-inflammatory behavior and mitochondria transfer activity [
]. Thus, understanding MSC characteristics and their underlying mechanisms in relation to their origin is essential in the search for the most potent cell source for a specific disease target of interest.
Diversity of tissue origin is just one of the challenges in MSC research. Even when the cells come from a particular tissue, many other parameters (e.g., isolation protocol, culture medium, oxygen tension, two- or three-dimensional system, manual culture or bioreactor) lead to heterogeneity in the final product, which is still generally considered to be MSCs. Culture medium significantly impacts the transcriptome of BM-MSCs and the properties of several MSC types [
]. Numerous culture medium variants have been using in pre-clinical and clinical studies. MSCs can be expanded with or without serum and in a xeno-containing or xeno-free medium, which can be homemade or commercial [
]. Because of the numerous medium variants, each MSC product resulting from a specific manufacturing protocol is unique. This might explain why it is so difficult to verify the efficacy of MSCs for targeted diseases and to identify subgroups of beneficial patients despite the long-term effort and expanding trial numbers. Notably, these trials are mostly in early phase 1 and 2 and have limited participants. In this case, meta-analysis is of importance. However, the bias of published data limits the power of such studies. Thus, more standardized MSC culture conditions will help in obtaining more reliable data and achieving a better understanding of MSC biology.
This study addresses some of these issues. First, the authors optimized a common xeno- and serum-free culture platform for MSCs derived from UC, AD and BM, which are the most frequently used MSC sources in clinical trials. Next, the authors characterized the expanded MSCs in early and advanced passages as well as after cryopreservation. Finally, the authors validated the platform by mimicking clinical-scale expansion of MSC products for potential therapeutic use.
Methods
Patient samples
A cohort of BM (n = 16), AD (n = 14) and UC (n = 30) samples obtained from healthy donors were collected in Vinmec International Hospital in 2019 after patients signed in an informed consent form. The authors also included cryopreserved samples of BM (n = 6) and AD (n = 8) that were obtained between 2017 and 2018 in our hospital with the agreement of patients in the form of written informed consent. Sample collection and data analysis were approved by the ethics committee of Vinmec Healthcare System and were carried out in accordance with the Declaration of Helsinki.
Isolation and culture of MSCs
UC samples were obtained in 0.9% sodium chloride (Bidiphar, Quy Nhon, Vietnam) and stored at 4°C. MSCs were isolated using enzymatic digestion methods. Briefly, the cord was cut into small fragments and incubated in 500 U/mL collagenase (Gibco, USA) at 37°C for 2.5 h in a gentleMACS dissociator (Miltenyi, Germany). The cells were seeded in treated cell culture flasks (Nunc; Thermo Fisher Scientific, USA).
Approximately 5 g of AD samples were collected in 0.9% sodium chloride (Bidiphar, Quy Nhon, Vietnam). The tissue was cut into small pieces and digested in 200 U/mL collagenase (Gibco, USA) for 1 h at 37°C in an Adi Plus multi-purpose medical centrifuge-VS-6030 (Vision Scientific Co, Ltd, Korea). Isolated cells were collected by centrifugation and plated in treated cell culture flasks (Nunc; Thermo Fisher Scientific, USA).
BM aspirates (40 mL) mixed with heparin were collected. Mononuclear cells were isolated using density-gradient centrifugation with Ficoll-Paque PREMIUM solution (GE Healthcare Life Sciences, USA). Harvested cells were seeded in treated cell culture flasks (Nunc; Thermo Fisher Scientific, USA).
Primary cells were cultured in MSC culture medium supplemented with 50 U/mL penicillin/streptomycin (Life Technologies, USA) at 37°C with 5% carbon dioxide. After 7 days, antibiotics were removed from the culture. MSCs were harvested when the cells reached 80% confluency. For long-term storage, MSCs were cryopreserved at passage (P) 0 and 1 in the serum-free, xeno-free and defined reagent CryoStor CS10 (STEMCELL Technologies, Canada) in the gas phase of liquid nitrogen in an automated Brooks system (Brooks Life Sciences, USA). The temperature was monitored and maintained at –196°C.
The authors performed a medium test for UC-MSCs employing commercial xeno- and serum-free media; namely, NutriStem MSC XF medium (Biological Industries, Israel), StemPro MSC serum-free medium (Gibco, USA), StemMACS MSC expansion media kit XF (Miltenyi, Germany), PowerStem MSC1 medium (PAN-Biotech, Germany) and MesenCult-ACF plus medium (STEMCELL Technologies, Canada). For BM and AD, the last four MSC expansion media were tested. The primary cells were cultured in the media of interest directly after isolation to maintain the same in vitro conditions for the cells.
Examination of population doubling time
Growth curves were constructed for UC-, BM- and AD-MSCs from P2 to P6. For each passage, MSCs were seeded at a concentration of 5000 cells/cm2 in triplicate in treated cell culture flasks (Nunc; Thermo Fisher Scientific) coated with the supplied attachment substrate for the MesenCult-ACF plus medium (STEMCELL Technologies, Canada) or with CELLstart coating substrate (Thermo Fisher Scientific, USA) for the other media and grown until the culture reached 80% confluency. The cells were harvested using TrypLE Select CTS enzyme (Gibco, USA). Cells were counted using a Neubauer improved C-chip disposable hematocytometer (INCYTO, Germany). Dead cells were stained with Trypan Blue. Population doubling time was calculated as described in the supplementary material. Each culture was performed in triplicate.
