Cryopreservation and resuscitation method, cryopreservation liquid and resuscitation liquid for pig islet cells

A technique for islet cells and cryopreservation, which is applied to animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inability to meet islet cells, affect the recovery rate, viability rate and function, and reduce the apoptosis rate. , the effect of increasing the recovery rate and reducing the fragmentation

Active Publication Date: 2019-06-18
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the temperature range of 0 to -60°C, the damage to the cell mass caused by ice crystals cannot be completely avoided only by the cryoprotectant, which will seriously affect the recovery rate, viability and function of the cells after recovery, and cannot meet the short-term acquisition requirements. Sufficient islet cell requirements

Method used

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  • Cryopreservation and resuscitation method, cryopreservation liquid and resuscitation liquid for pig islet cells
  • Cryopreservation and resuscitation method, cryopreservation liquid and resuscitation liquid for pig islet cells
  • Cryopreservation and resuscitation method, cryopreservation liquid and resuscitation liquid for pig islet cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, porcine islet cell microencapsulation wrapping;

[0061] Collect the isolated and cultured islet cells for 3-5 days, wash them with PBS for 2-3 times, centrifuge and discard the supernatant, mix the islet cell mass with 0.5-10% sodium alginate at a concentration of 5000-10000IEQ / mL in the mixed solution Add the microcapsule electrostatic droplet generating device after mixing the concentration in the mixture, adjust the equipment parameters, and select the appropriate parameters (microcapsule particle size range 300-700 μm) to prepare microcapsules; after the microcapsules are prepared, the microencapsulated islet cell cluster Equilibrate in a 50-200mM calcium chloride solution for 10-30 minutes. After the equilibrium is completed, let it stand and discard the supernatant, wash with PBS for 2-3 times, and perform subsequent experiments.

Embodiment 2

[0062] Embodiment 2, cryopreservation of microencapsulated porcine islet cells

[0063] Microencapsulated porcine islet cryopreservation solution, including A solution and B solution, is composed as follows: A solution is a low-concentration cryopreservation solution, containing 1M dimethyl sulfoxide, 0.5M ethylene glycol, 40μg / mL catalase, Trehalose 0.33M, β-aminoethanesulfonic acid (taurine) 3mM, pig serum 30%; liquid B is a high-concentration cryopreservation solution, containing dimethyl sulfoxide 3M, ethylene glycol 0.5M, catalase 40μg / mL, trehalose 0.33M, β-aminoethanesulfonic acid (taurine) 3mM, pig serum 30%.

[0064] 1. Set aside the microencapsulated islet cell mass to settle, and discard the supernatant;

[0065] 2. Add liquid A to the microencapsulated islet cell mass with the supernatant discarded, and equilibrate at 4°C for 30 minutes;

[0066] 3. Add an equal volume of liquid B, mix well, and place at 0°C for 20 minutes to balance;

[0067] 4. Divide the micr...

Embodiment 3

[0070] Example 3, cryopreservation of microencapsulated porcine islets

[0071] Microencapsulated porcine islet cryopreservation solution, including A solution and B solution, is composed as follows: A solution is a low-concentration cryopreservation solution, containing dimethyl sulfoxide 0.5M, ethylene glycol 1M, catalase 70μg / mL, Trehalose 0.78M, β-aminoethanesulfonic acid (taurine) 8mM, porcine serum 70%; liquid B is a high-concentration cryopreservation solution, containing dimethyl sulfoxide 2M, ethylene glycol 1M, catalase 70μg / mL, trehalose 0.78M, β-aminoethanesulfonic acid (taurine) 8mM, pig serum 70%;

[0072] 1. Set aside the microencapsulated islet cell mass to settle, and discard the supernatant;

[0073] 2. Add liquid A to the microencapsulated islet cell mass with the supernatant discarded, and equilibrate at 4°C for 30 minutes;

[0074] 3. Add an equal volume of liquid B, mix well, and place at 0°C for 20 minutes to balance;

[0075] 4. Divide the microenca...

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Abstract

The invention discloses a cryopreservation and resuscitation method, cryopreservation liquid and resuscitation liquid for pig islet cells. According to the method, before cryopreservation, sodium alginate is used to wrap the pig islet cells, a relatively-stable three-dimensional adhesion space is formed without having impact on normal metabolic level, and stable recycling rate (higher than 60%) and insulin secretion function are ensured after cryopreservation and resuscitation of the pig islet cells to achieve the objective of building an islet cell bank.

Description

technical field [0001] The invention belongs to the technical field of cryopreservation of animal islet cells, and in particular relates to a cryopreservation-resuscitation method for islet cells, as well as a cryopreservation solution and a resuscitation solution. Background technique [0002] Diabetes mellitus is an endocrine disease characterized by hyperglycemia, which has seriously plagued human health for a long time. The reason for its occurrence is the relative or absolute deficiency of insulin secretion (type Ⅱ diabetes) or absolute deficiency (type Ⅰ diabetes). According to the eighth edition of the global diabetes map released by the International Diabetes Federation (IDF) in 2017, the number of people with diabetes in the world has exceeded 400 million, and the number of people with diabetes in my country has reached 110 million. At present, the main method of treating diabetes is subcutaneous injection of exogenous insulin. Although this method has achieved rema...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/071
Inventor 王维王佳谷星石李桑徐畅
Owner HUNAN XENO LIFE SCI
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