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Culture medium of endometrial organ and culture method

A technology of endometrium and culture method, which is applied in the field of culture medium and culture of endometrium organoids, can solve the problem of lack of endometrial organoid research and reports, test procedures, operation steps, culture conditions, and culture medium formulations. Too many reports and other issues, to achieve the effect of rapid expansion, wide application range, and fast growth

Active Publication Date: 2022-01-21
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although a variety of human-derived other tissues (such as liver, intestine, etc.) can be successfully cultured into organoids in vitro using different methods and under different culture conditions, the current research and reports on the culture methods of endometrial organoids are limited. In particular, there are not many reports on the specific test process, operation steps, culture conditions, and medium formulations.

Method used

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  • Culture medium of endometrial organ and culture method
  • Culture medium of endometrial organ and culture method
  • Culture medium of endometrial organ and culture method

Examples

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Effect test

Embodiment 1

[0032] This embodiment provides an endometrial cancer organoid culture medium and its culture method. The endometrial cancer organoid culture medium is composed of: 80ng / ml EGF, 150ng / ml Noggin, 250ng / ml R- spondin 1, 150ng / ml Wnt3a, 25ng / ml FGF9, 60ng / ml KIAA1199, 20ng / ml sox2, 80nM isoflavones, 500nM CHIR99021, 8μM A83-01, 8μM RKI-1477, 100ug / ml of primary cell antibiotics. The methods for culturing endometrial cancer organoids are as follows:

[0033] 1) Sample washing: transfer the obtained endometrial cancer specimen into a centrifuge tube, then shake and wash with sterile normal saline for 30 seconds, remove the supernatant, and re-add sterile normal saline for washing. Repeated washing 3 times according to the above method to remove impurities on the tissue surface.

[0034] 2) Sample shearing: In a biological safety cabinet, transfer the sample to a 6cm culture dish, use sterilized surgical scissors on ice to cut the tissue to a size of 1-5mm 3 .

[0035] 3) Sampl...

Embodiment 2

[0041] This embodiment provides an endometrial cancer organoid culture medium and its culture method. The endometrial cancer organoid culture medium is composed of: 20ng / ml EGF, 400ng / ml Noggin, 80ng / ml R- spondin 1, 400ng / ml Wnt3a, 50ng / ml FGF9, 100ng / ml KIAA1199, 80ng / ml sox2, 20nM isoflavones, 1000nM CHIR99021, 20μM A83-01, 18μM RKI-1477, 50ug / ml of primary cell antibiotics. The method for culturing endometrial cancer organoids is the same as in Example 1. After 6 days of culture, endometrial cancer organoids can be obtained, and the tissue morphology and structure are observed under an ordinary optical microscope as figure 2 shown.

Embodiment 3

[0043] This embodiment provides an endometrial organoid culture medium and a culture method. The endometrial organoid culture medium is composed of: 50ng / ml of EGF, 100ng / ml of Noggin, and 500ng / ml of R-spondin 1 according to the final concentration , 100ng / ml Wnt3a, 50ng / ml FGF9, 30ng / ml KIAA1199, 50ng / ml sox2, 15nM isoflavones, 200nM CHIR99021, 15μM A83-01, 10μM RKI-1477, 100ug / ml Pro generation of cellular antibiotics. The endometrial organoid culture method is:

[0044]1) Sample washing: Transfer the obtained endometrium sample into a centrifuge tube, then shake and wash with sterile normal saline for 30 seconds, remove the supernatant, and add sterile normal saline again for washing. Repeated washing 3 times according to the above method to remove impurities on the tissue surface.

[0045] 2) Sample shearing: In a biological safety cabinet, transfer the sample to a 6cm culture dish, use sterilized surgical scissors on ice to cut the tissue to a size of 1-5mm 3 .

[00...

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Abstract

The invention provides a culture medium of endometrial organ and a culture method. The culture medium is prepared from the following components according to final concentration: EGF with the concentration of 10 to 100 ng / ml, Noggin with the concentration of 20 to 500 ng / ml, R-spondin 1 with the concentration of 20 to 500 ng / ml, Wnt3a with the concentration of 20 to 500 ng / ml, FGF9 with the concentration of 1 to 100 ng / ml, KIAA1199 with the concentration of 10 to 200 ng / ml, sox2 with the concentration of 1 to 100 ng / ml, isoflavone with the concentration of 10 to 100 nM, CHIR99021 with the concentration of 100 to 2000 nM, A83-01 with the concentration of 1 to 40 [mu] M, a ROCK inhibitor with the concentration of 2 to 50 [mu] M and primary cell antibiotic with the concentration of 50-200 [mu] / ml; and all the components are dissolved in a DMEM / F12 culture solution. When the culture medium is used for culturing endometrial organ, rapid amplification of endometrial stem cells and maturation and differentiation of the organ are facilitated, the number of passage times is remarkably increased, and the same tissue structure and genome characteristics of primary tissues can still be maintained after multiple passage times.

Description

technical field [0001] The invention relates to the technical field of organoid culture, in particular to a culture medium and a culture method for endometrial organoids. Background technique [0002] The endometrium undergoes a dynamic development process in which the functional layer of the endometrium undergoes menstrual cycle regeneration, differentiation, and shedding under the control of the hypothalamus-pituitary-ovarian axis during reproduction. The mucosa contains a single layer of glands lined by secretory columnar epithelium separated by stroma. The basic functions of the endometrium are luminal and glandular epithelium, a mixture of ciliated and secretory cells. The luminal epithelium is the site of embryo attachment. The epithelium covers the surface of the endometrium and invaginates from the endometrium to the stroma to form glands. Glandular secretions are rich in growth factors and lipids necessary for placental growth. [0003] Organoids are organ-specif...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2501/11C12N2501/415C12N2501/115C12N2501/999
Inventor 兰坚强朱宇张慧星刘丹黄敏
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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