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Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors

Abstract The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead‐to‐bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cell‐populated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead‐to‐bead cell transfer method for cell culture scale‐up from 4‐ to 50‐L cultures in single‐use bioreactors. We achieved a 242‐fold increase in the number of cells to 1.45 × 1010 after 27‐day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri‐lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead‐to‐bead transfer method for larger‐scale manufacturing of hMSCs for cell therapy.

culture. Subsequently the authors developed a bead-to-bead cell transfer process with intermittent agitation for cell culture scale-up in bioreactors, which maintained the proliferation, viability, and normal phenotype of hMSCs. The bead-to-bead cell transfer has great potential for large-scale manufacturing of hMSCs for cell therapy.

| INTRODUCTION
An increasing number of clinical studies are showing that human mesenchymal stem cells (hMSCs) have great potential for treating various diseases such as impaired tissue functions and immunological disorders. hMSCs can be differentiated into several types of cells such as osteoblasts, adipocytes, chondrocytes, and neurons. Their secretome includes pro-angiogenic factors and growth factors, such as vascular endothelial growth factor, fibroblast growth factor 2, and insulin-like growth factor, which can promote tissue vascularization and regeneration. [1][2][3] They also secrete extracellular vesicles that encapsulate microRNAs and cytokines, which have been shown to modulate inflammation and promote the repair of damaged tissues. [4][5][6] hMSCs are usually isolated from human tissues such as bone marrow, adipose tissue, or the umbilical cord and expanded in culture medium for clinical applications. However, in vitro aging of hMSCs happen and cells show limited proliferation, lose their differentiation capacity and immunosuppressive function after long-term expansion. 7,8 An ideal culture medium should maintain the characteristics of hMSCs, such as normal surface marker expression, and support their self-renewal after long-term culture. Although considered as nonessential supplements in a culture medium, nucleosides have been shown to influence the proliferation and differentiation of many mammalian cells including adipose-tissue-derived stem cells. [9][10][11] Whether nucleosides supplementation can affect the prolonged expansion and properties of hMSCs remains to be investigated.
Clinical applications of allogeneic hMSCs, which are derived from donors other than patients, require excessively large numbers of cells to treat hundreds or thousands of patients. In such cases, large-scale culture of hMSCs is necessary to obtain enough cells. 12 hMSCs for clinical studies are often cultured in multilayered culture flasks such as cell stack chambers. Although such planar culture platforms provide a much greater surface area for cell growth than single-layered tissue culture flasks, they have limited surface area, require intensive labor, and make process control, monitoring, and automation difficult. 13,14 Hollow fiber and fixed bed bioreactors can be used for high-density cell culture but have limited scalability for cell expansion and cause problems such as inhomogeneous nutrient supply and cell distribution. 12,[15][16][17][18] Stirred tank bioreactor-based cell expansion, where cells are cultured under a mixed and controlled culture environment, exhibits good scalability and has the potential for culturing a large number of cells with controlled quality attributes. 14,19,20 In stirred tank bioreactor-based cell expansion, hMSCs are often inoculated on microcarriers, which are suspended in the bioreactor and provide the surface for cell adhesion and proliferation. Some studies reported the expansion of hMSCs with microcarriers in bioreactors, and the harvested cells from bioreactors maintained normal surface marker expression and the potential of differentiation into adipocytes, osteoblasts, and chondrocytes. [21][22][23][24][25] An efficient method for the subculture of hMSCs in bioreactors is necessary for their large-scale manufacturing. 26 A common method involves removal of spent medium, microcarrier washing, proteolytic enzyme treatment for cell detachment from microcarriers, and separation of cells from microcarriers, followed by centrifugation, washing, and reinoculation of the harvested cells on fresh microcarriers. The whole process is laborious, susceptible to contamination, and potentially compromises cellular integrity by enzymatic digestion of cell membrane proteins and centrifugal forces. 27,28 Another method involves bead-to-bead cell transfer, which relies on the spontaneous migration of hMSCs from cell-populated microcarriers to fresh microcarriers that provide greater surface area for cell growth. Compared with the other methods, a bead-to-bead cell-transfer method can obviate the laborious process, risks of contamination, and cellular damage from proteolytic enzymes and physical stress during each subculture. However, after the supplementation of fresh microcarriers to cell-populated microcarriers, cell migration can be inefficient, resulting in a mixture of bare microcarriers and densely populated microcarriers or large microcarrier aggregates, which limited cell proliferation due to the shortage of the growth surface. 26,29,30 Large microcarrier aggregates can impede cell growth or cause cell death during expansion due to limited nutrient supply and encumber the enzymatic dissociation of hMSCs from microcarriers during cell harvest. 31,32 We developed an efficient bead-to-bead cell-transfer method for the large-scale expansion of hMSCs with microcarriers in bioreactors for cell therapy. We first studied whether a medium with or without nucleosides can affect the expansion of hMSCs. Subsequently, we used different agitation profiles during bead-to-bead cell transfer to develop the subculture process in bioreactors. Finally, we used the medium containing nucleosides and bead-to-bead cell transfer for hMSC culture scale-up from 4-to 50-L in single-use stirred-tank bioreactors. The cell expansion and metabolite concentrations in bioreactors were monitored and the cells after the expansion were characterized.

