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Improved micromanipulation liquid for mitochondrial replacement technology and preparation method of improved micromanipulation liquid

A technology of micromanipulation liquid and mitochondria, which is applied in the field of assisted reproduction, can solve the problems of mitochondrial replacement technology, the effectiveness of reconstructing embryo safety, the difficulty of expensive implementation, etc., and achieve the effect of improving effectiveness and safety, and simple operation

Pending Publication Date: 2022-07-29
SHANGHAI TONGJI HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We can't help but ask, will this non-physiological phenomenon affect the effectiveness of mitochondrial replacement technology and the safety of reconstituted embryos? Will the accumulation of mitochondria around the nucleus bring in more mitochondria containing disease-causing genes during the removal of the nuclear donor? To date, no research has definitively answered these questions
Since the severity of mitochondrial genetic diseases depends on the ratio of mutant and normal mtDNA, i.e. egg / embryo mitochondrial heterogeneity, this has prompted scientists to try various methods to minimize the mitochondrial heterogeneity in the patient's oocyte cytoplasm , including mitophagy, polar body transplantation, gene editing, improving micromanipulation mechanical technology, etc., but these methods are expensive and difficult to implement, and there is no research on how to improve the concentration of CB in the micromanipulation solution. To break through the research bottleneck of the effectiveness and safety of mitochondrial replacement technology

Method used

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  • Improved micromanipulation liquid for mitochondrial replacement technology and preparation method of improved micromanipulation liquid
  • Improved micromanipulation liquid for mitochondrial replacement technology and preparation method of improved micromanipulation liquid
  • Improved micromanipulation liquid for mitochondrial replacement technology and preparation method of improved micromanipulation liquid

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The present embodiment provides an improved micromanipulation fluid for mitochondrial replacement technology, the preparation method of which is as follows:

[0025] 10 mg of cytochalasin B (CB, C6762, Sigma) dry powder was dissolved in 1 mL of dimethyl sulfoxide (DMSO, D2650, Sigma) at room temperature and mixed to prepare a 10 mg / mL CB stock solution. Pipette 1μL of CB stock solution and add it to 10mL of Vitrolife assisted reproductive IVF-gamete treatment solution (G-GAMETE TM , 10126, Vitrolife) solution, prepared into a working solution with a final concentration of CB >0 μg / mL and <2.5 μg / mL, which is the improved micromanipulation solution.

Embodiment 2

[0027] In this example, the effects of micromanipulation solutions containing different concentrations of CB on egg fertilization rate, embryo development rate, egg mitochondria distribution, ultrastructure and spindle morphology were comprehensively evaluated on mouse oocytes. The specific experimental steps and The result is as follows:

[0028] (1) The effect of gradient concentration CB treatment on the distribution pattern of mitochondria in mouse ovum was observed under confocal microscope

[0029] 1) A total of 978 mouse MII eggs were co-stained in 250 nM of mitochondrial fluorescent dye MitoTracker Red (M7512, Life Technologies) and 1 mg / mL of chromosomal fluorescent dye Hoechst 33342 (R37605, Life Technologies) for 20 minutes.

[0030] 2) Take 1 μL of 10 mg / mL CB stock solution, add it to a centrifuge tube containing 1 mL of G-GAMETE solution, mix well, and mark it as No. 5 (5 μg / mL CB group); aspirate 500 μL of the solution from the No. 5 centrifuge tube, add Put 50...

Embodiment 3

[0055] This example explores the effect of the improved micromanipulation solution on the carrying of mtDNA in nucleosomes extracted from human oocytes / fertilized eggs and the mitochondrial heterogeneity of human reconstructed embryos compared with the traditional micromanipulation solution. The specific experimental steps and the result is as follows:

[0056] 1) Spindle transplantation (ST) procedure: 30 human MII eggs were transferred into microdroplets of manipulation solution containing different concentrations of CB (1 μg / mL and 5 μg / mL) for 5 minutes before micromanipulation. The spindle position was determined using the Oosight imaging system and adjusted to the 3 o'clock position. The oocytes were fixed with a hand-held pipette and then drilled with a laser in the zona pellucida at 3 o'clock. Spindle-chromosome complexes were aspirated and briefly exposed to HVJ-E before transfer to enucleated recipient oocytes for fusion. Reconstituted oocytes were washed several t...

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Abstract

The invention provides an improved micromanipulation solution for a mitochondrial replacement technology and a preparation method of the improved micromanipulation solution. The improved micromanipulation liquid for the mitochondrial replacement technology is prepared from cytochalasin B, dimethyl sulfoxide and a vitrolite assisted reproduction IVF-gamete treatment liquid, in the improved micromanipulation solution, the concentration of cytochalasin B is greater than 0 mu g / mL and less than 2.5 mu g / mL. According to the improved micromanipulation liquid for the mitochondrial replacement technology, the purpose of improving the effectiveness and safety of the mitochondrial replacement technology is achieved only by adjusting the concentration of cytochalasin B, operation is easy, and safety guarantee is provided for thoroughly blocking maternal-fetal heredity of mitochondrial diseases.

Description

technical field [0001] The invention relates to the technical field of assisted reproduction, in particular to an improved micromanipulation fluid for mitochondrial replacement technology and a preparation method thereof. Background technique [0002] Mitochondrial DNA (mtDNA) mutations in eggs cause maternal familial diseases, ie inherited mitochondrial diseases, for which there is currently no effective treatment. [0003] Mitochondrial replacement technology is a technology that replaces the cytoplasm of patients with mitochondrial genetic diseases with the cytoplasm of eggs from healthy donors. It is inherited between mother and fetus, but its efficacy and safety have been the focus of debate among domestic and foreign experts, and are also the key to hinder its wide clinical application. At present, the micromanipulation solution routinely used in this technology contains cytochalasin B (CB) at a concentration of 5 μg / mL to maintain the stability of the germ cytoplasm,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89
CPCC12N15/89
Inventor 李颖纪亚忠
Owner SHANGHAI TONGJI HOSPITAL
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