Culture and cryopreservation method for prostatic cancer organoid with tumor immune microenvironment

A prostate cancer and culture method technology, applied in the field of prostate cancer organoid culture and cryopreservation, can solve the problems of deviation from the real situation of patients, failure to represent the clinical disease spectrum, failure of new drug clinical trials, etc., to achieve improved success rate and low cost Effect

Inactive Publication Date: 2020-04-21
北京科途医学科技有限公司
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Problems solved by technology

However, the number of prostate tumor cell lines successfully constructed so far is less than 10, which cannot represent a huge spectrum of clinical diseases
Coupled with the establishment of immortalized tumor ce

Method used

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  • Culture and cryopreservation method for prostatic cancer organoid with tumor immune microenvironment
  • Culture and cryopreservation method for prostatic cancer organoid with tumor immune microenvironment
  • Culture and cryopreservation method for prostatic cancer organoid with tumor immune microenvironment

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Effect test

Embodiment

[0059] The first medium used in this example is the addition of 10% by volume of autologous serum, 2mM superglutamine I, 10mM 4-hydroxyethylpiperazineethanesulfonic acid, 20% Rspondin1, 10μM Y-27632, without vitamin A RPMI 1640 medium after B27 supplement, 1.25 mM acetylcysteine, 10 mM nicotinamide, 50 ng / mL EGF, 500 nM A83-01, 3 μM SB202190 and 10 nM prostaglandin E2.

[0060] 1. Preparation of stimulated monocytes

[0061] Freshly drawn peripheral blood was taken from the patient, centrifuged at 800g / min for 10min, the upper layer of plasma was inactivated and centrifuged, then the supernatant was absorbed and transferred to a centrifuge tube for later use. Physiological saline was added to the sediment of the lower layer of blood to form a saline-blood sample mixture. Take an equal volume of lymphocyte separation solution in a new centrifuge tube, slowly add the above-mentioned physiological saline blood sample mixture to the upper layer of the lymph separation solution, c...

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Abstract

The invention provides a culture and cryopreservation method for a prostatic cancer organoid with a tumor immune microenvironment. The culture and cryopreservation method comprises the following steps: separating mononuclear cells and autoserum from peripheral blood, and stimulating the mononuclear cells by using a CD28 antibody and IL-2; separating tumor cells from a prostatic cancer tissue sample; mixing the tumor cells with a culture medium and a temperature-sensitive hydrogel, and then carrying out culturing and subculturing to obtain a tumor organoid precursor; stimulating the tumor organoid precursor by using IFN-gamma, and then conducting dissociating to form stimulated tumor cells; mixing the stimulated tumor cells with the stimulated mononuclear cells, conducting stimulating witha PD-1 antibody, mixing the stimulated mixture with the temperature-sensitive hydrogel, and carrying out culturing; and cryopreserving the tumor organoid with a cryopreservation solution containing FBS and the temperature-sensitive hydrogel. With the method in the invention, the success rate of culturing of organoids with a tumor microenvironment can be greatly improved at low cost.

Description

technical field [0001] The present application relates to the technical field of biomedicine, and specifically relates to a method for culturing and freezing prostate cancer organoids with a tumor immune microenvironment. Background technique [0002] Tumor organoids (Organoids) are cell cultures derived from patient tumor tissue that can be grown in three dimensions (3D) in vitro. Tumor organoids preserve tumor heterogeneity, tissue characteristics, and gene mutation information, which can simulate the occurrence and development of cancer in vitro, construct disease models, and conduct fast and accurate cancer drug screening, effectively supplementing existing animal and two-dimensional The (2D) cell culture model provides a new technical platform for the rapid and effective development of tumor drugs. [0003] Prostate cancer is currently the second most deadly tumor among male malignant tumors. The current conventional treatment for advanced prostate cancer is anti-andro...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/0786A01N1/02
CPCA01N1/0221C12N5/0645C12N5/0683C12N5/0693C12N5/0697C12N2501/2302C12N2501/24C12N2501/51C12N2502/1157C12N2502/30C12N2509/00
Inventor 孙志坚康平李程肖金平
Owner 北京科途医学科技有限公司
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