Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method

A technology of cryoprotectant and ovary, which is applied in the field of cryomedicine of biotechnology, can solve problems such as difficulty in penetration and removal, toxicity of cryoprotectant, etc., and achieve good cryopreservation effect, good cryoprotective effect, and easy removal

Inactive Publication Date: 2015-11-18
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the problems of toxicity, penetration and removal of cryoprotectants in the prior art, the present ...

Method used

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  • Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method
  • Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method
  • Cryoprotectant free of DMSO (dimethyl sulfoxide) and ovary cryopreservation method

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Embodiment 1

[0025] The screening of embodiment 1 cryoprotectant formula

[0026] The integrity of cell morphology, or the integrity of cell membrane structure, is the basis for ensuring cell activity and function. During the freezing, drying and rehydration experiments of human umbilical cord blood MNC, the cell membrane structure will inevitably be damaged by solutes and ice crystals to varying degrees due to material phase transitions. DMSO, trehalose, PVP and other cell cryoprotectants can reduce the damage of solutes and ice crystals, thereby protecting the cell membrane structure. The previous experiments of my experimental group concluded that it is guessed that the prevalence of cell membrane structure will have a certain impact on cell activity during freezing, drying and rehydration. In view of this, the formulation of cryoprotectants is screened.

[0027] Experimental reagents:

[0028] Ficoll, Trehalose, PVP, Cholesterol, Lecithin, Trypan Blue, Apoptosis Detection Annexi...

Embodiment 2

[0056] (1) Experimental animals28 sheep ovaries with vascular pedicles with ovarian artery and aorta were taken as objects, placed in a mixture containing heparin sodium and antibiotics (final concentrations were heparin sodium 0.01IU / L, penicillin 1IU / L, streptomycin 1IU / L, respectively). , Amphotericin B 0.25mg / L) in normal saline, transported back to the laboratory within 1h in a 12°C incubator.

[0057] (2) Main reagents Trehalose was purchased from Sinopharm Chemical Reagent Co., Ltd., dimethyl sulfoxide (DMSO), polyvinylpyrrolidone 40 (Polyvinylpyrrolidone40, PVP40), and lecithin were purchased from Sigma Corporation in the United States. Cholesterol was purchased from Amresco, USA. Fetal Bovine Serum (FBS) was purchased from Hyclone, USA. Glycerin was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. The in situ cell apoptosis detection kit (InSituCellDeathDetectionKit, POD) was purchased from ROCHE Company in the United States, Trizol was purchased from LifeTec...

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Abstract

The invention relates to the field of hypothermal medicine of biotechnology, in particular to a cryoprotectant free of DMSO (dimethyl sulfoxide). The cryoprotectant comprises 200 mmol/L of trehalose, 0.3 g/mL of PVP (polyvinylpyrrolidone) 40, 200 mu g/L of cholesterol and 200 mu g/L of lecithin. The cryoprotectant is injected into a newly obtained ovary; all macroscopic follicles on the surface of the ovary are punctured and sucked, so that content in the follicles is removed; then the ovary is put in a freezing bag to be cooled. The cryoprotectant doesn't contain DMSO, does not have cytotoxicity and is easy to remove, simple to prepare and good in cryoprotective effect, raw materials are easy to obtained, the rate of normal primordial follicles is high, and the number of dead cells is small; an ovary cryopreservation method is simple and easy to operate and good in cryoprotective effect; the histological structure can be better conserved after the whole ovary of sheep is frozen.

Description

technical field [0001] The invention relates to the field of biotechnology low-temperature medicine, in particular to a DMSO-free cryoprotectant, and also relates to a method for cryopreserving ovaries using the cryoprotectant. Background technique [0002] With the advancement of diagnosis and treatment methods, the survival rate of cancer patients in reproductive women has increased significantly. However, cancer treatments such as chemotherapy and radiation may cause ovarian function to decrease or even fail. Deep cryopreservation of ovarian cortex tissue has been used clinically as a method to preserve fertility before radiotherapy and chemotherapy, but it is limited by tissue ischemia damage and a large number of follicle losses after transplantation, and complete ovarian transplantation can restore blood supply by anastomosis of blood vessels. In fact, this problem can be overcome, and there have been reports of complete sheep ovaries being transplanted and giving...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 迟令龙李栋
Owner SHANDONG QILU STEM CELL ENG
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