Flow cytometry analysis
MSC identity was examined by evaluating the expression of surface markers, specifically the positive markers CD73, CD90 and CD105 and the negative markers CD34, CD45, CD11b, CD19 and HLA-DR, using a human MSC analysis kit (Becton, Dickinson and Company, USA) and Navios (Beckmann Coulter, USA) and BD FACSCanto II (Becton, Dickinson and Company, USA) flow cytometers. Data analysis was performed using the Navios system and FlowJo software. The flow cytometry analysis was performed with fresh MSCs at P3 and P6 and cryopreserved MSCs after thawing at P3.
Colony-forming unit assay
UC-, AD- and BM-MSCs were cultured until P2 and were plated in triplicate at concentrations of 4, 20 and 100 cells/cm2, respectively. Cells were cultured in MSC expansion medium as described earlier, with medium exchange twice per week. After 14 days, cells were fixed with methanol and stained with Giemsa (Merck, Germany). Colony numbers were counted, and the morphology of colonies was observed under a microscope.
Osteogenic, adipogenic and chondrogenic lineage differentiation assays
The differentiation ability of isolated MSC samples was tested using a StemPro osteogenesis, adipogenesis and chondrogenesis differentiation kit (Gibco, USA) according to the manufacturer's instructions. The cells were cultured in the differentiation media for 14 days and then fixed with 4% paraformaldehyde (Sigma-Aldrich, USA). Differentiation of MSCs into osteogenic, adipogenic and chondrogenic lineages was detected using Alizarin Red S, Oil Red O and Alcian Blue (Sigma-Aldrich, Singapore), respectively. For details, see supplementary material.
Immunomodulation assay
To analyze the immunomodulatory capacity of MSCs, the authors performed co-culture assays between peripheral blood mononuclear cells (PBMCs) and MSCs. PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, USA) and activated using phytohemagglutinin (PHA) (Life Technologies, USA). After 4 days of co-culture, the cells were labeled with anti-CD3, anti-CD4 and anti-CD8 antibodies (clones BW264/56, VIT4 and REA734, respectively; Miltenyi) and 7-aminoactinomycin D (Miltenyi, Germany) and then analyzed with a BD FACSCanto II flow cytometer (Becton, Dickinson and Company, USA). Non-activated PBMCs served as the negative control, and PHA-activated PBMCs without MSCs were the positive control. For details, see supplementary material.
Karyotyping
MSCs were cultured until P3 and P6 for karyotyping. To test genome stability after cryopreservation, MSCs were thawed and used for karyotype analysis at P3. Cells in metaphase were arrested using KaryoMAX Colcemid solution (Life Technologies, USA). Cells were incubated in 0.56% potassium chloride and fixed with Carnoy's fixative before being applied on slides. The samples were heated at 60°C overnight, treated with 0.05% trypsin (Gibco, USA) and then stained with Giemsa (Merck). Metaphases were analyzed using the metaphase system and Ikaros software (MetaSystems, Germany). For details, see supplementary material.
Bacterial and fungal culture tests
Cell supernatant was collected for bacterial and fungal culture tests. These were performed using the ISO 15189 certified microbiological laboratory of the diagnostic department at the Vinmec Times City International Hospital, Hanoi, Vietnam. Bacterial and fungal contamination was detected using the BacT/Alert three-dimensional microbial detection system (Biomerieux, USA). Mycoplasma was detected using a MycoAlert PLUS Mycoplasma detection kit (Lonza, Switzerland) and measured using a Lucetta luminometer (Lonza, Switzerland) following the manufacturer's instructions.
Measurement of endotoxin
Endotoxin measurement was performed using Food and Drug Administration-licensed Limulus amebocyte lysate (LAL) reagents and an Endosafe nexgen-PTS spectrophotometer (Charles River, USA) following the manufacturer's instructions. Briefly, the tested samples were diluted 10 times with LAL reagents, vortexed for 30–60 seconds and inactivated at 80°C for 5 min. After centrifugation at 3000 g/min for 3 min, 25 µL of sample was added into an Endosafe LAL cartridge and measured in the Endosafe nexgen-PTS spectrophotometer.
Statistical analysis
If not otherwise indicated, data were analyzed using a two-sided Student's t-test with GraphPad Prism 8 software. Welch's correction was applied when significantly different standard deviations were observed. Analysis of variance (ANOVA) was performed to compare the means of more than two groups as indicated in the text. Statistical significance was defined as P < 0.05.
Results
Identifying standard xeno- and serum-free culture conditions for expansion of UC-, AD- and BM-MSCs
To standardize the culture conditions of all three MSC sources of interest, five commercially available media—namely, MesenCult-ACF plus medium (STEMCELL Technologies), NutriStem MSC XF medium (Biological Industries), PowerStem MSC1 medium (PAN-Biotech), StemMACS MSC expansion media kit XF (Miltenyi) and StemPro MSC serum-free medium (Gibco)—were tested for culture of newly isolated UC-MSCs (n = 4, except n = 2 for the second medium). The cells were successfully expanded in StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech), whereas the other media were unable to support the growth of the tested cell types. Therefore, the authors employed these two media for isolation and culture of UC-MSCs (n = 30 and 9, respectively). Comparison of the two media revealed that UC-MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi) required a longer time to reach confluence after isolation than those cultured in PowerStem MSC1 medium (PAN-Biotech) (P = 0.03) (see supplementary Figure 1A). Moreover, the cell numbers harvested in the StemMACS MSC expansion media kit XF (Miltenyi) at P0 and P1 were lower than the numbers harvested in the PowerStem MSC1 medium (PAN-Biotech) (P = 0.004 and 0.006, respectively) (Figure 1A).
