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Preparation process for insulin-secreting cells and special medium composition used therein

A technology of insulin secretion and culture medium, applied in animal cells, vertebrate cells, artificial cell constructs, etc., to achieve the effect of easy operation and high efficiency

Inactive Publication Date: 2013-07-03
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in practical application, it faces ethical challenges or limitations of materials

Method used

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  • Preparation process for insulin-secreting cells and special medium composition used therein
  • Preparation process for insulin-secreting cells and special medium composition used therein
  • Preparation process for insulin-secreting cells and special medium composition used therein

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Embodiment 1

[0055] Embodiment 1, the preparation of culture medium

[0056] DMEM / F12 medium (also known as DF12 medium) and high-glucose DMEM medium were purchased from GIBCO Company. Fetal calf serum (FCS) was purchased from GIBCO. Niacinamide was purchased from Sigma Company. Glutamine: GIBCO, catalog number 25030-081. Retinoic acid was purchased from Sigma Company, the product catalog number is R2625. Human Activin A (Activin A): British Pepro Tech Company, the product catalog number is 120-14. EGF (epidermal growth factor, epidermal growth factor): Pepro Tech Company, the product catalog number is 100-15. bFGF (basic fibroblast growth factor, basic fibroblast growth factor): Sigma Company, the product catalog number is F0291. exendin-4: Sigma Company, the product catalog number is E7144. B27: GIBCO Corporation, catalog number 17504-044. N2: GIBCO Corporation, catalog number 17502-048. Non-essential amino acids: GIBCO, catalog number 11140.

[0057] The medium is composed of h...

Embodiment 2

[0060] Example 2, Sequential induction culture of human insulin-secreting cells (application of medium combination A)

[0061] 1. Preparation of the third generation of mesenchymal stem cells

[0062] 1. Aseptically collect the isolated umbilical cord (the source is human), cut the umbilical cord about 10 cm long in a biological safety cabinet, and clean the blood stain with cell washing solution; remove the amniotic membrane, 2 umbilical veins and 1 umbilical artery with a scalpel , to obtain Walton's jelly (embryonic mucus-like connective tissue between arteries and veins).

[0063] 2. Cut Walton’s jelly into minced meat, transfer to a centrifuge tube, digest with 0.75g / LII type collagenase for 2-3 hours, filter through a 100-mesh sieve and collect the filtrate, and centrifuge at 1200rpm at room temperature for 10 minutes. Collect cells.

[0064] 3. Place the cells in step 2 at 37°C, 50ml / L CO 2 , in an incubator with a relative air humidity of 95%, and when the cells rea...

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Abstract

The invention discloses a medium composition for inducing mesenchymal stem cells into insulin-secreting cells. The medium composition comprises a medium A, a medium B and a medium C. The medium A comprises the following solutes: a fetal calf serum with a concentration of 4.75 to 5.25 ml / L and activin A with a concentration of 4.75 to 5.25ng / mL. The medium B comprises the following solutes: retinoic acid with a concentration of 0.95 * 10<-5> to 1.05 * 10<-5> mol / L, EGF with a concentration of 19 to 21ng / ml, bFGF with a concentration of 19 to 21ng / ml, glutamine with a concentration of 1.9 to 2.1 mmol / L, 0.95 to 1.05% of non-essential amino acids and 1.9 to 2.1% of B27. The medium C comprises the following solutes: a fetal bovine serum with a concentration of 4.5 to 5.5 ml / L, exendin-4 with a concentration of 19 to 21 ng / ml, activin A with a concentration of 9.5 to 10.5 ng / ml, nicotinamide with a concentration of 9.5 to 10.5 mmol / L, 1.9 to 2.1% of B27 and 0.95 to 1.05% of N2. An induction culture method provided by the invention is applicable to a variety of tissue-derived mesenchymal stem cells and has the advantages of no introduction of viruses, easy operation and high efficiency.

Description

technical field [0001] The invention relates to a method for preparing insulin-secreting cells and a special medium composition thereof. Background technique [0002] "Diabetes" is a disease in which glucose in the blood tends to accumulate excessively. The alias given to it abroad is "Silent Killer", especially "adult type diabetes". , that is, there is one "diabetic" patient in ten people. Once suffering from "diabetes", life expectancy will be reduced by as much as ten years, and possible complications can spread throughout the body. [0003] Over the years, the medical profession has been devoted to the study of diabetes. This is because once suffering from "diabetes", the human body's troubles will follow. Because the immune function is weakened, it is easy to be infected with various infectious diseases caused by colds, pneumonia, and tuberculosis, and it is not easy to cure. And selectively destroy cells, phagocytes. The defense function against cancer cells will...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/074
Inventor 赵春华李晶
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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