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Culture medium for separating and inducing human adipose stem cells into pancreatic beta cells and use method of culture medium

A technology for human adipose stem cells and culture medium, which is applied in the field of culture medium for separating and inducing human adipose stem cells into islet beta cells, can solve the problems of limitation and lack of cell sources, and achieves fewer induction steps, reduced induction time, and safety. high sex effect

Inactive Publication Date: 2015-08-26
GUIZHOU BEIKE FACTORR BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this therapy is limited by the scarcity of cell sources

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 of the present invention: a culture medium for separating and inducing human adipose stem cells into pancreatic beta cells, including DMEM medium, wherein activin is 3mmol / L, trans-retinoic acid is 1umol / L, and EGF is 100ng / ml, bFGF is 10ng / ml, glucagon-like peptide 1 is 10ng / ml, nicotinamide is 10mmol / L; the total volume is 1000ml; the above ingredients are mixed and filtered to sterilize to make the finished product.

Embodiment 2

[0031] Embodiment 2 of the present invention: a method for inducing human adipose-derived mesenchymal stem cells into insulin-secreting cells,

[0032] 1) Receiving adipose tissue: Wipe the outer wall of the container containing the adipose tissue collection bottle with 75% alcohol.

[0033] 2) Divide adipose tissue: Use a 10ml pipette to draw the lower layer of red liquid into the fat collection bottle and discard it. The remaining upper layer of fat is mixed and then divided into packages. Each T175 culture bottle is divided into 50ml of adipose tissue.

[0034] 3) Wash adipose tissue: remove red blood cells; add 100ml of normal saline injection to the T175 culture bottle, tighten the cap, shake vigorously (or vibrate with an oscillator) for 3 minutes to fully wash the adipose tissue, then stand still for 2 minutes to separate the different phases , absorb the lower aqueous phase; repeat the above operation three times until the lower layer is relatively clear.

[0035] 4) ...

Embodiment 3

[0045] Example 3 of the present invention: identification of islet cells after induction

[0046] 1. Identification of induced insulin-secreting cells by dithizone staining

[0047] Dissolve 50mg of DTZ dithizone in 5ml of dimethyl sulfoxide, mix well, filter and sterilize, set aside. DTZ staining was carried out after 7 days of cell induction. When staining, take 1ml of the induced cells and add them to a 12-well culture plate, add 10ul of DTZ staining solution, incubate in a 37°C incubator for 20 minutes, absorb a few cells in the stained wells, wash with PBS for 2 Second, observe under the microscope. Insulin-secreting cells are brown.

[0048] 2. Detection of islet-positive cells by flow cytometry: Take the induced 7-day cells, wash with PBS and centrifuge for 3 times, discard the supernatant, resuspend the cells with 100ulPBS, add 10ulFITC-labeled anti-insulin antibody, incubate at room temperature for 30 minutes in the dark, flow The positive cell rate of cytometer an...

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Abstract

The invention provides a culture medium for separating and inducing human adipose stem cells into pancreatic beta cells and a use method of the culture medium. The optimal culture medium is designed; gene transfection is not required, so that the risk of gene modification and cancers is avoided, few induction steps are used, the use is simple, and the induction time is short and is shortened by 30% in comparison with the prior art; the culture medium is safe, nontoxic and high in success rate of induction. Moreover, after autogenous adipose mesenchymal stem cells are induced and differentiated into insulin-secreting cells, rejection and ethical problems are avoided after autotransplantation, and the safety is high, so that the clinical application prospect is wide. The culture medium is simple, feasible, low in cost and good in use effect.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to a culture medium for separating and inducing human adipose stem cells into pancreatic islet β cells and a method for using the same. Background technique [0002] In the past ten years, the incidence of diabetes has been increasing day by day, and it is more and more seriously endangering human health. However, the traditional treatment methods failed to effectively and stably control blood sugar, so researchers tried to improve the treatment of diabetes and turned their attention to islet cell transplantation. However, this therapy is limited by the scarcity of cell sources. Facing the huge gap of cell sources, the most promising is to induce and differentiate a large number of islet-like cells from adipose-derived mesenchymal stem cells for transplantation. It has been reported that a variety of adult stem cells can be induced to differentiate into islet β-like ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 边曙光
Owner GUIZHOU BEIKE FACTORR BIOTECH CO LTD
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