Figure 1Isolation of MSCs from UC, AD and BM. (A) The number of UC-MSCs isolated after culture in SMACS was lower than that after culture in PStem medium (n = 30 and 9, respectively). (B) Similarly, AD-MSCs showed an increased yield at P0 and P1 in PStem compared with that seen in SMACS (n = 10 and 6, respectively). (C) BM-MSC cell numbers harvested at P0 and P1 in SMACS, ACF, StemPro and PStem media demonstrate that SMACS and ACF were significantly more potent for isolation of MSCs than StemPro (n = 10, 16, 8 and 2, respectively). (D–F) Morphology of isolated MSCs from UC (D), AD (E) and BM (F) in tested media, showing a spindle-shaped morphology of MSCs. (G) A representative example of BM-MSCs analyzed for MSC markers via flow cytometry, showing high expression of positive markers CD73, CD90 and CD105 and low expression of negative markers CD34, CD45, CD11b, CD19 and HLA-DR. (H) Expression analysis of MSC markers in UC-, AD- and BM-MSCs indicates a normal MSC phenotype in most of the tested cultures, except in AD- and BM-MSCs cultured in PStem, in which negative markers were upregulated. BM-MSCs cultured in PStem showed altered expression of CD90 and CD105. Dotted lines represent 95% and 2%, which are the cutoff values recommended by ISCT for positive and negative markers, respectively, in MSCs
Similarly, both StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech) strongly supported the generation and growth of AD-MSCs (n = 6 and 19, respectively). No significant difference in recovery time was observed between the two tested media (P = 0.9) (see supplementary Figure 1B). However, there was an increase in cell numbers in the first two passages propagated in the PowerStem MSC1 medium (PAN-Biotech) compared with the StemMACS MSC expansion media kit XF (Miltenyi) (P0, P = 0.04, P1, P = 0.003) (Figure 1B).
In terms of BM-derived cells, purified mononuclear cells were propagated in StemMACS MSC expansion media kit XF (Miltenyi), MesenCult-ACF plus medium (STEMCELL Technologies), StemPro MSC serum-free medium (Gibco) and PowerStem MSC1 medium (PAN-Biotech) for the enrichment of MSCs (n = 10, 22, 8 and 2, respectively). The cells successfully proliferated in all of the tested media (see supplementary Figure 1C). Furthermore, the numbers of harvested cells at P1 favored StemMACS MSC expansion media kit XF (Miltenyi) and MesenCult-ACF plus medium (STEMCELL Technologies) more than StemPro MSC serum-free medium (Gibco) (P = 0.01 and < 0.0001, respectively) (Figure 1C).
Regardless of culture medium and tissue source, isolated MSCs derived from UC, AD and BM showed the characteristic fibroblastic, spindle-shaped morphology of MSCs (Figure 1D–F). Taken together, these results illustrate that MSCs from UC, AD and BM can be isolated and expanded under the same xeno- and serum-free conditions and that StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech) are potential medium candidates for a standardized culture platform.
Flow cytometric characterization of MSCs under the tested xeno- and serum-free conditions
To check MSC identity, the authors analyzed the surface marker expression of the isolated MSC samples using flow cytometry. Here the authors investigated the expression of the characteristic positive MSC markers CD73, CD90 and CD105 and the negative markers CD34, CD45, CD11b, CD19 and HLA-DR (Figure 1G). The analysis revealed that the expression profiles of MSCs were dependent on the culture medium and tissue source (Figure 1H; also see supplementary Table 1). Particularly, in UC-MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech), more than 95% of the cells were positive for CD73, CD90 and CD105, and less than 2% of the cells expressed the negative markers (n = 30 and 8, respectively). These expression levels met the recommendation of the International Society for Cell & Gene Therapy (ISCT) for MSC identity evaluation [
By contrast, although AD-MSCs cultured in PowerStem MSC1 medium (PAN-Biotech) expressed high levels of positive markers, their expression of the negative markers was exaggerated, with a mean ± standard deviation (SD) value of 8.67% ± 12.80% (n = 19). Of note, 11 of the 19 tested samples (57.89%) showed more than 2% negative marker-expressing cells. By contrast, AD-MSCs in the StemMACS MSC expansion media kit XF (Miltenyi) culture exhibited the normal range of MSC markers recommended by the ISCT (n = 6). In the case of BM-MSCs, the PowerStem MSC1 medium (PAN-Biotech) induced negative marker expression in these cells (45.8% ± 54.0%), along with lower levels of CD90 (81.5% ± 15.1%) and CD105 (93.7% ± 0.6%) (n = 2) (Figure 1H; also see supplementary Figure 1D). By contrast, the cells expanded in StemMACS MSC expansion media kit XF (Miltenyi), MesenCult-ACF plus medium (STEMCELL Technologies) and StemPro MSC serum-free medium (Gibco) displayed normal expression levels (n = 10, 22 and 8, respectively) (Figure 1H).
It is worth noting that biological variations might occur. In the authors’ cohort, a UC-MSC sample in StemMACS MSC expansion media kit XF (Miltenyi) culture expressed a lower level of CD105, with 91.4% of cells positive, and a BM-MSC sample showed a slight increase in expression of negative markers, with 2.38% of cells positive, whereas the other analyzed markers remained within normal expression ranges.
