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Use of tetrahydropyridines in cell, tissue, organ cryopreservation

An ectoine and cryopreservation technology, which is applied in applications, preservation of human or animal bodies, preservation of microorganisms, etc., can solve problems such as dehydration, cell death, and pH changes

Inactive Publication Date: 2009-07-29
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during cryopreservation, the water in the cell and the external environment will form ice crystals, which can cause mechanical damage in the cell, increase in electrolyte, change in osmotic pressure, dehydration, pH change, protein denaturation, etc., and can cause cell death.
At present, the commonly used cryoprotectant is dimethyl sulfoxide, which can lower the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the permeability of biofilms to electrolytes, drugs, poisons and metabolites. Methyl sulfoxide has a strong toxic effect on cells at room temperature
So far, no relevant studies and reports have been found on the use of ectoine for cryopreservation of cells, tissues, and organs

Method used

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  • Use of tetrahydropyridines in cell, tissue, organ cryopreservation
  • Use of tetrahydropyridines in cell, tissue, organ cryopreservation
  • Use of tetrahydropyridines in cell, tissue, organ cryopreservation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Effect of ectoine on cryopreservation of pig hepatocytes

[0016] Healthy 1-7-day-old Chinese experimental miniature pigs, using the modified Seglen in vitro two-step collagenase perfusion method to separate liver cells, filter with double-layer sterile gauze, and then centrifuge at 50g for 3 minutes to obtain liver cells, and place them under an Olympus inverted phase-contrast microscope The number of hepatocytes was calculated with a hemocytometer, the survival rate was calculated by the 4g / L trypan blue exclusion method, and more than 85% were used in the experiment.

[0017] The cell culture medium was L-15 culture medium, added with 10ml / L FBS, hydrocortisone 0.1μg / L, insulin 100μg / L, penicillin 100kU / L and streptomycin 100mg / L, and the control group was added with 50mL / L DMSO , the experimental group added 50mL / L DMSO and 100mL / L ectoine.

[0018] Then divide into cryopreservation tubes, place at room temperature for 15 minutes, place at 4°C for 20 min...

Embodiment 2

[0023] Example 2: Effect of ectoine on cryopreservation of human hepatocytes

[0024] The separation of human hepatocytes was carried out according to the literature method (Wang Yuming, Chen Guozhi, Dong Jiahong, et al. In vitro perfusion method for isolating hepatocytes and its application. Chinese Journal of Experimental Surgery, 1994, 11 (Supplement): 45-46)). Separated cells were stained with trypan blue and counted. Hepatocytes were adjusted to a concentration of 1×10 7 -2×10 7 / ml was divided into 0.7ml cryopreservation tubes for later use, 50mL / L DMSO was added to the control group, and 50mL / L DMSO and 150mL / L ectoine were added to the experimental group. Freeze the cryopreservation tube at 0°C for 30 minutes, then freeze at -30°C for 1 hour, and then store it at -80°C for 4 hours, and finally transfer it to a liquid nitrogen tank, store it for 1 month, and then resuscitate. The survival rate and 24h adherence rate were calculated (see Table 2 for the results), and ...

Embodiment 3

[0033] Example 3: Effect of ectoine on rat ovary autotransplantation

[0034] The unmated female SD rats were selected, and the exfoliated cells were checked by vaginal smear to confirm the regular estrous cycle. They were randomly divided into two groups with 10 rats in each group. The control group was the ovary autotransplantation group after cryopreservation in DMSO. The bilateral ovaries were removed and frozen for 1 week before subcutaneous autologous transplantation. The experimental group was the ectoine cryopreservation group. In the ovary autotransplantation group, bilateral ovaries were removed and frozen for 1 week, and then autologous subcutaneous transplantation was performed.

[0035] Vaginal smears were taken every day from the second day after operation to observe the changes of vaginal exfoliated cells. For those with estrous cycle changes, stage according to the type of exfoliated cells; for those without estrous cycle, the proportion of keratinocytes was c...

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Abstract

The invention discloses new application of tetrahydropyridines, namely the application of the tetrahydropyridines to the cryopreservation of cells, tissues and organs. The effective concentration is 0.1g / L to 100g / L; and the preferred concentration is 0.5g / L to 20g / L. During use, the tetrahydropyridines can be used separately or can be jointly used with other chemical reagents for the cryopreservation of cells, tissues and organs. Through experiments, the inventor of the application verifies that: the tetrahydropyridines can help the cells in resisting the refrigeration conditions and improve the protective effect on the cryopreservation of cells, tissues and organs. If the tetrahydropyridines is jointly used with dimethyl sulfoxide, the tetrahydropyridines can reduce the dosage of the dimethyl sulfoxide, reduce the toxicity of the dimethyl sulfoxide on the cells, and improve the cryopreservation effect.

Description

technical field [0001] The invention relates to the application of ectoine in cryopreservation of cells, tissues and organs. Background technique [0002] In biological experiments and disease treatment, it is often necessary to cryopreserve microorganisms, animal cells, tissues, and organs, especially in the process of organ transplantation. The cryopreservation of organs is conducive to ensuring the quality of donor organs. However, during cryopreservation, the water in the cell and the external environment will form ice crystals, which can cause mechanical damage in the cell, increase in electrolyte, change in osmotic pressure, dehydration, pH change, protein denaturation, etc., and can cause cell death. At present, the commonly used cryoprotectant is dimethyl sulfoxide, which can lower the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the permeability of biofilms to electrolytes, drugs, poisons and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02A01N1/00C12N1/04
Inventor 厉保秋
Owner SHANDONG UNIV
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