Good Manufacturing Practice-compliant Isolation and Culture of Human Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into different kinds of mesenchymal cells such as osteoblasts, chondroblasts, and adipocytes. These cells were identified a long time ago and are considered the first discovered source of MSCs. BM-MSC transplantation is used to treat bone disease, myocardial infarction, stroke, and diabetes mellitus. This study aimed to provide a new method for the in vitro primary culture and secondary culture of BM-MSCs that is compliant with good manufacturing practices, for use in clinical applications. Bone marrow was aspirated from the hipbone by using special needles and syringes. Mononuclear cells (MNCs) were isolated from the marrow by using Ficoll-Hypaque gradient centrifugation. These MNCs were cultured in DMEM/F12 medium supplemented with 2.5%, 5%, and 10% activated platelet rich plasma (aPRP) in the experimental groups and 10% FBS in the control group to obtain BM-MSCs. The results showed that aPRP could be replaced with FBS for isolation and proliferation of BM-MSCs. BM-MSCs cultured in both DMEM/F12 medium supplemented with aPRP and DMEM/F12 medium supplemented with FBS exhibited the characteristic phenotype of MSCs, such as being positive for CD44, CD73, CD90, and CD105, and being negative for CD14, CD34, CD45, and HLA-DR. They also successfully differentiated into adipocytes and osteoblasts. More importantly, BM-MSCs strongly proliferated in medium supplemented with aPRP, maintained the normal karyotype, and non-tumorigenesis in the athymic mice. Thus, this study provides an advanced protocol that is beneficial for the clinical applications of BM-MSCs.

To increase the number of BM-MSCs, BM-MSCs are subjected to long-term culture in vitro.However, in most studies, MSCs were cultured in media containing xenogeneic additives such as fetal bovine serum (FBS) (Choudhery et al., 2013;Huang et al., 2014;Odabas et al., 2014).These media are associated with risks such as prion-mediated infections, viral transmission, and adverse immunological reactions.However, serum-free media are commercially available for use in BM-MSC culture.The main limitations of these media are their high cost and complexity.In fact, when grown in serum-free media, BM-MSCs hardly adhere to culture-flask surfaces; hence, all culture flasks must be pre-coated with adherent matrixes.
In this study, we established an animal productfree expansion protocol by using autologous activated platelet rich plasma.During the procedure, all components of animal origin, such as trypsin, were not used.Thus, because this protocol is free of animal products, it is safe and feasible for large-scale BM-MSC isolation and expansion for use in clinical applications.

Bone Marrow and Peripheral Blood Collection
The project was approved by the local ethics committee.Five donors participated in this study.In total, 20 mL of bone marrow was aspirated from each donor after obtaining informed consent.The collection was performed in accordance with the standards of the local ethics committee.Besides BM samples, 20 mL peripheral blood was also collected from each donor.Both BM and peripheral blood were anticoagulated by using CDPA solution.All samples were immediately transferred to the laboratory.

MNC Isolation and Activated PRP Preparation
Mononuclear cells (NMCs) were isolated from the bone marrow.The BM samples were diluted at a ratio of 1:1 with phosphate-buffered solution (PBS) and then subjected to density centrifugation using Ficoll-Hypaque (1.077 g/mL; Sigma-Aldrich, St Louis, MO, USA).BM samples were centrifuged at 3000 rpm for 30 min.MNCs were collected from the interphase of the centrifuge tube.
The collected MNCs were washed twice with PBS and then used for further experiments.
Peripheral blood samples were used to produce activated PRP (aPRP).ACD anti-coagulated peripheral blood samples were centrifuged in two steps to get PRP.
In the first step, these samples were centrifuged at 500 × g for 5 min to obtain plasma.In the second step, the plasma samples were centrifuged at 800 × g for 10 min to obtain platelet pellets at the bottom of the tubes.To prepare aPRP, a third of the plasma volume and the platelet pellet was collected and re-suspended, following which 100 μL CaCl 2 per 1 mL of PRP was added to activate growth factor release.The samples were then incubated at 37°C for 30 min or until clotting occurred.

Primary Culture
Primary culture was performed as described in a previously published study (Pham et al., 2014).Five BM samples were used for primary culture.MNCs were cultured in DMEM/F12 medium containing 1% antibioticantimycotic (Sigma-Aldrich) and various concentrations of autologous aPRP (2%, 5%, 7%, or 10%) or 10% fetal bovine serum (FBS) for the control.The cells were plated at a density of 5 × 10 4 cells/mL in T-75 flasks (Corning) and incubated at 37°C with 5% CO 2 .After three days of culture, 6 mL of fresh medium was added to each T-75 flask.The medium was replaced with fresh medium every 4 days until the cells reached 70-80% confluence.The efficiency of the media was evaluated by considering the time required for adherent cells to appear and reach 70-80% confluence for the first subculture.