Thus, although MSCs derived from various origins can be enriched in both StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech), the flow cytometric analysis suggests that StemMACS MSC expansion media kit XF (Miltenyi) enabled the expansion of MSCs with consistent expression of surface markers.
Characterization of cell growth and colony formation ability of MSCs under the tested xeno- and serum-free conditions
To determine whether different culture media could potentially promote the proliferation of MSCs derived from various sources in the same manner, a growth curve analysis was performed for the cells from P2 to P6. As MSCs are recommended to be infused at early passages to avoid culture-induced genome instability, the analyzed passages were of interest for therapeutic applications. The results demonstrated that UC-MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi) proliferated faster than their counterparts cultured in PowerStem MSC1 medium (PAN-Biotech) (P = 0.006), with a mean ± SD population doubling time (PDT) of 21.73 h ± 3.43 h versus 24.31 h ± 1.77 h (n = 29 and 8, respectively) (Figure 2A). Furthermore, analysis of cell growth dynamics indicated that UC-MSCs cultured in PowerStem MSC1 medium (PAN-Biotech) showed reduced growth capacity at later passages, whereas growth remained more consistent under the StemMACS MSC expansion media kit XF (Miltenyi) conditions (one-way ANOVA, P = 0.006 and 0.02, respectively) (Figure 2B).
Figure 2Growth ability of MSCs derived from UC, AD and BM. (A) Growth curve analysis of UC-MSCs revealed a decreased PDT of the cells in SMACS compared with their counterparts in PStem (n = 29 and 8, respectively). (B) The cells cultured in SMACS retained their proliferation rate during the analyzed time from P2 to P6, whereas cells expanded in PStem showed a reduced growth trend after passaging. (C) The average PDT of AD-MSCs between P2 and P6 did not indicate a significant difference in the two media of interest. (D) AD-MSCs initially grew more slowly in SMACS. However, their growth was maintained over time, whereas the MSCs cultured in PStem showed gradually reduced potency after each passage (n = 7 and 19, respectively). (E) The PDT of BM-MSCs was similar in SMACS and ACF but significantly higher in StemPro (n = 9, 21 and 8, respectively). (F) Cell growth dynamics indicated more consistent growth of BM-MSCs in SMACS (n = 9) than in ACF (n = 21). (G) Comparison of MSCs from different origins revealed a lower PDT of UC-MSCs than that seen with adult tissue-derived MSCs under the same culture conditions in SMACS. (H) In accordance with the decline in growth, AD-MSCs at P6 in PStem showed a round shape, in contrast to the spindle shape of AD-MSCs cultured in SMACS. UC-MSCs at P6 retained an MSC-specific morphology in both SMACS and PStem media. However, the cells in PStem required more time to become confluent. Scale bar, 50 μm. *P < 0.05, **P < 0.01, ** 0.001 ≤ P < 0.01, ****P < 0.0001. ACF, MesenCult-ACF plus medium; No., number; PStem, PowerStem MSC1 medium; SMACS, StemMACS MSC expansion media kit XF; StemPro, StemPro MSC serum-free medium. (Color version of figure is available online).
The trend was more prominent in the case of AD-MSCs. Although the cells were highly proliferative at the early passages in PowerStem MSC1 medium (PAN-Biotech), proliferation declined gradually at each subsequent passage (one-way ANOVA, P < 0.0001, n = 19) (Figure 2D). By contrast, AD-MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi) behaved similar to UC-MSCs (one-way ANOVA, P = 0.038, n = 7) (Figure 2). Of note, there was no difference in the PDT of AD-MSCs expanded in these two media (P = 0.33): the mean PDT was 35.67 h ± 4.16 h in StemMACS MSC expansion media kit XF (Miltenyi) and 38.20 h ± 6.21 h in PowerStem MSC1 medium (PAN-Biotech) (n = 19 and 7, respectively) (Figure 2C).
Similarly, the PDT of the BM-MSCs in StemMACS MSC expansion media kit XF (Miltenyi) and MesenCult-ACF plus medium (STEMCELL Technologies) was comparable (mean ± SD, 38.44 h ± 10.83 h and 37.98 h ± 8.64 h, n = 9 and 21, respectively), whereas cells grown in StemPro MSC serum-free medium (Gibco) required significantly longer for population duplication (120.20 h ± 35.56 h, n = 8) (Figure 2E). Similar to the other MSC types, StemMACS MSC expansion media kit XF (Miltenyi) maintained MSC growth over the analyzed time (P = 0.14), whereas decreased cell division was observed for the cells cultured in MesenCult-ACF plus medium (STEMCELL Technologies) (P = 0.01) (Figure 2F). Of note, when comparing the PDT of MSCs isolated from the three tested sources, the authors noticed that UC-MSCs showed the highest proliferative properties, whereas adult-derived AD- and BM-MSCs divided significantly more slowly (P < 0.0001 in both cases) (Figure 2G).
Additionally, observation of MSCs at P6 revealed that PowerStem MSC1 medium (PAN-Biotech) induced a change in AD-MSCs, which became round in shape, whereas AD-MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi) displayed the normal spindle-shaped morphology observed in earlier passages (Figure 2H). UC-MSCs retained the characteristic MSC morphology under both conditions. However, the cells in PowerStem MSC1 medium (PAN-Biotech) required a longer time to reach confluence (Figure 2H).
