Human mesenchymal stem cells

Abstract Mesenchymal stem cells (MSCs) have attracted great interest for cell therapy and tissue regeneration due to their self‐renewal capacity, multipotency and potent immunomodulatory effects on immune cells. However, heterogeneity of MSCs has become a prominent obstacle to limit their translation into practice, as cells from different tissue sources or each individual have great differences in their transcriptomic signatures, differentiation potential and biological functions. Therefore, there is an urgent need for consensus standard for the quality control and technical specifications of MSCs. ‘Human Mesenchymal Stem Cells’ is the latest set of guidelines on hMSC in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hMSC, which is applicable to the quality control for hMSC. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hMSC for clinical development and therapeutic applications.

This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mesenchymal stem cells (hMSC), which is applicable to the quality control for hMSC. The guideline of MSCs has been originally released by the China Society for Cell Biology on 9 January 2021. Now it is translated into an English version. We hope that publication of these guidelines will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hMSC for clinical development and therapeutic applications.  This standard specifies the technical requirements, test methods,   test regulations, instructions for use, labelling requirements, packaging requirements,   storage requirements, transportation requirements and waste disposal requirements for hMSC, which is applicable to the quality control for hMSC. It was originally released by the China Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hMSC for clinical development and therapeutic applications.

| Scope
This document specifies the technical requirements for hMSC, and requirements for test methods, test regulations, instructions for use, labelling, packaging, storage, transportation and waste disposal.
This standard is applicable for the production and testing of hMSC.

| Normative references
The following content constitute indispensable articles of this stand-

| Terms and definitions
For the purpose of this document, the terms and definitions in T/ CSCB 0001, T/CSCB 0002 and following terms and definitions apply to this document.

| Human mesenchymal stem cells
A type of stem cells exhibit a fibroblast-like morphology (spindle and fusiform) after adherent culture, and possess the ability to selfrenew and differentiate into the osteogenic, adipogenic and chondrogenic lineages in vitro.
Note 1 to entry: hMSC can not only be isolated from multiple tissues (such as bone marrow, umbilical cord, placenta, adipose tissue, umbilical cord blood et al) but also be obtained by differentiation or transdifferentiation. hMSC from different sources have differences in gene expression profile and differentiation potential.

| Abbreviations
The following abbreviations are applicable for this document.

Cell morphology
Cells under adherent culture shall exhibit a fibroblast-like morphology (spindle and fusiform), and uniform morphology.

Chromosome karyotype
The normal karyotype shall be 46, XX or 46, XY.

Cell viability
The cell viability shall be ≥90% prior to cryopreservation, and ≥70% after thawing.

Cell surface markers
The expressions of CD105, CD73 and CD90 shall be ≥95% of the cell population, and the expression of CD11b, CD19, CD31, CD34, CD45 and HLA-DR shall be ≤2% of the cell population.
Additionally, after co-cultured with T cells, these cells shall broadly suppress T cell proliferation and the secretion of IFNγ and TNFα.
6. Tri-lineage differentiation capacity Cells shall be able to differentiate to osteoblasts, adipocytes and chondroblasts under standardized differentiating conditions in vitro.

Tumorigenesis
Cells shall be negative for in vivo tumorigenicity testing using immunodeficient animal model (such as immunodeficient mice).

| Process control
1. The process of cell expansion, cryopreservation, cell thawing and other cell manufacturing shall follow the requirements of T/CSCB 0001-2020.
2. The STR test results of cell products shall be consistent with STR of the donor cell.

| Cell morphology
Observe the morphology of cells grown in 2D condition using a microscope.

| Chromosome karyotype
The method in the Pharmacopoeia of the People's Republic of China shall be followed.

| Cell viability
The method in Appendix A shall be followed.

| Cell surface markers
The method in Appendix B shall be followed. 2.6.5 | Immunomodulatory capacity 1. The induced expression of IDO The method in Appendix C shall be followed.

The suppression of T cell proliferation
The method in Appendix D shall be followed.
3. The suppression of secretion of IFNγ and TNFα by T cells The method in Appendix E shall be followed.

Osteogenic differentiation
The method in Appendix F shall be followed.

Adipogenic differentiation
The method in Appendix G shall be followed.

Chondrogenic differentiation
The method in Appendix H shall be followed.

| Tumorigenesis
The method in Appendix I shall be followed.

Fungi
The '1101 Sterility Inspection Method' in Pharmacopoeia of the People's Republic of China (Volume IV) shall be followed.

Bacteria
The '1101 Sterility Inspection Method' in Pharmacopoeia of the People's Republic of China (Volume IV) shall be followed.

Mycoplasma
The '3301 Mycoplasma Inspection Method' in Pharmacopoeia of the People's Republic of China (Volume IV) shall be followed.

HBV
The method in National Guide to Clinical Laboratory Procedures shall be followed.

HCV
The method in WS 293 shall be followed.

HIV
The method in WS 293 shall be followed.

HTLV
The method in National Guide to Clinical Laboratory Procedures shall be followed.

EBV
The method in National Guide to Clinical Laboratory Procedures shall be followed.

