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In-vitro induced pancreas-islet-like structure forming method

A pancreatic islet, inductive technology, applied in biochemical equipment and methods, artificial cell constructs, microorganisms, etc., can solve the problem of different islet size and efficiency, and achieve the formation of islet-like structures conducive to the formation of islet-like structures. , uniform size effect

Inactive Publication Date: 2012-06-27
CHINA JAPAN FRIENDSHIP HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing reports have shown that using this method to induce the formation of islet-like structures varies from 2 days to 14 days (Endocr J. 2004, 51(3):381-6; Am J Physiol Endocrinol Metab. 2005, 288(3 ):E502-9; Exp Clin Endocrinol Diabetes. 1995;103 Suppl 2:118-22.), the size and efficiency of the formed islets vary

Method used

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  • In-vitro induced pancreas-islet-like structure forming method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Induction of islet endocrine cells derived from pancreatic adult stem cells to induce the formation of islet-like structures

[0031]Adult pancreatic stem cells (the source and differentiation steps of which can be obtained from the content disclosed in the prior art, and can also be referred to Chinese Journal of Diabetes, 2007, 15(3):171-175) were first inoculated in T 75 In the culture flask, adhere to the wall and culture for 30min. Then collect non-adherent cells, suspend culture at a density of 40,000 cells / ml in a 50-ml sterile siliconized centrifuge tube (Corning, USA); the medium uses M199 medium containing 20% ​​heat-inactivated fetal bovine serum, and Contains 4.5g / L of glucose, 1mmol / L of Ca 2＋ and 0.01 mmol / L adenosine triphosphate, laminin (0.1 μg / ml), type IV collagen (0.1 μg / ml) and fibronectin (0.1 μg / ml) (the above reagents were purchased from Sigma, Germany). at 50%O 2 , 5%CO 2 , in a 37°C environment, the adult pancreatic stem cells wer...

Embodiment 2

[0032] Example 2 Inducing iPS cell-derived islet endocrine cells to induce the formation of islet-like structures

[0033] For the sources of iPS cells and the induction and differentiation steps, please refer to the reference (Cell Res. 2009, 19(4):429-438). The islet endocrine cells obtained after iPS cells were induced and differentiated were inoculated in T 75 In culture flasks, adhere to the wall for 3 h. Then collect non-adherent cells, suspend culture at a density of 40,000 cells / ml in a poly-HEMA-coated 10cm dish; the medium uses 1640 medium containing 20% ​​heat-inactivated fetal bovine serum, and contains 4.5g / L of glucose, 1mmol / L of Ca 2＋ and 0.01 mmol / L adenosine triphosphate, laminin (20 μg / ml), type IV collagen (20 μg / ml) and fibronectin (20 μg / ml) (the above reagents were purchased from Sigma, Germany). ; at 50%O 2 , 5%CO 2 , in an environment of 37°C, the cells were cultured in this suspension culture condition for 24 hours. Under a stereo microscope, a...

Embodiment 3

[0034] Example 3 Inducing endocrine cell lines derived from islets to induce the formation of islet-like structures

[0035] The rat insulinoma cell line-INS-1 cells preserved in our laboratory were suspended and cultured at a density of 40,000 cells / ml in non-treated culture flasks on the surface; the medium used contained 20% heat-killed embryo The DMEM medium of bovine serum contains 4.5g / L glucose, 1mmol / L Ca 2＋ and 0.01 mmol / L adenosine triphosphate, laminin (2 μg / ml), type IV collagen (5 μg / ml) and fibronectin (3 μg / ml) (the above reagents were purchased from Sigma, Germany). ; at 50%O 2 , 5%CO 2 , in an environment of 37°C, the cells were cultured in this suspension culture condition for 6 hours. Under a stereomicroscope, it was observed that about 90% of the cells formed islet-like structures, and their sizes were relatively uniform, mostly with a diameter of about 150-200 μm, which was very similar to the islet-like structures formed in Examples 1 and 2.

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Abstract

The invention discloses an in-vitro induced pancreas-islet-like structure forming method. Pancreas-islet endocrine cells prepared by committed induction of adult stem cells or induced pluripotent stem cell (iPS cells) or pancreas-islet endocrine cells from a pancreas-islet source are subjected to suspension culture in a culture medium which is added with similar components of natural i pancreas-islet extracellular matrix in a high-oxygen culture environment with 50% of O2, so that the in-vitro induced pancreas-islet-like structure is formed quickly and efficiently. The method has the advantages of high speed (4-24 hours) and high efficiency (the formation rate of the pancreas-islet-like structure is 90%), and the pancreas-islet-like structures formed by the method are uniform in size (like the natural pancreas-islet) and fine in function (the insulin secretion function thereof is superior to that of the non-islet-like structure). In addition, the in-vitro induced pancreas-islet-like structure forming method is a key technical method for preparing the pancreas islet according to the dry cell technology.

Description

technical field [0001] The present invention relates to a method for inducing the formation of islet-like structures in vitro, in particular to an islet endocrine cell prepared from in vitro induced adult stem cells or induced pluripotent stem cells (induced pluripotent stem cells, iPS) through a directional induction differentiation technique, and pancreatic islets A method of inducing the formation of islet-like structures from endocrine cell lines derived from pancreatic islets. Background technique [0002] The morbidity and mortality of diabetes are increasing year by year, and it has become one of the major diseases that endanger human health. The current use of insulin therapy can improve the patient's glucose metabolism disorder to a certain extent, but it cannot effectively prevent or reverse the microangiopathy and its complications caused by diabetes. Pancreatic islet transplantation is an effective method for treating insulin-dependent diabetes mellitus, and exp...

Claims

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Application Information

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IPC IPC(8): C12N5/02C12N5/071
Inventor 娄晋宁蔡寒青许世清张文健
Owner CHINA JAPAN FRIENDSHIP HOSPITAL
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