Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method

Abstract

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Label‐free quantitative proteomic analysis of human periodontal ligament stem cells by high‐resolution mass spectrometry

“…The hBMSCs used in this study were kindly provided by the Translational Research Center at the School of Medicine, Inha University. The samples were human clonal BMSC lines generated from human bone marrow aspirates using subfractionation culturing method which was based on clonal selection and expansion of clonal MSCs from a single colony‐forming unit‐derived colonies . Each homogenous single colony of the hBMSC lines was confirmed in its stem cell characteristics beforehand.…”

“…Each homogenous single colony of the hBMSC lines was confirmed in its stem cell characteristics beforehand. The protocol to isolate, identify and cultivate the hBMSCs has been described previously . In brief, hBMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing low glucose with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin and incubated in a humidified atmosphere as described above in culturing the hPDLSCs.…”

“…The samples were human clonal BMSC lines generated from human bone marrow aspirates using subfractionation culturing method which was based on clonal selection and expansion of clonal MSCs from a single colony-forming unit-derived colonies. 16 Each homogenous single colony of the hBMSC lines was confirmed in its stem cell characteristics beforehand. The protocol to isolate, identify and cultivate the hBMSCs has been described previously.…”

“…The protocol to isolate, identify and cultivate the hBMSCs has been described previously. 16,17 In brief, hBMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing low glucose with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin and incubated in a humidified atmosphere as described above in culturing the hPDLSCs. The hPDLSCs and hBMSCs from passages three to five (P3 to P5) and seven (P7), respectively, were used in the following experiments.…”

An Update on the Progress of Isolation, Culture, Storage, and Clinical Application of Human Bone Marrow Mesenchymal Stem/Stromal Cells

“…Yi et al developed and optimized GMP compatible culture protocol, which is suitable for producing BMSCs for clinical uses. Clinical-grade BMSCs are stable in terms of genomic integrity [18]. Even that, manufacturing systems hardly meet a standard requirement because of the differences between donors, harvesting methods, devices, and cultivation conditions [19].…”