Colony formation ability of MSCs under the tested xeno- and serum-free conditions
In addition to growth curve analysis, the authors examined the colony formation ability of MSCs at P2 (Figure 3A). The colony-forming unit (CFU) numbers of UC-MSCs were similar in the StemMACS MSC expansion media kit XF (Miltenyi) and PowerStem MSC1 medium (PAN-Biotech) (P = 0.32, n = 29 and 8, respectively) (see supplementary Figure 1E), whereas AD-MSCs showed a higher colony formation potential in StemMACS MSC expansion media kit XF (Miltenyi) than in PowerStem MSC1 medium (PAN-Biotech) (P = 0.0011, n = 7 and 12, respectively) (see supplementary Figure 1F). The CFU numbers of BM-MSCs tended to be higher in StemMACS MSC expansion media kit XF (Miltenyi) than in MesenCult-ACF plus medium (STEMCELL Technologies); however, the difference was not statistically significant (P = 0.13, n = 9 and 19, respectively) (see supplementary Figure 1G). The results also revealed that UC-MSCs had the highest CFU numbers (P < 0.0001), with 366 CFUs ± 154 CFUs (n = 29), followed by AD-MSCs (97 CFUs ± 18 CFUs, n = 7) and BM-MSCs, which had the lowest CFUs (16 CFUs ± 3 CFUs, n = 9) (Figure 3B).
Figure 3Colony formation and differentiation ability of MSCs from different origins cultured in SMACS. (A) Representative examples of colonies derived from UC-, AD- and BM-MSCs at P2. Cryopreserved UC-, AD- and BM-MSCs were seeded at a concentration of 4, 20 and 100 cells/cm2, respectively. (B) Under the same culture conditions, UC-MSCs were the most potent MSC type in terms of colony formation potency, followed by AD-MSCs, whereas BM-MSCs showed the lowest CFU numbers (n = 29, 7 and 9). (C–E) All tested MSC types derived from UC- (C), AD- (D) and BM-MSCs (E) were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. White scale bar, 200 μm. Black scale bar, 50 μm. SMACS, StemMACS MSC expansion media kit XF. (Color version of figure is available online).
Taken together, these data illustrate that although cells had longer recovery times and lower yields at the first two passages in StemMACS MSC expansion media kit XF (Miltenyi) than their counterparts in PowerStem MSC1 medium (PAN-Biotech), the cells expanded using the StemMACS MSC expansion media kit XF (Miltenyi) exhibited normal surface marker expression and high colony formation capacity and maintained their long-term proliferative bioactivity, findings observed in all three main sources: UC, AD and BM.
Differentiation capacity of MSCs under the tested xeno- and serum-free conditions
To investigate the effects of different media on the differentiation capacity of AD-, BM- and UC-MSCs, tri-lineage differentiation assays were performed, and the results demonstrated that all the analyzed MSC types were able to differentiate into osteogenic, adipogenic and chondrogenic lineages (Figure 3C–E, Table 1). In comparison to AD- and BM-MSCs, UC-MSCs exhibited lower adipogenesis potential (see supplementary Figure 2A). Furthermore, UC-MSCs required a longer time for osteogenic differentiation (see supplementary Figure 2B).
Table 1Differentiation of UC-, AD- and BM-MSCs into osteogenic, adipogenic and chondrogenic lineages.
Medium
SMACS
PStem
ACF
Results
Positive
Negative
Positive
Negative
Positive
Negative
UC-MSCs
n = 29 (%)
n = 8 (%)
Osteogenesis
28 (96.55)
1 (3.45)
7 (87.5)
1 (12.5)
Adipogenesis
29 (100)
0 (0)
8 (100)
0 (0)
Chondrogenesis
29 (100)
0 (0)
8 (100)
0 (0)
AD-MSCs
n = 4 (%)
n = 14 (%)
Osteogenesis
4 (100)
0 (0)
14 (100)
0 (0)
Adipogenesis
4 (100)
0 (0)
14 (100)
0 (0)
Chondrogenesis
4 (100)
0 (0)
14 (100)
0 (0)
BM-MSCs
n = 10 (%)
n = 20 (%)
Osteogenesis
10 (100)
0 (0)
20 (100)
0 (0)
Adipogenesis
10 (100)
0 (0)
20 (100)
0 (0)
Chondrogenesis
10 (100)
0 (0)
20 (100)
0 (0)
MSCs were cultured in SMACS, PStem and ACF media until P3, and expansion media were replaced with differentiation media for 14 days. The cells were fixed and stained with Alizarin S, Oil Red O and Alcian Blue to observe osteogenic, adipogenic and chondrogenic differentiation, respectively. The number of analyzed samples and positive/negative tested samples and their frequencies are indicated in the table.
ACF, MesenCult-ACF plus medium; PStem, PowerStem MSC1 medium; SMACS, StemMACS MSC expansion media kit XF.
Karyotyping was performed with fresh MSCs at P3 and P6 and with cryopreserved samples at P3 (n = 30, 6 and 10, respectively). Overall, the samples showed a normal karyotype. Of note, inv(9)(p11q13) was observed in one AD-MSC sample. The pericentric inversion on chromosome 9 is a common population inversion variant and is not counted as a mutation. For each sample, 20 metaphases were counted, if not mentioned otherwise. In some samples, less than 12 metaphases were detected, and thus no results were available. These samples and samples that were not analyzed are marked as NA.