| Expansion of hMSCs on planar surface
Bone-marrow derived hMSCs at passage 2 were purchased from Lonza, Inc. Alpha modification of minimum essential media with nucleosides (MEM alpha, nucleosides) (Gibco) was supplemented with 10% fetal bovine serum (FBS) (Biological Industries) and used as culture medium.
hMSCs were plated on tissue culture flasks at a density of 3000 cells/cm 2 and culture in a 37 C incubator with 5% carbon dioxide for 6-7 days. Cells were dissociated from the culture vessels with TrypLE Select (Thermo Fisher Scientific). hMSCs at passage 3 were plated on 10-layer Nunc EasyFill Cell Factory (Thermo Fisher Scientific) vessels at the same plating density and cultured for 6-7 days, followed by enzyme treatment. The harvested cells (passage 4) were cryopreserved in liquid nitrogen for inoculation in spinner flasks or bioreactors.
To compare the effect of nucleosides in medium on the expansion of hMSCs, cells from two donors were cultured in tissue culture flasks (175 cm 2 ) from passage 3 to passage 6 in alpha modification of minimum essential media with (MEM alpha, nucleosides, Thermo Fisher Scientific) and without nucleosides (MEM alpha, no nucleosides, Thermo Fisher Scientific) supplemented with 10% FBS. hMSCs were also expanded in these two media in spinner flasks, as discussed in Section 2.2. The nucleosides included adenosine, cytidine, guanosine, uridine, 2 0 deoxyadenosine, 2 0 deoxycytidine HCl, 2 0 deoxyguanosine, and thymidine, all of which had a concentration of 10 mg/L in the medium. Cells were plated in tissue culture flasks at a density of 3000 cells/cm 2 and cultured for 7 days during each passage and harvested for cell count and analysis of surface marker expression.

| Subculture of cells in spinner flasks
In a biosafety cabinet, the hMSCs cultured on microcarriers in the spinner flasks were swirled several times and 20 mL was transferred into new spinner flasks. Fresh microcarriers (400 cm 2 ) in the culture medium (80 mL) were added into the new spinner flasks. The new spinner flasks were placed in the incubator and agitated intermittently for 24 or 6 hours followed by continuously agitation or agitated continuously right after microcarrier addition, as shown in Table S1 and  Afterwards, 2.27 g Cytodex 1 microcarriers and 3 Â 10 7 hMSCs at passage 4 were inoculated into each bioreactor and intermittently agitated with 6 cycles of 5-minute agitation and a 25-minutes static period followed by overnight static phase without agitation. One liter of culture medium was added into each bioreactor and the working volume of each bioreactor increased to 2 L. From day 1 to day 8, the agitation rate was increased from 70 to 85 rpm to keep the microcarriers from settling. Fifty percent medium exchange was carried out on day 8.

