Universal cancer organoid in-vitro culture medium

An in vitro culture and medium technology, applied in the field of cell engineering, can solve the problems of using the same medium for various tumor samples, increasing the cost of culture, etc., and achieve the effect of maintaining cell viability and improving the efficiency of enzymatic hydrolysis

Active Publication Date: 2020-01-31
陈璞
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

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 invention puts forward a universal cancer organoid in-vitro culture medium, which consists of a DMEM/F-12K (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12K) basic culture medium and an adding factor, wherein the adding factor consists of HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), L-Glutamine, EGF (epidermal growth factor), Noggin, FGF (fibroblast growth factor)-10, A83-01 and Y27632. According to the culture medium of the embodiment of the invention, the success rate of organoid passage culture can be greatly improved, in addition, the universal culture of cancer samples, including gastric carcinoma, rectal carcinoma, lung carcinoma and the like, can be realized, culture cost is greatly lowered, the long-term culture of the organoid can be realized, and abiobank is established.

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 Applications(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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