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Combined culture medium for islet culture and preparation method thereof

A culture medium and islet technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve the problems of islet cell damage and not being used clinically, achieve low immunogenicity, protect survival and function, reduce effect of risk

Active Publication Date: 2020-12-11
THE FIRST HOSPITAL OF CHINA MEDICIAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Basic research shows that CMRL1066+10%FBS is more suitable for islet culture in vitro, but it is not used clinically due to animal origin
Cytokines are contained in human serum, but at the same time there are cytokines that have an immune reaction with islet cells, which can easily lead to damage to islet cells

Method used

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  • Combined culture medium for islet culture and preparation method thereof
  • Combined culture medium for islet culture and preparation method thereof
  • Combined culture medium for islet culture and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation of the combined medium for islet culture of the present invention

[0029] The present invention provides a novel combined medium for culturing islets, including the following components: CMRL1066 medium, 0.625 wt% BSA and amniotic membrane extract. Wherein, the concentration of amniotic membrane extract protein in the combined medium is 0.1-1.0 mg / mL.

[0030] The preparation method of the novel combined medium for culturing islets of the present invention comprises the following steps:

[0031] 1. Wash the amniotic membrane with a PBS solution containing 1000 IU / ml penicillin and 0.1 mg / ml streptomycin until there is no blood clot;

[0032] 2. Cut the washed amniotic membrane into pieces, and immerse it in liquid nitrogen for manual grinding until the amniotic membrane is ground into fine particles of amniotic membrane;

[0033] 3. Weigh the amniotic membrane fine particles, add the basal medium CMRL1066 at a weight-to-volume ratio of 1:2 (1:2...

Embodiment 2

[0038] Example 2. Effects of different concentrations of amniotic membrane extract on the activity of primary pancreatic islet cells in C57 / BL6 mice

[0039] Take an ultra-low adhesion 6-well plate, and group the normally extracted mouse primary islets according to 75 islets per well, and divide them into a control group and an experimental group. The composition of the combined medium is CMRL1066+0.625 wt% BSA+amniotic membrane extract, wherein, in the combined medium, the protein concentration of the amniotic membrane extract is 0.5 mg / mL, and 2 mL of medium is added to each well, placed at 37 ° C, 5% CO 2 cultured in a cell incubator for 48 h. After the incubation, add AO / EB to the culture wells containing islets at the ratio of adding 20 μl of AO / EB 1:1 mixture per ml of culture solution, and perform fluorescence color development within 3-5 minutes after mixing. The results are as follows: Figure 1-2 shown, medium green is for live cells, orange is for dead cells, figu...

Embodiment 3

[0040] Example 3. Effects of different concentrations of amniotic membrane extract on the function of primary islet cells in C57 / BL6 mice

[0041]Take the ultra-low adhesion 6-well plate, and divide the normal extracted mouse primary islets into groups according to 75 islets per well, and divide them into a control group and an experimental group. The composition of the combined medium is CMRL1066+0.625%BSA+amniotic membrane extract, wherein, in the combined medium, the protein concentration of the amniotic membrane extract is 0.1-1.5mg / mL, and each well is added with 2mL of medium, placed at 37°C, 5% CO 2 in a cell incubator for 48 h.

[0042] After the culture, 10 islets were randomly selected from each group for ELISA test to detect the insulin secreted by the islets. The selected islets were incubated in Krebs-Ringer-bicarbonate buffer (KRBB) solution containing 2.8mM glucose for 30min, and the supernatant was discarded; then the islets were placed in 1mL Krebs-Ringer-bi...

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Abstract

The invention discloses a combined culture medium for islet culture and a preparation method thereof. The combined culture medium for the islet culture is prepared from the following raw materials: aCMRL1066 culture medium, BSA, and an amnion extract. The novel combined culture medium disclosed by the invention is prepared by the following steps: performing liquid nitrogen grinding on human amnion; carrying out ultrasonic homogenization; carrying out gradient centrifugation; and carrying out filtration and sterilization so as to obtain the culture medium with the amnion extract; and the prepared novel combined culture medium containing the amnion extract is capable of effectively protecting in-vitro survival and functions of pancreas islet.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combined culture medium for islet culture and a preparation method thereof. Background technique [0002] Diabetes is a syndrome of glucose, protein and lipid metabolism disorders in which insulin metabolism is defective due to the destruction of autologous islet cells, resulting in insufficient insulin production or receptor cell resistance to insulin. It is mainly divided into type 1 diabetes and type 2 diabetes. diabetes. Since the release of the Edmonton Protocol, islet transplantation has been regarded as one of the effective methods for the treatment of type 1 diabetes mellitus and some type 2 diabetes mellitus because it is less invasive than adult organ transplantation and can achieve reliable transplantation results. [0003] At present, in clinical islet transplantation, in order to further purify islets, detect islet quality, transport and transfer islets, and induce im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0677C12N2500/84C12N2501/998
Inventor 杨召铭张佳林郭庭维林建贞李峰张城硕
Owner THE FIRST HOSPITAL OF CHINA MEDICIAL UNIV
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