Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunomodulatory function from prepuce culture of children

A technology of high quality stem cells and differentiation potential, applied in the field of stem cell culture, can solve the problems of strong time limitation, unsatisfactory curative effect, low rate of adipose tissue stem cell acquisition, etc., achieve fast proliferation speed, strong immune regulation function, and inhibit inflammation The effect of the reaction

Active Publication Date: 2020-08-04
SHENZHEN CHILDRENS HOSPITAL
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  • 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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  • Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunomodulatory function from prepuce culture of children
  • Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunomodulatory function from prepuce culture of children
  • Method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunomodulatory function from prepuce culture of children

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0063] (2) After arriving at the laboratory, wash with sterile PBS and transfer to a Petri dish. Then, the FSK was sectioned longitudinally to spread the tissue, the epidermis and dermis were separated, and the dermis was then cut into small pieces to obtain a tissue homogenate.

[0064] (3) Evenly inoculate the above-mentioned tissue homogenate into a T75 culture flask, place the culture flask flat so that the tissue pieces are evenly distributed on the entire bottom surface as much as possible, and add 50 U / mL penicillin / streptomycin (Lonza) mesenc...

Embodiment 2

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

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

[0069] (2) After arriving at the laboratory, wash with sterile PBS and transfer to a Petri dish. Then, the FSK was sectioned longitudinally to spread the tissue, the epidermis and dermis were separated, and the dermis was then cut into small pieces to obtain a tissue homogenate.

[0070] (3) The above-mentioned tissue homogenate was inoculated evenly in a T75 culture bottle, and the culture bottle was placed flat so that the tissue pieces were evenly distributed on the entire bottom surface as far as possible, and DMEM / f12 medium (Lonza) supplemented w...

Embodiment 3

[0100] This example uses single-cell sequencing and enzyme-linked immunosorbent assay to study the immune characteristics of 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 for 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 expressed chemotaxis related markers: CXCL1, CXC...

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Abstract

The invention provides a method for obtaining mesenchymal stem cells with multidirectional differentiation potential and immunomodulatory function from children prepuce culture, comprising the following steps of: cleaning prepuce cut by circumcision with sterile PBS, and putting into a culture dish; longitudinally slicing the prepuce to diffuse tissues, separating epidermis from dermis, and then cutting the dermis into small pieces to form a tissue homogenate; uniformly inoculating the tissue homogenate into a culture bottle, horizontally placing the culture bottle to enable tissue blocks to be uniformly distributed on the whole bottom surface as much as possible, and adding a culture medium for primary culture; and finally, subculturing to the third generation to establish a mesenchymal stem cell bank. The culture method is high in speed, high in yield and rich in raw material source, and the cultured mesenchymal stem cells are high in purity, have multidirectional differentiation potential and immunomodulatory functions, can express more immunosuppression related genes and chemotactic genes, and can be used for treating various sepsis (infectivity (bacteria and viruses) and immunity) caused by inflammatory reaction.

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 multidirectional differentiation potential and immune regulation function from children's foreskin culture. Background technique [0002] Prior to this, staged results have been achieved in the treatment of sepsis with stem cells at home and abroad, and the safety of MSCs in the treatment of sepsis has been preliminarily confirmed through human clinical research, and it has shown a certain curative effect. Studies have found that MSCs also play an active role in the treatment of virus-related diseases such as human immunodeficiency virus (HIV) immune abnormalities, hepatitis B virus (HBV) chronic hepatitis, and influenza virus acute lung injury (ALI). [0003] Mesenchymal stem cells (MSCs) are multipotent stromal cells that exist in various tissues and can differentiate into different types of cells, including osteoblas...

Claims

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Application Information

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