Development of hMSC production kit and optimization of quality management standards and establishment of quality management system

A kit and standard technology, applied in the field of stem cell therapy, can solve problems such as the lack of research on cell quality stability, the establishment of cell quality evaluation and subject evaluation systems, and the difficulty of forming quality standards, so as to optimize primary tissue processing Improvement of technology, yield of primary cells, reduction of raw material consumption and production cost

Inactive Publication Date: 2021-02-09
路春光
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, no matter in the preclinical or clinical research stage, the existing research results still have different degrees of problems, and all the problems converge to different degrees on the quality of hMSCs
Among the problems in preclinical research are that there are great differences in hMSCs sources, culture systems, preparation processes, quality standards, and evaluation techniques used by different developers, and there are also problems such as the content of quality evaluation and the rationality of evaluation technologies. It is difficult to form a relatively uniform quality standard; the stability of cell quality in the clinical research stage is lacking, and the cell quality evaluation and subject evaluation system has not yet been established, etc.

Method used

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  • Development of hMSC production kit and optimization of quality management standards and establishment of quality management system
  • Development of hMSC production kit and optimization of quality management standards and establishment of quality management system
  • Development of hMSC production kit and optimization of quality management standards and establishment of quality management system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 Study on the quality of industrialized production process of human umbilical cord MSC

[0063] 1. Cell Identification

[0064] (1) Cell morphology:

[0065] It is generally believed that under standard culture conditions, the morphology of human umbilical cord mesenchymal stem cells is fibroblast-like or spindle-shaped, and grows adherently. Under a high-power microscope, the cells should have a strong three-dimensional sense and fewer intracellular granules. See figure 1 .

[0066] (2) Cytogenetic identification:

[0067] The source of human umbilical cord mesenchymal stem cells should be single to ensure the stability and controllability of cell function. When determining the source, use STR map for genetic analysis to confirm that the source of the cells is a single source of cell donors. See figure 2 ;

[0068] STR profile analysis shows that after the isolated cells are amplified on 21 autosomal allele loci, the test results are all single-peak or...

Embodiment 2

[0136] Preparation and use of the culture kit of embodiment 2 hMSC

[0137] The present invention provides a kind of MSC induction culture kit, comprises container and the clinical MSC cell serum-free expansion medium MSC- and cell culture supplement MSC- :

[0138] 1) The present invention provides a clinical MSC cell expansion complete culture medium MSC- , PH7.2~7.4, balanced salt Earle's salt, nucleoside, L-glutamine 2mM, NaHCO3 2200mg / L, D-glucose 1000mg / L, sodium pyruvate 1mM, phenol red indicator 10mg / L, inorganic salt , amino acids, vitamins, ribonucleosides, deoxyribonucleosides, etc., add 2mM GlutaMAX™-I and 10% Prime serum during use, and the present invention does not exclude the use of medium other than the above-mentioned medium for cultivation;

[0139] 2) The present invention provides a MSC cell culture additive MSC- , which contains 2-50ng / mL bFGF, 2-50ng / ml EGF;

[0140] The preparation of described complete medium: MSC- Add 1 / 1000 MSC- , 2mM Gl...

Embodiment 3

[0145] Example 3 hMSC cell culture supplement MSC- The formula is further optimized

[0146] The experiment is divided into 6 groups:

[0147] Group A: α-MEM +3ng / mL bFGF+3ng / ml EGF+10%FBS+Gln;

[0148] Group B: α-MEM+6ng / mL bFGF+6ng / ml EGF+10%FBS+Gln;

[0149] Group C: α-MEM+6ng / mL bFGF+6ng / ml EGF+6ng / ml VEGF+6ng / ml PDGFP+10%FBS+Gln;

[0150] Group D: α-MEM +3ng / mL bFGF+3ng / ml EGF+3ng / ml VEGF+3ng / ml PDGF+10%FBS+Gln;

[0151] Group E: α-MEM +3ng / mL bFGF+3ng / ml EGF+6ng / ml VEGF+6ng / ml PDGF+10%FBS+Gln;

[0152] Prime serum group: α-MEM +6ng / mL bFGF+6ng / ml EGF+56ml Prime+5ml Gln

[0153]Under the six groups of culture conditions, the umbilical cord MSCs were continuously cultured to the P3 generation, and the growth curve was drawn in Figure 5 :

[0154] Statistical analysis of six groups of data, there was no significant difference between groups, P=0.62, P>0.05;

[0155] The results of flow cytometry detection of umbilical cord MSC cultured under six groups of culture ...

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Abstract

The invention relates to the technical field of stem cell therapy, and particularly relates to an optimized industrial production system and quality management standard optimization and system establishment for isolating mesenchymal stem cells (MSC) and human mesenchymal stem cells (hMSC) from fetal appendages (umbilical cord blood, umbilical cord, placenta, etc.), hair follicles, fat, dental pulp, etc.; and the invention also discloses an MSC multiplication culture kit, an identification kit and application thereof. According to contents specified by "Guidelines for Stem Cell Preparation Quality Control and Preclinical Research (Trial)", "Guiding Principles for Cell Products Research and Evaluation Technology" (Draft for Soliciting Comments), and General Rule 9203 of "Chinese Pharmacopoeia (2015 Edition)"-Guiding Principles for Pharmaceutical Microbiology Laboratory Quality Management, quality standards of raw material and auxiliary material control, umbilical cord receiving control and mesenchymal stem cell preparation control are established. According to the invention, the process and quality standards are optimized and established, the yield is increased, and conditions are created for clinical application and realizing industrial production.

Description

technical field [0001] The present invention relates to the technical field of stem cell therapy, in particular to an optimized method for isolating mesenchymal stem cells (MSCs), human mesenchymal stem cells ( hMSC) industrialized production system and quality management standard optimization and system establishment; MSC expansion culture kits and identification kits and their applications. Background technique [0002] Human mesenchymal stem cells (human MSCs, hMSCs) are a type of adult stem cells with certain self-replication and renewal and multi-lineage differentiation potential, widely present in fetal and adult tissues, such as bone marrow, fat, skin, dental pulp, tendon, And newborn accessory tissues, such as umbilical cord, placenta, amniotic membrane and other tissues. In addition to a certain degree of differentiation potential, hMSCs also have unique immune regulation and tissue regeneration functions, participate in the regulation of abnormal immune responses,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775G01N33/569
CPCC12N5/0665C12N5/0668G01N33/56966C12N2501/115C12N2501/11C12N2501/998G01N2469/10
Inventor 路春光
Owner 路春光
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