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Method for preparing pancreatic beta cells and application of pancreatic beta cells

A cell and pancreas technology, applied in the field of medicine and biology, can solve problems such as affecting the activity and function of encapsulated cells, lack of immune isolation function, fibrotic encapsulation, etc., and achieve the effects of maintaining expression, increasing success rate, and maintaining karyotype stability.

Pending Publication Date: 2021-06-18
BETACURE MEDICAL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many of these materials will have severe fibrosis and no immune isolation function in the transplanted body, which will seriously affect the activity and function of the wrapped cells.

Method used

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  • Method for preparing pancreatic beta cells and application of pancreatic beta cells
  • Method for preparing pancreatic beta cells and application of pancreatic beta cells
  • Method for preparing pancreatic beta cells and application of pancreatic beta cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1, planar culture, in vitro 3D culture and domestication of hPSCs cells

[0095] 1. Planar culture of hPSCs cells

[0096] Required reagent preparation method:

[0097] mTeSR1 pluripotent stem cell medium (STEMCELL, 85850);

[0098] Accutase digestive enzyme (STEMCELL, 07920);

[0099] Rho kinase inhibitor Y27632 (Abcam, ab120129): use DMSO to make a 10mM storage solution, and use it at a final concentration of 10μM, that is, use it at 1000X;

[0100] mTeSR1+Y27632 medium: 15mL of mTeSR1 needs to be added with 15μL of 10mM Y27632, mix well, and store at 4°C when not in use. It is best to use up within two weeks after preparation.

[0101] hPSCs cells (clinical-grade human embryonic stem cells or human induced pluripotent stem cells, sourced from Beijing Stem Cell Bank).

[0102] 1. Recovery and culture of hPSCs

[0103] 1.1. Matrigel-coated culture plate: take out the 6-well plate, add 0.5-2mL Matrigel (Matrigel, Corning, 354277) to each well, shake the 6-w...

Embodiment 2

[0125] Example 2. Differentiation of hPSCs cells into pancreatic beta cells

[0126] The name and formula of the medium used in the whole differentiation process are as follows,

[0127] S1 medium: MCDB131 basal medium (Thermofisher, 10372019) + 2-20mM glucose (Glucose, Sigma, G7528-250G) + 1-5g / L sodium bicarbonate (NaHCO 3 , Sigma, S5761-500G)+0.5-5% recombinant human serum albumin (Human serum albumin, HSA, He Yuan Bio, HYC002M01)+1:10000-1:100000 insulin-transferrin-selenium-ethanolamine (InsulinTransferrinSeleniumEthanolamine, ITS-X, Thermofisher, 51500056)+1-5mM glutamine substitute (GlutaMAX TM Supplement, Glutamax, Thermofisher, 35050061) + 0.1-0.5mM Vitamin C (L-Ascorbic acid, Vitamin C, Sigma, A4544-25G) + 1% Penicillin-Streptomycin (Penicillin-Streptomycin, Pen / Strep, Thermofisher, 15140122 ).

[0128] S2 medium: MCDB131+2-20mM Glucose+1-3g / L NaHCO 3 +0.5-5%HSA+ITS-X 1:10000-1:100000+1-5mM Glutamax+0.1-0.5mM Vitamin C+1%Pen / Strep.

[0129] S3 / S4 medium: MCDB13...

Embodiment 3

[0214] Example 3. Differentiated pancreatic beta cells are packaged with improved APA microencapsulation

[0215] 1. Preparation of improved ultrapure sodium alginate solution

[0216] The dissolved sodium alginate is filtered through 0.8μm, 0.45μm and 0.22μm PES sterile filtration devices in sequence; the viscosity of sodium alginate is 50-200cP; the prepared sterile sodium alginate can be stored in a 4-degree refrigerator 1-4 weeks.

[0217] 2. APA microencapsulated differentiated beta cells - microfluidic method or high voltage electrostatic method

[0218] 2.1 Microfluidic method (microfluidic plate purchased by Suzhou Wenhao Microfluidic Technology Co., Ltd., model WH-SP-01):

[0219] Mix 1%-3% sodium alginate solution with beta cells, 1mL sodium alginate corresponds to 1-10*10^6 cells;

[0220] After mixing, add one of the pipelines, and add 0.1-2g / L calcium chloride solution to the other pipeline;

[0221] Cross-linked into a gel after passing through the 1-10cm col...

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PUM

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Abstract

The invention relates to a method for preparing differentiated pancreatic beta cells and application of the differentiated pancreatic beta cells. The method comprises the following steps: (1) performing three-dimensional suspension domestication culture on pluripotent stem cells; and (2) inducing the domesticated cells to differentiate into pancreatic beta cells. The invention further relates to a method for preparing a sodium alginate-polylysine-sodium alginate (APA) microencapsulated artificial pancreas islet from the differentiated pancreas beta cells. The method comprises the step of preparing the microencapsulated artificial pancreas islet by using a sodium alginate solution. The invention also relates to application of the pancreatic beta cells and the artificial pancreas islet.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to a method for preparing pancreatic beta cells and an application thereof. Background technique [0002] Diabetes is a disease in which the dysfunction or failure of pancreatic islets leads to the disorder of glucose metabolism in the body. According to the latest statistics from the International Diabetes Federation (IDF), in 2019, more than 463 million people worldwide suffered from diabetes, and by 2045, 700 million people will suffer from diabetes; China ranks first in the world with 116 million diabetic patients , and is expected to reach 147 million people by 2045. In 2019, about 4.2 million people (20-79 years old) died of diabetes or its complications around the world, and one person died of diabetes every 6 seconds, accounting for about 11.3% of all deaths worldwide. [0003] Type 1 diabetes accounts for 5-10% of all diabetes and is caused by a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0735C12N5/10A61K35/39A61P3/10
CPCC12N5/0676C12N5/0677A61K35/39A61P3/10C12N2506/02C12N2506/45C12N2513/00C12N2500/25C12N2500/46C12N2500/32C12N2500/60C12N2500/34C12N2501/998C12N2501/91C12N2501/415C12N2501/16C12N2501/117C12N2501/727C12N2501/999C12N2501/11C12N2500/22C12N2533/32C12N2501/115C12N2501/165C12N2533/74Y02A50/30
Inventor 杨子江王浩周围苏茵
Owner BETACURE MEDICAL INC
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