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Cculture medium and culture method for ovarian cancer organoids

A culture medium and ovarian cancer cell technology, applied in the field of biomedicine, can solve the problems of low success rate of initial establishment, unsatisfactory culture success rate, and high culture cost

Pending Publication Date: 2021-04-20
汪雪 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it faces the problems of high culture cost and unsatisfactory culture success rate (mainly manifested as difficult passage and low success rate of initial establishment)

Method used

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  • Cculture medium and culture method for ovarian cancer organoids
  • Cculture medium and culture method for ovarian cancer organoids
  • Cculture medium and culture method for ovarian cancer organoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: A case of establishing ovarian cancer organoids from the ascites of a patient with stage III epithelial ovarian cancer.

[0028] Step 1: Achieve separation of cells. Collect 50ml of ascites sample from the operation, filter it through a sterile pore sieve to remove non-cellular components, centrifuge at a speed of 100-200g for 5-10 minutes at a low temperature, and discard the supernatant.

[0029] Step 2: Aggregation of tumor cells: Lyse the cells obtained by centrifugation with 2-5ml red blood cell lysate to remove red blood cells, add RPMI 1640 medium to neutralize and centrifuge to remove the lysate. Add cleaning solution ① (Add penicillin 100U / ml and streptomycin 0.1mg / ml to sterile PBS solution) 8-10ml resuspends the cells obtained by centrifugation, and transfers to a 15ml centrifuge tube for 50-200g centrifugation for 5 minutes, discards the supernatant, repeats this step 2 -5 times. Before the final step of centrifugation, take a little solution a...

Embodiment 2

[0032] Example 2: A case of establishing ovarian cancer organoids from an orthotopic tumor of a patient with stage III epithelial ovarian cancer.

[0033] Step 1: Tumor tissue pretreatment: Cut the in situ tissue samples of epithelial ovarian cancer patients into pieces, wash them with cleaning solution (sterile PBS solution with penicillin 100U / ml, streptomycin 0.1mg / ml) for several times, and in a sterile Environment The tissue samples were minced into minced meat of about 1-mm3 size.

[0034] Step 2: Preparation of ovarian cancer single cells, add 5-8ml of Advanced DMEM / F12 medium containing 5mg / ml collagenase type II and 5μM Y-27632, and then place in a 37°C constant temperature incubator for 0.5- 2 hours; after enzymatic hydrolysis, sieve with a 100μm cell sieve, add Advanced DMEM / F12 medium to stop the digestion, and centrifuge at 100-200g for 5-10min, then discard the supernatant (repeat once), and the precipitate obtained by centrifugation is the ovary Cancer cells; b...

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Abstract

The invention relates to a biomedical technology, in particular to a culture medium and culture method for ovarian cancer organoids. The culture medium comprises a basal culture medium DMEM / F12, B27, glutamine, HEPES, N-acetylcysteine, FGF2, A83-01, nicotinamide, forskolin, beta-estradiol, hydrocortisone, R-spondin-1, noggin, FGF10, EGF and Heregulin beta 1 and at least one selected from PFBS, PFOA and PFOS. According to the culture medium and the culture method, the establishment success rate of an ovarian cancer culture medium is improved, the amplification efficiency of the ovarian cancer organoids is improved, the proliferation efficiency after passage is stable, the drug screening process is accelerated, and the cost of culture materials is reduced.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically relates to a culture medium and a culture method of ovarian cancer organoids. Background technique [0002] Organoids are an in vitro three-dimensional culture system in a mixed cell state. It has a self-renewing stem cell population with individual differences and tissue specificity. It has consistent spatial characteristics with the source tissue and can reproduce the source tissue. part of the functionality, thereby providing a highly physiologically relevant simulation system. Based on the above characteristics, the construction of tumor organoids (Patient-derived organoids, PDO) from tumor tissues can be applied to the elucidation of tumorigenesis mechanisms, high-throughput drug screening, research and development of therapeutic targets, and individualized treatment. Scientific research value and clinical value. [0003] Ovarian cancer is a common disease that th...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02
Inventor 汪雪徐小雅金柳王贵成
Owner 汪雪
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