Maintaining quality of MSCs from different origins after cryopreservation and prolonged in vitro cultivation
To assess the viability of the freshly harvested cells upon cryopreservation, MSC samples were subjected to a Trypan Blue assay (n = 30, 4 and 10 for UC-, AD- and BM-MSCs, respectively) (Figure 4A). Overall, the fresh cells at P1 showed high viability, with living cell frequencies (mean ± SD) of 98.45% ± 0.92%, 97.83% ± 3.21% and 98.8% ± 2.49% in UC-, AD- and BM-MSCs, respectively. The cryopreservation process might reduce the viability of cells; however, a reasonable survival rate was observed in all the analyzed samples (mean ± SD), with 90.65% ± 5.75% UC-MSCs, 96.22% ± 4.36% AD-MSCs and 90.33% ± 7.62% BM-MSCs. Additionally, prolonged in vitro expansion until P6 did not affect cell survival compared with the younger P1 counterparts (see supplementary Figure 3A).
Figure 4Maintaining MSC quality after cryopreservation. (A) The viability of both fresh and cryopreserved MSCs derived from UC, AD and BM was higher than 70% (dotted line), which is the minimal value required by the FDA for therapeutic cell products (n = 29, 4 and 10, respectively). (B) Cryopreserved MSCs showed high levels of positive markers CD73, CD90 and CD105 and low levels of negative markers CD34, CD45, CD11b, CD19 and HLA-DR (n = 29, 4 and 10, respectively). Dotted lines represent 95% and 2%, which are the cutoff values recommended by ISCT for positive and negative markers, respectively, in MSCs
. (C) The growth rate of cryopreserved MSCs from UC and AD was similar to that of fresh samples, whereas BM-MSCs showed compromised cell growth. (D,E) CFU numbers of UC-MSCs (D) and AD-MSCs (E) before (n = 26 and 4, respectively) and after cryopreservation (n = 29 and 7, respectively), suggesting a similar colony formation capacity of the cells. (F–H) Representative examples of the karyotype of UC- (F), AD- (G) and BM-MSC (H) samples. 0.01 ≤ *P < 0.05. Cryo, cryopreserved; FDA, Food and Drug Administration; Neg., negative; No., number. (Color version of figure is available online).
Furthermore, the authors observed a normal marker profile in cryopreserved MSCs and P6 cells, in which more than 95% of the cells expressed CD73, CD90 and CD105 and as low as 2% of the cells showed negative expression of the CD34, CD45, CD11b, CD19 and HLA-DR markers (Figure 4B; also see supplementary Figure 3B).
Next, the authors analyzed the influence of cryopreservation on cell growth and colony formation ability of MSCs. For this purpose, the authors divided fresh cells into two parts. The first part was further expanded to calculate growth ability and used for the CFU assay at P2. The other part was cryopreserved. After 1 month, cells were thawed and tested for cell growth and CFU capacity at the same passage. The cell growth ability of UC- and AD-MSCs was comparable between the fresh and cryopreserved/thawed samples, but thawed BM-MSCs grew more slowly than the fresh cells (Figure 4C). In addition, cryopreservation did not alter the CFU numbers of UC- and AD-MSCs (Figure 4D,E). CFU data of fresh BM were not available in this study. The authors also tested the influence of prolonged in vitro culture on colony formation, which showed that UC- and BM-MSCs maintained their potential at P6 (CFU of P2 compared with P6, P = 0.2 and P = 0.75, respectively) (see supplementary Figure 3C,E). AD-MSCs at P6 showed lower CFU numbers than their counterparts at P2 (P = 0.01) (see supplementary Figure 3D).
To evaluate the genetic stability of MSCs cultured in StemMACS MSC expansion media kit XF (Miltenyi), the authors performed karyotype analysis at P3 and P6 of both freshly cultured and cryopreserved samples (UC, AD and BM, n = 30, 6 and 10, respectively) Table 2. The results showed that all tested samples maintained the normal karyotypes (Figure 4F–H). In total, 117 karyograms of over 133 tested samples (88%) met the analysis standard, with no mutations or major alterations in chromosome number and structure (see supplementary Table 2). One AD-MSC sample showed pericentric inversion on chromosome 9—inv(9)(p11q13), which is a common population inversion variant—at both P3 and P6. In 16 of 133 samples (12%), the number of metaphases was too low for the analysis. Overall, the results indicated that the cultured MSCs showed a normal karyotype at P3 and P6 in fresh samples and after thawing of cryopreserved samples.
Expansion of MSCs from UC and AD under optimized conditions
To demonstrate that the authors’ xeno- and serum-free platform might support large-scale production of MSCs, UC- and AD-MSCs were expanded to clinically relevant numbers. For this purpose, one vial of two million cryopreserved UC-MSCs at P1 was thawed and manually cultured until P5 (n = 2) (Figure 5A). The results suggested that expansion of UC-MSCs as much as 5976 ± 966 times was feasible within 14 days. Similarly, fresh AD-MSCs were tested for in vitro expansion capacity from P0 to P3 (n = 3) (Figure 5B), and the cells were able to expand exponentially during the analyzed time.
