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Universal cancer organoid culture medium in vitro

An in vitro culture and culture medium technology, applied in the field of cell engineering, can solve the problems of increasing culture cost and using the same culture medium for multiple tumor samples

Active Publication Date: 2021-09-24
陈璞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, in the existing organoid in vitro culture technology, the culture medium is aimed at a certain type of tumor sample, and it is impossible to use the same medium for multiple tumor samples, resulting in the frequent preparation of different culture media during the culture process, which increases the cost of culture

Method used

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  • Universal cancer organoid culture medium in vitro
  • Universal cancer organoid culture medium in vitro
  • Universal cancer organoid culture medium in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The optimization experiment of embodiment 1 culture medium composition

[0051] In this protocol, colon adenocarcinoma organoid samples were used to investigate the effects of common components of the prior art medium on the growth of organoids.

[0052] The specific protocol is as follows: count the organoids, use Matrigel to resuspend the organoids according to the concentration of 10 μL / 50 organoids, plant them in a 96-well plate, 10 μL / well, and use the culture method minus different single components after seeding the plate. After 5 days of culture, the growth pictures were recorded and the effects of different medium conditions on the growth of organoids were quantitatively detected.

[0053] The result is as figure 1 As indicated, CM represents complete medium (including 10 mM HEPES, 2 mM L-Glutamine, 50 ng / mLEGF, 100 ng / mL Noggin, 500 ng / mL LR-Spondin 1, 100 ng / mL WNT-3A, 10 ng / mL FGF-10, 0.5 μM A83-01 , 5uM SB202190, 4mM Nicotinamide and 10μM Y27632 in DMEM / F...

Embodiment 2

[0055] In this protocol, samples from patients with bone metastases of gastric cancer were used for culture using the culture medium optimized in Example 1.

[0056] Transfer the tumor sample (the tumor sample can come from the tumor sample taken after the patient's operation or puncture, which belongs to medical waste) to a centrifuge tube containing transfer buffer (transfer Buffer) within ten minutes, and put it into a biological container containing an ice box. In the sample transfer box (maintain temperature 4-8°C), and the ice box in the sample transfer box can be maintained at 4-8°C for a long time. Under this condition, the biological activity of the sample can be maintained for up to 4 days.

[0057] Among them, transfer Buffer contains 15mM HEPEs, 100μM glycine, 100μM L-alanine, 100μM L-asparagine, 100μM L-aspartic acid, 100μM L-glutamic acid, 100μM L-proline and 100μM L-serine Advanced DMEM / F-12K medium.

[0058] Sample pretreatment: This sample is a surgical sampl...

Embodiment 3

[0064] In this protocol, samples from patients with rectal cancer were used for culture using the culture method described above.

[0065] Refer to Example 2 for the specific process. Take photos and record the growth status of organoids on the 1st day, 4th day and 6th day of culture respectively. The results are as follows: image 3 shown.

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Abstract

The present invention proposes a culture medium. The medium consists of DMEM / F-12K basal medium and supplementary factors consisting of HEPES, L-Glutamine, EGF, Noggin, FGF-10, A83-01, Y27632. The culture medium according to the embodiment of the present invention not only greatly improves the success rate of subculture of organoids, but also can realize the universal culture of cancer samples such as gastric cancer, rectal cancer and lung cancer, which greatly reduces the culture cost and can Realize long-term organoid culture and establish biological sample bank.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular, the invention relates to a universal culture medium for cancer organoids in vitro. Background technique [0002] The three-dimensional organ structure obtained by culturing the stem cells existing in the tissue under suitable conditions in vitro is called an organoid. Organoids are differentiated from tissues and contain the same genetic information characteristics as tissue cells. Due to its three-dimensional structural characteristics, it retains the physiological characteristics in vivo to the greatest extent in an in vitro environment. [0003] However, in the existing organoid in vitro culture technology, the culture medium is aimed at a certain type of tumor sample, and it is impossible to use the same medium for multiple tumor samples, resulting in the frequent preparation of different culture media during the culture process, which increases the cost of culture. [0004] T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0679C12N5/0688C12N5/0693C12N2500/32C12N2500/60C12N2501/11C12N2501/119C12N2501/15C12N2501/23C12N2501/727C12N2509/00
Inventor 曹志鹏陈璞卓朗曹文荣闫鹏
Owner 陈璞
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