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Porcine islet cell frozen stock solution and cryopreservation method

A technology of islet cells and cryopreservation solution, which is applied in the field of cryopreservation solution and cryopreservation of porcine islet cells, can solve the problems of low viability of islet cell clusters, inability to effectively protect islet cell clusters, and inability to preserve long-term storage, and achieve High activity rate and the effect of reducing apoptosis

Active Publication Date: 2017-07-28
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cryopreservation solution of conventional single-cell suspension cannot effectively protect the islet cell mass, and the survival rate of the frozen islet cell mass is extremely low, and it cannot be stored for a long time

Method used

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  • Porcine islet cell frozen stock solution and cryopreservation method
  • Porcine islet cell frozen stock solution and cryopreservation method
  • Porcine islet cell frozen stock solution and cryopreservation method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 Cryopreservation experiment of porcine islet cells

[0031] Islet cell counting: take 50 μl of samples from the cell suspension each time, count DTZ-positive cell clusters under the microscope and repeat the sampling 3 times, and count islets according to the following formula: islet yield = (sum of 3 positive islet values / 3) × [Total amount of sample (ml) / 50 μl].

[0032] Divide the isolated and extracted porcine islet cells into two parts for cryopreservation: the first part of porcine islet cells is resuspended with ordinary freezing medium (50% serum + 10% DMSO + 40% basal medium) at a density of 10,000 IEQ / mL; In cryopreservation tubes, about 1 mL per tube; cool down to -80°C, and store in liquid nitrogen for long-term storage. The second porcine islet cells were resuspended with improved freezing medium (50% serum + 10% DMSO + 40% basal medium + trehalose 0.05 mml / L + tocopherol 20 μg / ml) at a density of 10000 IEQ / mL; In cryopreservation tubes, abou...

Embodiment 2

[0033] Example 2 Detection of Viability of Porcine Islet Cells Cryopreserved for 1 Month After Recovery

[0034] The normal cryopreservation solution, the improved cryopreservation solution and the cryopreservation solution of the present invention were respectively cryopreserved for 1 month and resuscitated. One day after recovery, the islet cells were counted to calculate the cell recovery rate, then stained with Annexin V-PI, and the apoptosis of the cells was observed by FACSCalibur flow cytometer. The experimental results showed that most of the pig islet cells in the conventional cryopreservation group were frozen for one month, and only 22.07% of the surviving cells remained. 41.69%, the survival rate of the pig islet cells after one month of cryopreservation using the cell cryopreservation liquid and the cryopreservation method of the present invention is greatly improved, reaching 62.62%. It shows that the cryopreservation solution and the cryopreservation method of ...

Embodiment 3

[0037] Example 3 Functional detection of porcine islet cells frozen for 1 month after resuscitation

[0038] The porcine islet cells frozen in the common freezing solution described in Example 1 and the cells frozen in the freezing solution of the present invention were respectively frozen for one month and then resuscitated. Immediately after resuscitation, the insulin release ability of the cells was detected and the insulin release ability of the cells was detected after one day of culture. The experimental results show that the glucose stimulation index of the porcine islet cells recovered by the cryopreservation method group of the present invention is 2.56>1.8, indicating that the porcine islet cells recovered by the cryopreservation solution and the cryopreservation method of the present invention have functions, while the porcine islet cells frozen by conventional methods have glucose Stimulation index 1.24<1.8 is not functional. It shows that the use of the cryoprese...

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Abstract

The invention belongs to the technical field of cell biology, and more specifically discloses a porcine islet cell frozen stock solution and a cryopreservation method. The porcine islet cell frozen stock solution is composed of a basic medium, porcine serum, DMSO, catalase, tocopherol, trehalose, and Z-VAD-FMK. The porcine islet cell frozen stock solution is capable of reducing apoptosis of porcine islet cells in freeze preservation process, improving resistance of porcine islet cells on severe environment, and increasing cell survival rate. According to the freeze preservation method, a programmable cooler is used for cooling, wherein when the temperature is higher than -25 DEG C, the cooling speed is controlled to be 1 DEG C / min, when the temperature is -40 DEG C or higher, and -25 DEG C or lower, the cooling speed is controlled to be 0.25 DEG C / min, and when the temperature is -80 DEG C or higher and is lower than -40 DEG C, the cooling speed is controlled to be 5 DEG C / min. The cryopreservation method is capable of reducing generation of ice crystals, and protecting porcine islet cells more conveniently.

Description

technical field [0001] The invention relates to the field of cryopreservation of cells, and belongs to the field of biotechnology, in particular to a cryopreservation solution and a cryopreservation method of porcine islet cells. Background technique [0002] Diabetes mellitus is a metabolic disease caused by defective insulin secretion or insulin dysfunction. Diabetes can easily cause long-term metabolic disorders, causing dysfunction and failure of tissues and organs throughout the body, especially the eyes, kidneys, cardiovascular and nervous systems. Severe cases cause acute complications such as dehydration, electrolyte disturbance and acid-base imbalance, ketoacidosis and hyperosmolar coma. Insulin injection is currently an effective means of controlling blood sugar in diabetic patients, but exogenous insulin cannot achieve physiological regulation of blood sugar, so it is difficult to improve diabetic complications, which greatly reduces the quality of life of patien...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0226A01N1/0284
Inventor 王维易受南范立青
Owner HUNAN XENO LIFE SCI
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