Figure 5Expansion of MSCs derived from UC and AD. (A) UC-MSCs were expanded from P1 to P5 (n = 2). Cell numbers were counted at each passage, and the results demonstrated exponential growth of the cells. (B) AD-MSCs were isolated and expanded to P3 (n = 3). Similar to UC-MSCs, the number of AD-MSCs increased quickly within the culture period. (C) Schematic view of quality control tests that were performed for the expanded MSC products. (D) The identity of the cell products was confirmed by flow cytometry, in which high expression of CD73, CD90 and CD105 and absent expression of CD45, CD34, CD11b, CD19 and HLA-DR were observed. Dotted lines represent 95% and 2%, which are the cutoff values recommended by ISCT for positive and negative markers, respectively, in MSCs
. (E,F) Both UC- (E) and AD-MSCs (F) were capable of multi-lineage differentiation into osteogenic, adipogenic and chondrogenic lineages. Scale bars, 50 μm. (G) The immunomodulation ability of UC-MSCs was analyzed using a CFSE assay, and a representative example is depicted. (H) CD3+CD4+CD8− T-helper cells and CD3+CD4−CD8+ cytotoxic T cells were stimulated by PHA. UC-MSCs were able to significantly suppress the proliferation of both cell subsets (circles, squares and triangles represent PHA– UC-MSC–, PHA+ UC-MSC– and PHA+ UC-MSC+ cells, respectively) (n = 6). (I) Table depicts important parameters of cellular product: viability, sterility and purity. Karyotype was examined to assess the genetic stability of the in vitro cultured cells. Negative results for bacterial, fungal and Mycoplasma infection indicate that no micro-organisms were detected. *P < 0.05, ** 0.001 ≤ P < 0.01. CFSE, carboxyfluorescein succinimidyl ester; FITC, fluorescein isothiocyanate; NA, not applicable; Neg., negative. (Color version of figure is available online).
MSC products must undergo stringent quality control tests before application in humans, as depicted in Figure 5C. Here the authors analyzed cell identity using flow cytometry and assessed the potency of differentiation and the immunomodulatory ability of UC-MSCs. Furthermore, the authors recorded cell viability using Trypan Blue, sterility (bacteria, fungi, Mycoplasma), purity via endotoxin measurement and karyotype. All expanded MSC samples showed normal marker expression (Figure 5D). UC- and AD-MSCs were capable of multi-lineage differentiation: osteogenesis, adipogenesis and chondrogenesis (Figure 5E,F). A second tested potency assay was related to immunomodulation of UC-MSCs. For this assay, PBMCs (n = 6) were activated with PHA and cultured with UC-MSCs. After 4 days, the authors analyzed frequencies of proliferating fractions, which included cells with low levels of carboxyfluorescein succinimidyl ester among the viable CD3+CD4+ T-helper cells and CD3+CD8+ cytotoxic T cells (Figure 5G). Indeed, UC-MSCs significantly inhibited proliferation of both CD3+CD4+CD8− T-helper cells and CD3+CD4−CD8+ cytotoxic T cells (Figure 5H). Similarly, AD-MSCs tended to reverse the activation of these T-cell subsets by PHA (see supplementary Figure 3F). Finally, the harvested cells showed high viability and no contamination with bacteria, fungi or Mycoplasma, and endotoxin levels were under the detection limit, which was 0.05 EU/mL. Karyotype analysis confirmed a normal karyotype (Figure 5I). Overall, the authors’ optimized culture platform enabled a clinically relevant scale expansion of MSCs that met all critical quality control requirements of therapeutic cellular products.
Discussion
Because of the regenerative and immunoregulatory features of MSCs derived from either perinatal or adult tissues, MSCs are a valuable resource for clinical trials and regenerative medicine [
]. The majority of published culture conditions for MSCs in both research and clinical settings utilize fetal bovine serum (FBS) or human platelet-derived supplement as the main source of growth factors to support MSC proliferation and stemness [
]. However, their limitations, which include batch-to-batch variation, non-defined components and potential side effects due to contamination with micro-organisms and endotoxin, have been recently addressed [
Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum.
Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells.
]. Here, because of the current need for more standardized and safer MSC therapeutic products, the authors established a xeno- and serum-free culture protocol for MSCs derived from UC, AD and BM. Xeno- and serum-free culture conditions have been investigated in previous studies [
Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton's jelly derived mesenchymal stem cells? A comparative study.
]. Most of these studies compared MSC properties under xeno- and serum-free conditions with those of their counterparts cultured in the presence of FBS [
Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton's jelly derived mesenchymal stem cells? A comparative study.
Human mesenchymal stem cells possess different biological characteristics but do not change their therapeutic potential when cultured in serum free medium.
], and the results support potential replacement of xenogenic and serum-derived components in the MSC culture, especially for clinical applications. A recent study also tested a newly developed xeno- and serum-free medium for UC-, AD- and BM-MSCs, which were obtained from third-party suppliers [
Massive Clonal Selection and Transiently Contributing Clones During Expansion of Mesenchymal Stem Cell Cultures Revealed by Lentiviral RGB-Barcode Technology: Clonal Selection During Expansion of MSC Cultures.
]. This suggests the fundamental importance of standardized conditions for the initial culture to reduce bias and heterogeneity in subsequent observations. To the best of the authors’ knowledge, we are the first to intensively evaluate commercial xeno- and serum-free culture reagents and report a common platform for isolation and propagation of MSCs from all three of the mentioned tissue sources.