| Scale-up to 20-and 50-L cultures
A Flexsafe STR 50-L culture bag (Sartorius Stedim Biotech) was set into a bag holder skid and 15 L of culture medium was transferred into the bag. The medium's temperature (37 C), pH (7.4), and DO (50%) were controlled using a BIOSTAT STR control tower for overnight.
One-liter suspension of fresh microcarriers (18.2 g) in a culture medium and 4 L of culture on day 9 were transferred into the culture bag, and the culture volume became 20 L. Intermittent agitation was carried out for 24 hours (12 cycles of 5 minutes of agitation and 2 hours of static culture) to promote bead-to-bead cell transfer followed by continuous agitation of the culture for 4 days.
Subsequently, a 1-L suspension of fresh microcarriers (34.1 g) in the culture medium and 29 L of warmed fresh medium were added to the culture bag on day 13. The culture volume became 50 L, and intermittent agitation was carried out for 24 hours, similar to the 20-L culture.
The 50-L culture was then agitated continuously for 14 days, during which 50% medium exchange was carried out on day 20. The agitation speed of the 50-L bioreactor was 50 rpm, which was converted into volumetric power inputs (W/m 3 ) according to a previous study. 33 Triplicate samples (3-5 mL) were taken from the bioreactor daily for cell count and live/dead double staining or stored in a À80 C freezer for metabolites analysis.

| Characterization of expanded cells
To confirm their growth potential, expanded cells were thawed,

| Metabolite analysis
Frozen medium samples from bioreactors were thawed to room temperature and centrifuged at 500g for 5 minutes, and the supernatant was used for metabolite analysis. All the medium samples were ana-

| Statistical analysis
Cell expansion experiments in monolayer culture and spinner flasks were run in triplicate. Results were presented as mean of three samples or mean ± SD. One-way or two-way analysis of variance (ANOVA) followed with Tukey post hoc tests was used to analyze the difference between the means of more than two groups. P values less than .05 were considered statistically significant. (85.2% vs 99.1%) and higher expression of hMSCs' negative surface markers (4.80% vs 0.06%) compared with those cultured with nucleosides ( Figure 1B). The suspension culture of hMSCs with Cytodex 1 microcarriers in spinner flasks also showed a higher cell proliferation rate in the medium with nucleosides than those in the medium without nucleosides ( Figure 1C). These results suggest that supplementation of nucleosides in medium can promote proliferation and maintain the immunophenotype of hMSCs during their prolonged culture. 3.2 | Effect of agitation mode on bead-to-bead cell transfer Next, we used different agitation profiles during bead-to-bead cell transfer to develop the expansion process in bioreactors. We hypothesized that if cell-populated microcarriers are in contact with fresh microcarriers for a sufficient amount of time, cell migration between them can be promoted. Therefore, we checked whether intermittent agitation or continuous agitation affects cell transfer, as described in Materials and Methods and Table S1. Fresh microcarriers were added to cell-populated microcarriers that had been cultured in spinner flasks, as shown in Figure 2A.

| Cell expansion and subculture in bioreactors
As intermittent agitation after microcarrier addition could promote bead-to-bead cell transfer, it was adopted for the expansion of cell culture in bioreactors, as shown in Figure 4A. The agitation rates of bioreactors were converted into volumetric power inputs and shown in supplemental Table S2. After hMSCs were expanded with microcarriers in the 4-L culture for 9 days, the cell-populated microcarriers along with the medium in the culture were transferred into a stirred tank bioreactor with a maximum working volume of 50 L, followed by adding fresh Fresh microcarriers were added on days 9 and 13 followed by 24-hour intermittent agitation to facilitate bead-tobead cell transfer showed faster cell proliferation than in our study, possibly because cells at lower passage (passage 2), collagen-coated microcarriers, and human platelet lysate were used for cell expansion. In our study, we carried out serial subculture of passage 4 hMSCs from 4-to 50-L in bioreactors with bead-to-bead cell transfer and obtained much greater increase during cell expansion. Since our serial subculture in spinner flasks achieved more than a 10 000-fold increase in cell number, we believe that we can further increase the scale of bioreactor culture with our bead-to-bead cell-transfer method. We also used higher microcarrier densities such as 6.8 g of Cytodex 1 per liter for cell expansion and found that the migration of cells to microcarriers occurred at higher microcarrier densities. These results suggest that our bead-to-bead cell-transfer method can be used for high-density cell expansion in stirred bioreactors.

