A kind of endometrial organoid culture medium and culture method

An endometrial and culture method technology, which is applied in the field of endometrial organoid culture medium and culture, can solve the problem of lack of research and reports on endometrial organoids, test procedures, operation steps, culture conditions, and medium formulas. There are too many reports and other problems to achieve the effect of being conducive to rapid expansion, wide application, reducing cytotoxicity and inhibitors

Active Publication Date: 2022-07-05
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.

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  • A kind of endometrial organoid culture medium and culture method
  • A kind of endometrial organoid culture medium and culture method
  • A kind of endometrial organoid culture medium and culture method

Examples

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Embodiment 1

[0032] The present embodiment provides an endometrial cancer organoid culture medium and a 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. Endometrial cancer organoid culture methods are:

[0033] 1) Sample washing: transfer the obtained endometrial cancer sample 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. Repeat 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 petri dish, and use sterilized surgical scissors on ice to shred the tissue to a size of 1-5mm 3 .

[0035] 3) Sample digestion: add ...

Embodiment 2

[0041] This embodiment provides an endometrial cancer organoid culture medium and a 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 ml of primary cell antibiotics. The method for culturing endometrial cancer organoids is the same as that in Example 1. Endometrial cancer organoids can be obtained after 6 days of culture. figure 2 shown.

Embodiment 3

[0043] The present embodiment provides an endometrial organoid culture medium and a culture method. The endometrial organoid culture medium is composed of: 50ng / ml EGF, 100ng / ml Noggin, 500ng / ml 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 original Generation cell antibiotics. Endometrial organoid culture methods are:

[0044]1) Sample washing: transfer the obtained endometrial sample 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. Repeat 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 petri dish, and use sterilized surgical scissors on ice to shred the tissue to a size of 1-5mm 3 .

[0046] 3) Sam...

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Abstract

The invention provides an endometrial organoid culture medium and a culturing method. The culture medium comprises, according to the final concentration, EGF at 10-100ng / ml, Noggin at 20-500ng / ml, and R-spondin at 20-500ng / ml. 1, 20‑500ng / ml Wnt3a, 1‑100ng / ml FGF9, 10‑200ng / ml KIAA1199, 1‑100ng / ml sox2, 10‑100nM isoflavones, 100‑2000nM CHIR99021, 1‑40µM A83‑01, 2‑50μM ROCK inhibitor, 50‑200ug / ml primary cell antibiotic; the above components are all dissolved in DMEM / F12 medium. This medium is used for endometrial organoid culture, which is conducive to the rapid expansion of endometrial stem cells and the maturation and differentiation of organoids, and the number of passages is significantly increased, and the same tissue as the primary tissue can still be maintained after multiple passages. Structural and genomic properties.

Description

technical field [0001] The invention relates to the technical field of organoid culture, in particular to an endometrial organoid culture medium and a culture method. Background technique [0002] The endometrium undergoes a process of dynamic development. During reproduction, the functional layers of the endometrium undergo regeneration, differentiation, and shedding during the menstrual cycle under the control of the hypothalamic-pituitary-ovarian axis. The mucosa contains a single layer of glands lined by a secretory columnar epithelium separated by a stroma. The basic functions of the endometrium are the luminal and glandular epithelium, consisting of a mixture of cilia 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] Organ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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