Comprehensive analysis of both fresh cultured cells and preserved cells grown in StemMACS MSC expansion media kit XF (Miltenyi), in combination with CELLstart coating substrate (Thermo Fisher Scientific) and TrypLE (Gibco), demonstrated a number of findings: (i) all tested AD-, BM- and UC-MSCs cultured under the authors’ conditions exhibited basic MSC characteristics as defined by the ISCT [
]; (ii) cells maintained karyotype normality after six consecutive passages; (iii) UC-MSCs had the highest proliferation rate compared with their AD and BM counterparts, and similar results were observed in the CFU assay; (iv) cryopreservation of these MSCs under xeno- and serum-free conditions did not alter cell quality or characteristics; and (v) the authors’ platform competently supported clinical-scale production and quality control requirements according to the current guidelines [
Office of Medical Products and Tobacco Center for Devices and Radiological Health, Office of Medical Products and Tobacco, Center for Biologics Evaluation and Research.
Regulatory Considerations for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use Guidance for Industry and Food and Drug Administration Staff.2017;
Another challenge—but also a great opportunity—in the MSC research field is related to the large number of potential tissue donors. Although MSCs derived from adult sources, such as BM and AD, are commonly used [
], the invasiveness of the procedures required for obtaining these tissues, the risk of complications and the age-dependent reduction in cell quality have become potential obstacles in MSC application [
]. Recently, UC-MSCs—a perinatal MSC source—have emerged as an alternative cell source, allowing the development of allogeneic transplantation for various diseases because of several advantages, such as ease of obtainment, non-invasive retrieval method and absence of ethical barriers. Although differently originated MSCs show some similarities, such as morphology, marker identity and multi-lineage differentiation capacity, they largely differ in many other bioactivities (e.g., growth ability, differentiation into certain lineages, anti-inflammatory and immunomodulatory capacity and genetic and epigenetic signature) [
]. In this study, the authors have shown that UC-MSCs exhibit more potent in vitro expansion and CFU formation than their adult counterparts. Although MSCs from all analyzed sources were capable of differentiation, UC-MSCs showed compromised adipogenic and osteogenic differentiation compared with AD- and BM-MSCs. These results confirm previous findings showing that MSC characteristics depend on tissue origin. Further comparative study of MSCs from different tissues is of interest to identify the best and most potent MSC source for each disease entity. The authors’ standardized platform is critical for this approach, helping MSC applications be more precise and targeted.
Since 2018, allogeneic AD-MSCs have been authorized in the European Union for treatment of complex perianal fistulas in Crohn disease [
]. As allogeneic MSCs are increasingly used, Good Manufacturing Practice (GMP)-compliant, large-scale production has been of interest. By using a commercial medium, the authors might reduce bias between MSC manufacturing centers. The StemMACS MSC expansion media kit XF (Miltenyi) is not a GMP product. However, the related GMP product, MSC-Brew GMP medium (Miltenyi), is based on the formulation of the StemMACS MSC expansion media kit XF (Miltenyi), and thus translation of this product into GMP manufacturing for clinical applications would be feasible [
In sum, the authors have developed a standardized platform for xeno- and serum-free culture of MSCs from different origins. The authors demonstrated that the quality of expanded MSCs was conserved after in vitro culture and cryopreservation. Moreover, the standardized culture platform enables further comparative studies to understand the biology of MSCs depending on their origin. These studies would be beneficial for application of MSCs in a clinical setting and for investigation of the underlying mechanisms.
Funding
This work was funded by a Vingroup research grant (project nos. PRO.19.48 and PRO.19.49).
Declaration of Competing Interest
The authors of the study are employed by the not-for-profit Vinmec Healthcare System.
Author Contributions
Conception and design of the study: DMH, LTN, VTH, DTMP and QMT. Acquisition of data: DMH, VTH, DTMP, QMT, HTHB, LMH, NTHN, NTTA, PYN, TTHN, HTL, TDN and LNT. Analysis and interpretation of data: VTH, DMH, DTMP and QMT. Drafting or revising the manuscript: VTH, DMH, DTMP, QMT, LTN, HTHB, LMH, NTHN, NTTA, PYN, TTHN, HTL and TDN. All authors have approved the final article.
Acknowledgments
The authors thank our collaborating clinicians at the Vinmec Healthcare System for collecting samples for this study and the microbiology unit of the laboratory department at the Vinmec Times City International Hospital for performing micro-organism tests. The authors thank Bui Viet Anh, MSc, and Associate Professor Tran Thi Thanh Huong, MD, and their colleagues at the HiTech Center, Vinmec Healthcare System, for their support with the quality control analysis. The authors appreciate the scientific input and support of the Vinmec Scientific Committee and the Vinmec Ethics Committee. Finally, the authors’ special gratitude goes to all volunteers who donated primary materials for the research. The manuscript was edited by AJE under certificate number 4037-A8A0-CBD9-A7F1-E375.
Unique molecular signatures influencing the biological function and fate of post-natal stem cells isolated from different sources: gene expression of mesenchymal stem cells from various sources.
Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.
Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum.
Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells.
Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton's jelly derived mesenchymal stem cells? A comparative study.
Human mesenchymal stem cells possess different biological characteristics but do not change their therapeutic potential when cultured in serum free medium.
Massive Clonal Selection and Transiently Contributing Clones During Expansion of Mesenchymal Stem Cell Cultures Revealed by Lentiviral RGB-Barcode Technology: Clonal Selection During Expansion of MSC Cultures.
Center for Devices and Radiological Health, Office of Medical Products and Tobacco, Center for Biologics Evaluation and Research.
Regulatory Considerations for Human Cells, Tissues, and Cellular and Tissue-Based Products: Minimal Manipulation and Homologous Use Guidance for Industry and Food and Drug Administration Staff.2017;
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