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A method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immunomodulatory function from children's foreskin culture

A quality stem cell and foreskin technology, applied in the field of stem cell culture, can solve the problems such as the yield and purity can not meet the multiple clinical doses, the low yield of adipose tissue stem cells, and restrict the clinical application of MSCs, and achieve strong immune regulation and inhibition of inflammation. The effect of reaction, fast growth rate

Active Publication Date: 2022-07-08
SHENZHEN CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Studies have proved that although these different MSC populations have the same basic characteristics and properties, there are some differences in their immune properties and reactivity to inflammation. The clinical application is an urgent problem to be solved
[0005] At present, MSCs derived from adipose tissue and human umbilical cord are used in clinical cell therapy and have achieved certain results, but the curative effect is not satisfactory.
Moreover, the adipose tissue is obtained by invasive extraction, and the adipose tissue stem cell acquisition rate is low; the umbilical cord is the source of mesenchymal stem cells, but it can only be obtained at birth, and the time is limited, and the adipose tissue and human umbilical cord are derived from MSCs The yield and purity of mesenchymal stem cells cannot meet the standards for multiple clinical doses

Method used

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  • A method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immunomodulatory function from children's foreskin culture
  • A method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immunomodulatory function from children's foreskin culture
  • A method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immunomodulatory function from children's foreskin culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The present embodiment provides a method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immune regulation function from children's foreskin culture, and the specific steps are as follows:

[0062] (1) After circumcision, the foreskin sample was collected into a sterile sample container containing sterile phosphate buffered saline (PBS, Lonza) supplemented with penicillin / streptomycin (Lonza).

[0063] (2) Upon arrival in the laboratory, wash with sterile PBS and transfer to a petri dish. The FSK was then sectioned longitudinally to diffuse the tissue, the epidermis and dermis were separated, and the dermis was cut into small pieces to obtain a tissue homogenate.

[0064] (3) The above tissue homogenate was evenly inoculated into a T75 culture flask, and the culture flask was placed horizontally so that the tissue pieces were evenly distributed on the entire bottom surface as much as possible, and serum-free mesenchymal stem cel...

Embodiment 2

[0067] The present embodiment provides a method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immune regulation function from children's foreskin culture, and the specific steps are as follows:

[0068] (1) After circumcision, the foreskin sample was collected into a sterile sample container containing sterile phosphate buffered saline (PBS; Lonza) supplemented with penicillin / streptomycin (Lonza).

[0069] (2) Upon arrival in the laboratory, wash with sterile PBS and transfer to a petri dish. The FSK was then sectioned longitudinally to diffuse the tissue, the epidermis and dermis were separated, and the dermis was cut into small pieces to obtain a tissue homogenate.

[0070] (3) The above-mentioned tissue homogenate was evenly inoculated into a T75 culture flask, and the culture flask was flattened to make the tissue pieces evenly distributed on the entire bottom surface as much as possible, and DMEM / f12 medium (Lonza) supplemented...

Embodiment 3

[0100] In this example, single-cell sequencing and enzyme-linked immunosorbent assay were used to carry out immune characteristics research on the mesenchymal stem cells provided in Example 1. The specific steps and results are as follows:

[0101] 1. Single-cell sequencing and results

[0102] The method of single-cell sequencing is the same as the operation in Verification Example 2, and the results are as follows Figure 10-15 shown.

[0103] Depend on Figure 11 It can be seen that HuMSCs express immune-related genes: TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS1.

[0104] Depend on Figure 12 It can be seen that HuMSCs express chemotaxis-related markers: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL12, PDGFRA, PDGFRB.

[0105] Depend on Figure 13 It can be seen that FSK-MSCs express immune-related genes: TGFB1, TNFAIP6, IL10, HMOX1, IDO1, LIF, IL6, CD274, PTGS1.

[0106] Depend on Figure 14 It can be seen that FSK-MSCs express chemotaxis-related mark...

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Abstract

The invention provides a method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immune regulation function from children's foreskin culture, comprising the steps of: washing the foreskin cut by circumcision with sterile PBS, and placing it in a cultured The foreskin was longitudinally sliced ​​to diffuse the tissue, the epidermis and dermis were separated, and then the dermis was cut into small pieces to form tissue homogenate; the tissue homogenate was evenly inoculated into a culture flask, and the culture flask was placed flat to make tissue pieces Try to distribute evenly on the entire bottom surface, add medium for primary culture, and finally subculture to the third generation to establish a mesenchymal stem cell bank. The culture method has high speed, high yield, abundant raw material sources, and the cultured mesenchymal stem cells have high purity, multi-directional differentiation potential and immune regulation functions, and can express more immunosuppression-related genes and chemotactic genes. For the treatment of various types of sepsis (infectious (bacterial and viral) and immune) to suppress the inflammatory response.

Description

technical field [0001] The present invention relates to the field of stem cell culture, in particular to a method for obtaining mesenchymal stem cells with multi-directional differentiation potential and immune regulation function from children's foreskin culture. Background technique [0002] Before this, staged results have been achieved in the treatment of sepsis with stem cells at home and abroad. Human clinical studies have preliminarily confirmed the safety of MSCs in the treatment of sepsis, and showed a certain curative effect. Previous studies have found that MSCs also play an active role in the treatment of human immunodeficiency virus (HIV) immune abnormalities, hepatitis B virus (HBV) chronic hepatitis, influenza virus acute lung injury (ALI) and other virus-related diseases. [0003] Mesenchymal stem cells (MSCs) are multipotent stromal cells that exist in various tissues and can differentiate into different types of cells, including osteoblasts, adipocytes, cho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00C12N2509/10
Inventor 马廉
Owner SHENZHEN CHILDRENS HOSPITAL
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