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A kind of neonatal porcine islet cell culture method

A culture method and islet cell technology, applied in the directions of cell culture active agents, pancreatic cells, biochemical equipment and methods, etc., can solve the problems of increasing production costs, large number of newborn pigs, and high labor costs, reducing labor costs and saving Reagent consumables, the effect of reducing production costs

Active Publication Date: 2022-02-15
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has the following disadvantages: ①It requires a large number of newborn pigs: Newborn pig islet cell clusters cannot be expanded when cultured in vitro, and the number of cells will decrease greatly over time. If 500,000IEQ pig islet cells are transplanted per patient According to calculations, about 15,000IEQ islet cells are obtained from each newborn pig, and at least 34 newborn pigs are needed; ②The operation is cumbersome and the workload is heavy: this method needs to change the medium every other day, and a 15cm non-adherent cell culture dish can only culture 10,000IEQ For pancreatic islet cells, since the number of cells is gradually decreasing, the initial number of cells needs to be about 2,000,000 IEQ, and the medium needs to be changed for 200 dishes of cells every other day. The labor cost is too high; ③The cost of reagent consumables is high and the risk of contamination is high: clinical grade reagents The cost of consumables is generally higher than the cost of scientific research, and the safety detection of microbial contamination is more stringent. Frequent liquid changes will increase such risks and increase production costs

Method used

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  • A kind of neonatal porcine islet cell culture method
  • A kind of neonatal porcine islet cell culture method
  • A kind of neonatal porcine islet cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, newborn pig islet cell culture experiment of the present invention

[0057] The present invention carries out "1+N+1" purification culture for the isolated newborn pig islet cells: (1) the separated and extracted newborn pig islet cells are placed in ordinary medium (Ham's F10 medium with 10% pig serum added), 37°C, 5% CO 2 , cultivated for 1 day; collected the cells, discarded the supernatant; (2) adding medium: containing 30% hypoxic porcine mesenchymal stem cell conditioned medium, 5% human serum albumin, 10% porcine Serum, 20mM taurine, 4°C, cultivated for 3 days, 5 days, 7 days, 9 days; collected cells, discarded supernatant; (3) adding culture medium: adding 10% porcine serum to EGM-2 medium, 30% Hypoxic Porcine Mesenchymal Stem Cell Conditioned Medium, 37°C, 5% CO 2 Incubate for 24 hours. Count and do relevant testing.

Embodiment 2

[0059] The present invention carries out "3+N+1" purification culture for isolated newborn pig islet cells: (1) the newborn pig islet cells after separation and extraction are placed in ordinary culture medium (Ham's F10 medium with 10% pig serum added), 37°C, 5% CO 2 , cultivated for 3 days; collected the cells, discarded the supernatant; (2) adding medium: containing 30% hypoxic porcine mesenchymal stem cell conditioned medium, 5% human serum albumin, 10% porcine Serum, 20mM taurine, 4°C, cultured for 1 day, 3 days, 5 days; collected cells, discarded supernatant, (3) adding medium: adding 10% porcine serum, 30% deficient Oxygen porcine mesenchymal stem cell conditioned medium, 37°C, 5% CO 2 Incubate for 24 hours. Count and do relevant testing.

Embodiment 3

[0061] The present invention carries out "5+N+1" purification culture for the isolated newborn pig islet cells: (1) the separated and extracted newborn pig islet cells are placed in ordinary medium (10% pig serum is added to Ham's F10 medium), 37°C, 5% CO 2 , cultivated for 5 days; collected the cells, discarded the supernatant; (2) adding medium: containing 30% hypoxic porcine mesenchymal stem cell conditioned medium, 5% human serum albumin, 10% porcine Serum, 20mM taurine, 4°C, cultured for 1 day and 3 days; collected cells, discarded supernatant; (3) adding medium: adding 10% porcine serum, 30% hypoxic porcine interstitial medium to EGM-2 medium Mesenchymal Stem Cell Conditioned Medium, 37°C, 5% CO 2 Incubate for 24 hours. Count and do relevant testing.

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Abstract

The invention discloses a method for cultivating medical-grade newborn pig islet cells. The three-stage culture method is used to increase the yield of newborn pig islet cells and promote the maturation of islet cells; by using Ham's F10 medium to prepare porcine mesenchymal stem cell conditioned medium under hypoxic conditions, it is beneficial to the nutrient supply of islet cells and effectively improves cell growth. Injury in low temperature state; through low temperature (4°C) and high concentration of taurine, reduce cell metabolism and reduce cell apoptosis.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a newborn pig cell culture method that can be used for clinical xenotransplantation. Background technique [0002] The official website of the International Diabetes Federation (IDF) (http: / / www.diabetesatlas.org / ) has released the latest global diabetes map (IDF Diabetes Atlas) (version 9). According to the latest report, about 463 million adults aged 20-79 suffer from diabetes worldwide in 2019 (1 in every 11 people has diabetes); it is estimated that by 2030, the number of diabetic patients will reach 578.4 million; it is estimated that by 2045, diabetic patients will Reached 700.2 million. The way of treating diabetes by injecting insulin and oral medicine can only control hyperglycemia in diabetes, but cannot completely cure diabetes. Diabetic complications need to cost hundreds of thousands to millions of dollars for treatment, and 20% of the famil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0676C12N2523/00C12N2500/84C12N2501/998C12N2500/33
Inventor 王维李桑谷星石王佳方倩
Owner HUNAN XENO LIFE SCI
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