Secondary Culture
After successful primary culture, the samples were sub-cultured to evaluate the effects of various media.The proliferation rate was evaluated by the XCELLIgence system (Roche Applied Science, Indianapolis, IN, USA).A total of 1 × 10 3 cells were seeded into each well of a 96-well E-plate in triplicate.The culture plates were placed into the XCELLIgence system and incubated at 37°C in the presence of 5% CO 2 .Cell proliferation was monitored for 300 h, with the medium being replaced every third day.Both the cell doubling time and slope value were determined by a software of the XCELLIgence system.As a positive control, the mice were also injected with breast cancer cells at a different site.Tumor formation in mice was followed up for three months.In fact, these cells were positive for CD44, CD73, CD90 and CD105 and negative for CD14, CD34, CD45, and HLA-DR.This phenotype also agreed with that reported in previously published studies (Iudicone et al., 2014;Narbona-Carceles et al., 2014;Robey et al., 2014).
Although culture medium with 10% FBS excellently enhanced cell adherence to the flask surface in a manner similar to that observed in culture medium with 10% PRP during the primary culture, during the secondary culture, PRP efficiently stimulated BM-MSC growth.In fact, at 2.5% PRP concentration in the culture medium, proliferation rates of BM-MSCs in both 2.5% PRP medium and 10% FBS medium were similar.Moreover, their proliferation rates were significantly different between 10% FBS medium and 5% PRP and 10% PRP media.
These results showed that PRP is a rich source of natural Flow CytometryCell markers were analyzed by following a previously published protocol.Briefly, cells were washed twice in PBS containing 1% bovine serum albumin (Sigma-Aldrich).The cells were then stained with anti-CD13-FITC, anti-CD14-FITC, anti-CD34-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD73-FITC, anti-CD90-PE, anti-CD105-FITC, anti-CD106-PE, anti-CD166-PE, or anti-HLA-DR-FITC antibodies (all purchased from BD Biosciences, San Jose, CA, USA).Stained cells were analyzed by FACSCalibur flow cytometer (BD Biosciences).Isotype controls were used in all analyses.In Vitro Differentiation For differentiation into adipogenic cells, BM-MSCs were differentiated as described previously.Briefly, passage five cells were plated at a density of 1 × 10 4 cells/well in 24-well plates.At 70% confluence, the cells were cultured for 21 days in DMEM/F12 containing 0.5 mmol/L 3-isobutyl-1-methyl-xanthine, 1 nmol/L dexamethasone, 0.1 mmol/L indomethacin, and 10% FBS (all purchased from Sigma-Aldrich).Adipogenic differentiation was evaluated by observing lipid droplets in cells, stained with Oil Red, under a microscope.For differentiation into osteogenic cells, BM-MSCs were plated at a density of 1 × 10 4 cells/well in 24well plates.At 70% confluence, the cells were cultured for 21 days in DMEM/F12F12 containing 10% FBS, 10 - 7 mol/L dexamethasone, 50 μmol/L ascorbic acid-2 phosphate, and 10 mmol/L β-glycerol phosphate (all purchased from Sigma-Aldrich).Osteogenic differentiation was confirmed by Alizarin red staining.Tumorigenicity Assay The tumorigenicity of BM-MSCs was examined in athymic nude mice.All manipulations of mice were approved by the Local Ethics Committee of Stem Cell Research and Application, University of Science (Ho Chi Minh City, Vietnam).Each mouse was injected subcutaneously with 5 × 10 6 cells (three mice per group).
fig a w in 1 a co th ev ce Fig ex FB human growth factors.Moreover, these growth factors triggered BM-MSC growth.PRP also strongly stimulated the proliferation of MSCs that originated from other tissues such as umbilical cord blood(Murphy et al., 2012; Pham   et al., 2014), adipose tissue(Atashi et al., 2014; Van   Pham et al., 2014), and human dental stem cells(Lee et   al., 2011).The last criterion evaluated was the differentiation potential of BM-MSCs into mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes.The BM-MSCs cultivated in PRP medium and FBS medium could differentiate into adipocytes and osteoblasts.BM-MSCs cultured in PRP-supplemented medium were also found to maintain the MSC phenotype.To satisfy the stem cells for transplantation, BM-MSCs cultured in PRP medium were examined for changes in the normal karyotype and for their tumorigenicity.The results demonstrated that PRP did not affect the karyotype of BM-MSCs.At the 10 th passage, BM-MSCs in both PRP-or FBS-supplemented media maintained the normal karyotypes (2n = 46).Owing to the normal karyotype, these cells also could not form tumors in the athymic mice.Considering these analysis results, PRP could replace FBS in BM-MSC culture and satisfy the criteria of MSCs used for clinical transplantation.