HCMV
The method in National Guide to Clinical Laboratory Procedures shall be followed.

TP
The method in WS 293 shall be followed.

| Inspection rules
2.7.1 | Sampling method 1. Cells produced from the same production cycle, same production line, same source, same passage and same method are considered to be the same batch.
2. Three smallest units of packaging shall be randomly sampled from the same batch. 2.7.2 | Quality inspection and release 1. Each batch of products shall be subject to the qualify inspection before release, and inspection reports shall be attached.
2. The quality inspection items shall include all the attributes specified in 2.5.2.

| Review inspection
Review inspection shall be performed by professional cytological testing institutions or laboratories as necessary.

| Decision rules
1. Products that pass all requirements in 2.5.2 for the quality inspection for release are considered to be qualified. Products that fail to pass one or more requirements in 2.5.2 for the quality inspection for release are considered to be unqualified.
2. Products that pass all requirements in 2.5.2 for the quality review inspection are considered to be qualified. Products that fail to pass one or more requirements in 2.5.2 for the review inspection are considered to be unqualified.

| Instructions for usage
The instructions for usage shall include, but not limited to:

| Labels
The label shall include but not limited to: 2.10.3 | Transportation 1. T/CSCB 0001-2020 shall be followed.
2. Cryopreserved cell products shall be transported in dry ice or at a temperature below −130℃. Non-frozen cells are recommended for refrigerated transportation (2℃ ~ 8℃).

| Waste disposal
The waste generated during manufacturing and testing of hMSC shall be disposed following the requirements of T/CSCB 0001-2020.

ACK N OWLED G EM ENTS
The

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest are disclosed.

A.2 Reagents
Unless otherwise stated, all reagents used shall be analytical grade. The water used for testing shall be deionized water. The viability of cells is the mean of two duplicate samples.

A.5 Accuracy
The absolute difference value between the two independent tests by the independent inter-examiner, under the same conditions, shall not exceed 10% of their arithmetic mean.

FACE M A R K E R S (FLOW C Y TO M E TRY )
B. wash solution, antibody dilution solution.

B.3 Sample storage
The wash solution and fixed samples shall be stored at 2 ~ 8°C.

B.4.2 Antibody incubation
Incubate the samples with the diluted antibodies according to the manufacturer's instructions. Wash the cell samples with an appropriate volume of wash solution for 2 times, then centrifuge at 300 g for 4 min and discard the supernatant.

B.4.3 Filter and detection
Resuspend the samples with wash solution and then transfer the cell suspension into flow cytometry tube passed through a 40µm mesh filter. Load the samples into the flow cytometer and perform detection according to the manufacturer's instruction.

B.4.4 Gating
Gate the population of target cells based on particle size and granularity, excluding cell debris and other irrelevant particles.
The gating of positive staining cells shall be determined by the fluorescence intensity using isotype controls as a reference.
Both positive and negative experimental controls shall be set up for gating and the following analysis.

B.5 Analysis of results
Analyse the results using software according to manufacturer's instructions.

A PPE N D I X C N O R M ATI V E A PPEN D IX : D E TEC TI O N O F I N D U CED I D O E X PR E SS I O N (P CR A SSAY )
C.

C.4 Analysis of results
The expression of IDO is detected in hMSC cultured with IFNγ or IFNγ + TNFα, while it is undetectable in hMSC without stimulation.

D.4 Analysis of results
Analyse the results using software according to manufacturer's instructions and calculate the inhibition rate of hMSC on T-cell proliferation.
Inhibition rate is calculated according to equation (D1): In this equation: A-the percentage of the proliferating T cells without hMSC. C-the percentage of the proliferating T cells that cultured with hMSC.

E.2 Reagents
Unless otherwise stated, all the reagents used shall be analytical grade. The water used in the experiment shall be Grade 1 water as stipulated in GB/T 6682.

E.4 Analysis of results
Analyse the results using software according to manufacturer's instructions and calculate the inhibition rate of hMSC on the IFNγ and TNFα secretion of T cells.
Inhibition rate is calculated according to equation (E1)

F.2 Reagents
Unless otherwise stated, all the reagents used shall be analytical grade. The water used in the experiment shall be Grade 1 water as stipulated in GB/T 6682.

F.3.2 Cell seeding and induction
Choose the appropriate cell seeding method, seeding density and induction procedure according to the manufacturer's instructions, and osteogenic differentiation is induced for 14 ~ 21 days.

F.3.3 Calcium deposits staining
Stain the extracellular calcium deposits by Alizarin Red S staining according to the manufacturer's instructions.

F.4 Analysis of results
A large number of bright orange-red calcium deposits can be seen under the microscope.

G.4 Analysis of results
Orange-red lipid droplets can be seen under the microscope, and fat cells contain lipid droplets of varying sizes.

TI ATI O N A SSAY ( A LCI A N B LU E S TA I N I N G)
H.

H.4 Analysis of results
The dark blue extracellular matrix of chondrocytes can be seen under the microscope.

I.4 Analysis of results
Tumour formation rate is calculated according to equation (I1) In this equation: M-the total number of injected mice. N-the number of tumour-bearing mice.