Kit for obtaining lung stem cells through separation and method

A stem cell and kit technology, applied in the field of lung stem cell isolation and transplantation, can solve the problems of difficulty in separating and obtaining lung stem cells and need to be improved, and achieve the effects of easy promotion, easy mass production and utilization, and no bacterial and mycoplasma contamination.

Active Publication Date: 2017-10-20
REGEND THERAPEUTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to technical limitations for a long time, it has been difficult to isolate and obtain lung stem cells that can be expanded in large quantities from adult tissues and organs
[0004] Therefore, the current methods for the isolation and acquisition of lung stem cells still need to be improved

Method used

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  • Kit for obtaining lung stem cells through separation and method
  • Kit for obtaining lung stem cells through separation and method
  • Kit for obtaining lung stem cells through separation and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Lung stem cells are isolated from a biological sample containing lung stem cells as follows:

[0109] 1. Sample: The sample comes from a patient with pulmonary fibrosis, and an informed consent is required.

[0110] Sample collection process: Arrange the patient into the endoscopic examination room for distal tracheal examination. After clear observation of the distal bronchus (approximately at level 3-8) through the endoscope, biopsy in the bronchoscope Insert a disposable cell brush into the hole, select the normal part, keep away from the lesion as much as possible, extend the protective brush out of the end of the bronchoscope, then push out the inner cannula, push off the protective plug at the end of the brush, and then extend the inner cannula out of the end of the outer cannula Push out the brush, brush gently on the tracheal wall for one to five times, then withdraw the cell brush into the protective tube, then take the bronchoscope and the cell brush out of th...

Embodiment 2

[0137] According to the method of Example 1, lung stem cells were isolated from a biological sample containing lung stem cells, wherein the only difference was:

[0138] Tissue Digestion contains:

[0139] 99% by volume of PBS;

[0140] 1U / ml DNase;

[0141] 0.15mg / ml type I collagenase;

[0142] 0.15mg / ml type IV collagenase;

[0143] 0.07mg / ml dispase;

[0144] 0.3 mg / ml Protease XIV;

[0145] 2mg / ml streptolysin E; and

[0146] 0.35mg / ml trypsin,

[0147] Lung Stem Cell Isolation Medium contains:

[0148] 90% by volume of RPMI1640 medium;

[0149] 10% by volume of fetal bovine serum;

[0150] 2mmol / LL-Glutamine;

[0151] 0.1mmol / Lβ-mercaptoethanol;

[0152] 500U / ml leukemia inhibitory factor; and

[0153] 3.5 ng / ml EGF growth factor.

[0154] As a result, lung stem cells were successfully obtained, and the isolated lung stem cells had good cell activity, high purity, and no bacteria and mycoplasma contamination.

Embodiment 3

[0156] The lung stem cells obtained in Example 1 were continuously amplified, and then the lentivirus expressing the green fluorescent protein marker was added to the cell culture for 24 hours, and the virus was washed away with phosphate buffer and the cells were continued to be cultured. After 72 hours, the cells could be It was observed that almost all cells expressed GFP, indicating that the cells had been successfully labeled with GFP. Then, the lung stem cells expressing green fluorescent protein were resuspended in 30-70ul of phosphate buffered saline, and then infused into lung-injured immunodeficient mice through the airway (8 days before cell transplantation, the Bray Mycin was injected into the respiratory tract of NOD-SCID mice to cause lung damage in mice, and immunodeficiency mice with lung damage were obtained. Among them, in the lungs of NOD-SCID mice (purchased from Shanghai Slack Experimental Animal Co., Ltd.), half One month later, the mouse lungs were taken...

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Abstract

The invention discloses a kit for obtaining lung stem cells through separation and a method. The lung stem cell separation kit comprises tissue digestive juice, embryo fibroblasts subjected to radiation treatment and a lung stem cell separation culture solution. With the adoption of the kit, a large quantity of amplified lung stem cells can be obtained effectively, and the obtained lung stem cells are good in activity, high in purity and free of bacterium and mycoplasma contamination.

Description

technical field [0001] The invention relates to the technical field of separation and transplantation of lung stem cells, in particular to a kit and method for separating and obtaining lung stem cells. Background technique [0002] With the increase of air pollution and other factors such as viral infection, some lung diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease have more and more affected human health and even survival. For the treatment of these diseases, the most effective treatment at present is lung transplantation. However, due to practical reasons such as limited available donors and immune rejection between allogeneic donors, the number of patients who can receive lung transplantation is very rare. Apart from lung transplantation, there is currently no effective treatment. [0003] Lung stem cells belong to adult stem cells and are widely distributed in the body. They have organ targeting in cell transplantation therapy, strong rege...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073C12N13/00
CPCC12N5/0689C12N13/00C12N2501/11C12N2500/32C12N2500/44C12N2501/235C12N5/0656A61K35/42A61P11/00C12N2502/1323C12N2509/00Y02A50/30
Inventor 张婷
Owner REGEND THERAPEUTICS CO LTD
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