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. 2022 Jun 21;13(1):268.
doi: 10.1186/s13287-022-02946-5.

Xeno-free protocol for GMP-compliant manufacturing of human fetal pancreas-derived mesenchymal stem cells

Zahra Jabbarpour #  1 , Sajjad Aghayan #  1 , Babak Arjmand  2 , Khadijeh Fallahzadeh  2 , Sepideh Alavi-Moghadam  2 , Bagher Larijani  3 , Hamid Reza Aghayan  4
Affiliations

Xeno-free protocol for GMP-compliant manufacturing of human fetal pancreas-derived mesenchymal stem cells

Zahra Jabbarpour et al. Stem Cell Res Ther. .

Abstract

Background: Mesenchymal stem cells (MSCs) have been suggested as an appropriate source for diabetes cell-based therapies. The high proliferation and differentiation capacity of fetal MSCs and the role of fetal pancreatic-derived MSCs (FPMSCs) in islet generation make them good candidates for diabetes treatment. To manufacture clinical-grade MSCs, animal-free culture protocols are preferred. The current study aimed to establish a xeno-free/GMP-compliant protocol for FPMSCs manufacturing. The focus was on the effects of fetal bovine serum (FBS) replacement with pooled human serum (HS).

Material and methods: FPMSCs were isolated and expanded from the pancreas of legally aborted fetuses with few modifications in our previously established protocol. The cells were expanded in two different culture media, including DMEM supplemented with 10% FBS or 10% pooled HS. A side-by-side comparison was made to evaluate the effect of each serum on proliferation rate, cell cycle, senescence, multi-lineage differentiation capacity, immunophenotype, and tumorigenesis of FPMSCs.

Results: Flow cytometry analysis and three-lineage differentiation ability demonstrated that fibroblast-like cells obtained from primary culture had MSCs' characteristics. The FPMSCs displayed similar morphology and CD markers expression in both sera. HS had a higher proliferative effect on FPMSCs than FBS. In FBS, the cells reached senescence earlier. In addition to normal karyotypes and anchorage-dependent growth, in vivo tumor formation was not seen.

Conclusion: Our results demonstrated that HS was a better serum alternative than FBS for in vitro expansion of FPMSCs. Compared with FBS, HS increased FPMSCs' proliferation rate and decreased their senescence. In conclusion, HS can effectively replace FBS for clinical-grade FPMSCs manufacturing.

Keywords: Cell therapy; Diabetes; Fetal pancreas; GMP; Mesenchymal stem cell; Xeno-free.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Fig. 1
Fig. 1
FPMSCs’ characterization at the third subculture: A CD marker expression of FPMSCs isolated from different donors, B three-lineage differentiation of FPMSCs. The expression of mesenchymal CD markers and HLA-DR in both study groups C. The effect of different sera on FPMSCs’ differentiation to osteocytes D and adipocytes E
Fig. 2
Fig. 2
A FPMSCs’ morphology in different sera. B Growth curve of FPMSCs in HS- and FBS-supplemented media. C The results of cell cycle analysis. D and E SA-βG assay of FPMSCs from two batches
Fig. 3
Fig. 3
ICC assay of FPMSCs in both study groups. A Vimentin, B HLA-ABC, C Ki67, and D Nestin. The white arrows show the Nestin expression in FBS-cultured cells
Fig. 4
Fig. 4
Tumorigenicity assay of FPMSCs in HS- and FBS-supplemented media. A G-banding karyotyping, B soft agar colony formation assay, C tumor formation in nude mice. The white arrows show early colony formation of HEK 293 T cells
See this image and copyright information in PMC

References

    1. Bluestone JA, Buckner JH, Fitch M, Gitelman SE, Gupta S, Hellerstein MK, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Sci Transl Med. 2015 doi: 10.1126/scitranslmed.aad4134. - DOI - PMC - PubMed
    1. Shin J-Y, Jeong J-H, Han J, Bhang SH, Jeong G-J, Haque MR, et al. Transplantation of heterospheroids of islet cells and mesenchymal stem cells for effective angiogenesis and antiapoptosis. Tissue Eng Part A. 2015;21(5–6):1024–1035. doi: 10.1089/ten.tea.2014.0022. - DOI - PMC - PubMed
    1. Chhabra P, Brayman KL. Stem cell therapy to cure type 1 diabetes: from hype to hope. Stem Cells Transl Med. 2013;2(5):328–336. doi: 10.5966/sctm.2012-0116. - DOI - PMC - PubMed
    1. Wei X, Yang X, Han ZP, Qu FF, Shao L, Shi YF. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol Sinica. 2013;34(6):747–54. doi: 10.1038/aps.2013.50. - DOI - PMC - PubMed
    1. Yeung TY, Seeberger KL, Kin T, Adesida A, Jomha N, Shapiro AJ, et al. Human mesenchymal stem cells protect human islets from pro-inflammatory cytokines. PLoS ONE. 2012;7(5):e38189. doi: 10.1371/journal.pone.0038189. - DOI - PMC - PubMed

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