Selective medium of liver stem cells, method for selectively separating and amplifying liver stem cells as well as medicinal composition for treating diabetes

A technology for selecting medium and hepatic stem cells, which is applied in the fields of selective medium for hepatic stem cells, selective separation and expansion of hepatic stem cells and pharmaceutical compositions for treating diabetes, and can solve the problem of poor safety of hepatic stem cells and low purity of hepatic stem cells , easy differentiation and degeneration, etc., to overcome diabetes-dependent insulin therapy, strong selectivity, high-efficiency separation and large-scale amplification

Inactive Publication Date: 2010-10-06
北京弘润天源基因生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the complicated operation and high cost of the existing methods for separating and expanding hepatic stem cells, the isolated hepatic stem cells have low purity, low amplification efficiency, easy differentiation and denaturation, and the cultivation methods used in the separation and expansion process The base contains many harmful drugs to the human body, which makes the isolated and expanded hepatic stem cells less safe when prepared into a pharmaceutical composition. A selective medium for hepatic stem cells is provided, and the isolation and expansion of the medium is used. The method of liver stem cells is simple to operate, low in cost, high in separation purity, high in amplification efficiency and stable in properties of passaged cells

Method used

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  • Selective medium of liver stem cells, method for selectively separating and amplifying liver stem cells as well as medicinal composition for treating diabetes
  • Selective medium of liver stem cells, method for selectively separating and amplifying liver stem cells as well as medicinal composition for treating diabetes
  • Selective medium of liver stem cells, method for selectively separating and amplifying liver stem cells as well as medicinal composition for treating diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This example is used to illustrate the method of isolating hepatic stem cells from the isolated tissue umbilical cord using the selection medium of the present invention.

[0038] Umbilical cords were collected from 25-year-old healthy pregnant women after delivery. An informed consent was signed with the pregnant woman and the hospital to collect her umbilical cord after delivery.

[0039] The isolated tissue umbilical cord was cut into small pieces of about 1 mm3 with ophthalmic scissors. Gained shredded tissue fragments were digested with 0.25% trypsin solution at a weight-to-volume ratio of 1 g: 0.5 mL for 5 minutes, and then added fetal bovine serum (U.S. Hyclone Company) that was one-fifth of the trypsin solution volume to terminate the digestion. Centrifuge at 1000rpm for 5 minutes to collect the digested tissue fragments, pour off the supernatant of the trypsin solution, and then add 0.01% DNAse (DNase, Shanghai Sangong) in HBSS (Hans buffer, Shanghai Sangong) ...

Embodiment 2

[0044] This example is used to illustrate the method of isolating hepatic stem cells from the isolated tissue umbilical cord blood using the selection medium of the present invention.

[0045] Umbilical cord blood was collected from 25-year-old healthy pregnant women after delivery. An informed consent was signed with the pregnant woman and the hospital to collect her umbilical cord blood after delivery.

[0046] Dilute the isolated tissue umbilical cord blood with an equal volume of 1×PBS to obtain a umbilical cord blood dilution. The volume ratio of the umbilical cord blood dilution: 1.077 kg / m3 Ficoll-Hypaque is 2: 1 was added to a 50ml centrifuge tube, and density gradient centrifugation was carried out at 1500rpm and 25 degrees for 10 minutes. After centrifugation, the cells in the middle layer were collected and centrifuged with 1×PBS 1000rpm for 10 minutes. The cells were washed three times, and complete medium was added, containing 10 % fetal bovine serum, 30 ng / mL hu...

Embodiment 3

[0049] This example is used to illustrate the method of using the selection medium of the present invention to subculture, expand and culture seeded hepatic stem cells in vitro, and the method for cryopreserving the obtained seeded hepatic stem cells.

[0050] Take the hepatic stem cells obtained in Example 1 as the first generation. When the hepatic stem cells adhere to the wall and grow to reach 90% of the bottom surface of the culture bottle, the cells are digested with trypsin, centrifuged at 800rpm for 5 minutes, and collected in the epidermis-containing The growth factor is 50ng / ml, the basic epidermal growth factor is 40ng / ml, and subcultured in DMEM / F12 (1:1) medium containing 10% fetal bovine serum. Similarly, when the adherent growth of the hepatic stem cells reached 90% of the bottom surface of the culture bottle, the above subculture process was repeated. When the hepatic stem cells are expanded to the 4th passage, a bottle of hepatic stem cells is digested with tr...

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Abstract

The invention provides a selective medium of liver stem cells. The selective medium comprises a liquid-based medium, and the volume of the liquid-based medium is used as a standard. The selective medium also comprises 1-20vol% of fetal calf serum, 5-100ng / mL of human epidermal growth factors and 5-100ng / mL of basic fibroblast growth factors. The invention also provides a method for selectively separating and amplifying liver stem cells from in vitro tissues by using the selective medium. The invention also provides a medicinal composition for treating diabetes, which contains the liver stem cells prepared by using the method. The method using the selective medium can separate and amplify liver stem cells in high efficiency and in a large amount, and the liver stem cells subcultured and amplified through 10 generations still maintain the same properties with the primary liver stem cells. The medicinal composition for treating diabetes can restore damaged islet beta cells of a person with diabetes, and can eliminate insulin resistance.

Description

technical field [0001] The present invention relates to a selection medium for stem cells, a method for selectively isolating and expanding stem cells using the medium, and a pharmaceutical composition for treating diabetes comprising the stem cells obtained by the aforementioned method. More specifically, the present invention relates to a selective medium for hepatic stem cells, a method for selectively isolating and expanding hepatic stem cells using the medium, and a pharmaceutical composition for treating diabetes comprising the hepatic stem cells obtained by the aforementioned method. Background technique [0002] The existing methods for isolating and expanding hepatic stem cells are complex and costly, and the isolated hepatic stem cells have low purity, low expansion efficiency, and easy differentiation and denaturation, and the culture medium used in the isolation and expansion process contains many Harmful drugs make the isolated and expanded hepatic stem cells le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074A61K35/44A61K35/28A61K9/08A61P3/10A61K35/51
Inventor 沈丽黄启明郝丽敏王亚军马艳玲
Owner 北京弘润天源基因生物技术有限公司
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