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. 2022 Sep 1;11(17):2732.
doi: 10.3390/cells11172732.

Manufacture and Quality Control of Human Umbilical Cord-Derived Mesenchymal Stem Cell Sheets for Clinical Use

Juan Wang  1 , Shuang Gao  1 , Yufei Zhao  1 , Taibing Fan  2 , Mingkui Zhang  3 , Dehua Chang  4
Affiliations

Manufacture and Quality Control of Human Umbilical Cord-Derived Mesenchymal Stem Cell Sheets for Clinical Use

Juan Wang et al. Cells. .

Abstract

Human umbilical cord-derived mesenchymal stem cell (UC-MSC) sheets have attracted much attention in cell therapy. However, the culture media and coating matrix used for the preparation of UC-MSC sheets have not been safe enough to comply with current clinical drug standards. Moreover, the UC-MSC sheet preservation systems developed before did not comply with Good Manufacturing Practice (GMP) regulations. In this study, the culture medium and coating matrix were developed for UC-MSC sheet production to comply with clinical drug standards. Additionally, the GMP-compliant preservation solution and method for the UC-MSC sheet were developed. Then, quality standards of the UC-MSC sheet were formulated according to national and international regulations for drugs. Finally, the production process of UC-MSC sheets on a large scale was standardized, and three batches of trial production were conducted and tested to meet the established quality standards. This research provides the possibility for clinical trials of UC-MSC sheet products in the development stage of new drugs and lays the foundation for industrial large-scale production after the new drug is launched.

Keywords: large-scale production; preservation; umbilical cord mesenchymal stem cell sheet.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
The umbilical cord mesenchymal stem cell (UC−MSC) sheet production process conformation. (A) Morphology of the UC−MSC sheets. (B) Statistics of diameters of the UC−MSC sheets. (C) Statistical analysis of total cell amount per UC−MSC sheet. (D) Statistical analysis of cell viability of the UC−MSC sheets.
Figure 2
Figure 2
The UC−MSC sheet preservation solution selection. (A) Morphology of the UC−MSC sheet. (B) Statistics of cell sheet diameters. (C) Statistical analysis of total cell amount per UC−MSC sheet. (D) Statistical analysis of cell viability of the UC−MSC sheets.
Figure 3
Figure 3
Schematic diagram of the large-scale production of UC−MSC sheets.
Figure 4
Figure 4
Structure of the UC−MSC sheet. (A) Morphology of the UC−MSC sheet. (B) Enlarged morphology of the UC−MSC sheet. (C) Fibronectin and Integrin β1 staining of the UC−MSC sheet.
Figure 5
Figure 5
Characteristic tests of UC−MSC sheets. (A) Statistics of cell sheet diameters. (B) Statistical analysis of cell amount per sheet. (C) Statistics of cell viability. (D) Statistical analysis of cell apoptosis.
Figure 6
Figure 6
Cell characteristic tests of UC−MSC sheets. (A) Cell surface marker expression of freshly and 24 h preserved cell sheets. (B) Representative results of induced adipogenic (white arrows point to lipid droplets stained with oil red), osteogenic (white arrows point to calcium nodules stained with alizarin red), and chondrogenic (white arrows point to cartilage stained with alcian blue) differentiation of freshly and 24 h preserved cell sheets.
Figure 7
Figure 7
Detection of residues of high-risk substances. (A) Bovine serum albumin (BSA). (B) Human serum albumin (HSA). (C) Gentamicin. (D) Fibrinogen. (E) TrypLE. (F) Basic fibroblast growth factor (bFGF).
Figure 8
Figure 8
Growth factor detection of UC−MSC sheet. (A) Hepatocyte growth factor (HGF) (B) Vascular endothelial growth factor (VEGF) (C) Interleukin-6 (IL-6) (D) Interleukin-8 (IL-8).
Figure 9
Figure 9
Immunomodulatory effect test of the UC−MSC sheet. (A) Detection of lymphocyte proliferation inhibition ability of UC−MSC sheet. (B) Detection of lymphocyte tumor necrosis factor α (TNFα) secretion inhibition ability of UC−MSC sheet. (C) Detection of Th1 lymphocyte inhibition ability of UC−MSC sheet.
Figure 10
Figure 10
Proangiogenic function test of UC−MSC sheet. (A) ImageJ Angiogenesis Analyzer was used to quantify endothelial tube formation. Images were taken using an inverted microscope and analyzed using an angiogenesis analyzer. Yellow lines represent tubes that connect together to different junctions; green lines represent branches; dark blue represents isolated tubes; red circles represent the master junction points. (B) Statistical results of Nb junctions. (C) Statistical results of total length. *** represent p < 0.01.
Figure 11
Figure 11
Anti-apoptotic function test of the cell sheet. (A) Apoptosis results of H9C2 cells cultured in cell sheet-forming conditioned medium or negative control medium after induction of apoptosis by cobalt chloride. (B) Statistical results of apoptosis results of H9C2 cells cultured in cell sheet-forming conditioned medium or negative control medium after apoptosis was induced by cobalt chloride. *** represent p < 0.01.
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Grants and funding

This study is supported by Beijing Municipal Science and Technology Committee (No. Z191100001519002).

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