| Metabolite concentration during cell expansion
The concentrations of glutamine, glutamate, glucose, and lactate of the culture medium in bioreactors were analyzed using the BioProfile Medium samples taken from 20-and 50-L cultures (day [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] were also analyzed using the Cell Culture Media Analysis Platform to quantify 95 components in the medium including sugars, nucleic acidassociated compounds, amino acids, vitamins, and other metabolic intermediates. The changes in glucose, glutamine, and lactate concentrations detected with this platform correlated very well with those measured from the former analyzer ( Figure 6B). Interestingly, we found that the decrease in the concentrations of cytidine, thymidine, deoxycytidine, and adenosine showed a similar trend with those of glucose and glutamine, and depletions of cytidine and deoxycytidine within 4 days after adding fresh medium or medium exchange, suggested the consumption of nucleosides by cells ( Figure 6B and Figure S2). The specific rates of nucleoside consumptions for these four nucleosides in the 50-L bioreactor were similar, as shown in Table S3. Although other four nucleosides were contained in the culture medium, 2 0 deoxyadenosine and 2 0 deoxyguanosine were not quantified because parameters for their detection were not provided by the analyzer manufacturer. Uridine and guanosine were not detected in all samples from the whole culture period, possibly because their concentrations in medium was below the detection limit. However, it is possible that these undetected nucleosides were also used by cells during their expansion in bioreactors. The consumption of the nucleosides by hMSCs, along with the previous data that showed superior cell proliferation rates in medium with nucleosides, further suggests that nucleosides should be beneficial for the expansion of hMSCs.

| Cell harvest and characterization
We harvested hMSCs after their expansion in 50-L culture in bioreactors to check if they maintained their characteristics. Figure 7A shows images of microcarrier samples taken before and after cell harvest.
Before harvest, cells reached confluence on most of the microcarriers.
After enzyme treatment and separation of cells from the microcarriers, few cells could be observed on the surface of the microcarriers. When the harvested cells were plated and cultured on tissue culture flasks, they attached and proliferated during the 8-day culture ( Figure 7B). Flow cytometry analysis showed that more than 98.1% of the expanded cells had the expression of CD90, CD73 and CD105, and less than 0.2% had the expression of CD34, CD11b, CD19, CD45 or HLA-DR ( Figure 7C). After cell expansion, 95.7% and 94.0% of the cells expressed CD44 and CD146, respectively ( Figure S3). The hyaluronan receptor CD44 can facilitate the migration of MSCs to wound sites for tissue regeneration. 44 Expression of CD146 on hMSCs correlated with higher multipotency, immunomodulatory and secretory potency. 45,46 After 2 weeks of adipogenic and osteogenic differentiation of the  47 On the other hand, studies showed that nucleotide deficiency in cells could promote genomic instability F I G U R E 6 Concentration of metabolites measured using two medium analyzers. A, BioProfile FLEX Automated Cell Culture Analyzer, B, Cell Culture Media Analysis Platform. Days 0-9:4-L culture; days 9-13:20-L culture; days 13-27:50-L culture. Black arrows mark addition of fresh microcarrier suspension (microcarrier concentration = 5000 cm 2 /L). Gray arrows mark 50% medium exchange. Data points represent average metabolite concentrations measured from three medium samples during DNA replication while exogenously supplied nucleosides increased the nucleotide pool and rescued DNA damage. 48 A recent study showed that nucleosides supplementation in reprogramming medium can reduce the DNA damage and genomic rearrangements on induced pluripotent stem cells. 49 Therefore, it is possible that the nucleosides in our culture medium reduced the genetic aberrations in hMSCs during their expansion. Future work will be done to confirm the genomic stability of hMSCs after their expansion with exogenous nucleosides and the enzymes that involve with nucleoside metabolism.

| CONCLUSION
We investigated the effect of nucleosides in medium on the proliferation of hMSCs and the effect of different agitation modes on bead-tobead cell transfer in suspension culture. We also developed the serial subculture process toward 50-L suspension culture of hMSCs in bioreactors. Planar and suspension cultures showed that the culture medium with nucleosides promoted cell proliferation and maintained the immunophenotype of hMSCs compared with those without nucleosides. Our bead-to-bead cell transfer method, which involved adding

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
S.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; Y. Sato, Y. Suzuki: collection and/or assembly of data; Y.T., R.T., M.O., K.N.: data analysis and interpretation, administrative support.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.