Tumor sample pretreatment method

A sample and tumor technology, applied in the field of pretreatment of tumor samples, can solve the problems of culture failure, microbial contamination, and low number of clones, and achieve the effect of improving the success rate of culture and reducing the probability of microbial contamination.

Active Publication Date: 2021-10-29
NANOPEPTIDE DEPOT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In the existing tumor organoid culture methods, the main focus is on sample enzymatic hydrolysis and medium formulation, etc., and there are few detailed plans for sample pretreatment, but the sample pretreatment process is one of the main reasons for culture failure
[0003] For example, in the existing tumor organoid culture technology, the activity of the sample is likely to decrease during the transfer process, and the reduction in the number of living cells will lead to culture failure; One of the main reasons for the failure; after the sample is obtained, the existing technology lacks the standard for selective elimination, and often uses tissue parts with less tumor cell content for culture, resulting in a small number of successfully cultured clones
[0004] The above reasons are the main reasons for failure in the pretreatment process of tumor organoid culture, and this will greatly increase the time and price cost of tumor organoid culture

Method used

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Effect test

Embodiment 1

[0035] In this protocol, the inventors cultured a total of 34 tumor samples, of which the first 9 samples used the conventional sample processing method, and the other 26 samples used the pretreatment method of the present invention.

[0036] The specific method of common pretreatment: After the sample is obtained, put it into the transfer buffer, transport the sample to the laboratory at 4-8°C in the sample transfer box, and then wash it with HBSS buffer + 1% double antibody for 5 times, each time for 5 minutes, and then use Cut into 1-2mm tissue pieces with scissors, add 5ml of enzymatic solution for treatment, and repeat the digestion several times until the tissue is completely digested, then centrifuge the collected cell fluid, wash 3 times with HBSS buffer, count, seed the plate, and matrix After the gel was solidified, the corresponding medium was added for cultivation.

[0037] The pretreatment method of the present invention: after the sample is obtained, put it into ...

Embodiment 2

[0041] In this protocol, samples from patients with lung squamous cell carcinoma were used, and the transfer buffer proposed by the present invention was used to store them at 4°C for 4 days, and then cultured. The result is as figure 2 As shown, the results show that the transfer buffer proposed by the present invention can effectively maintain the activity of the sample for up to 4 days at 4°C, and can still successfully culture organoids.

Embodiment 3

[0043] In this program, 10 samples were selected and treated with washing buffer in different sequences.

[0044] 10 cases in the control group:

[0045] 1) Perform the second washing treatment on the fresh tumor samples in the washing buffer, that is, wash the fresh tumor samples in the washing buffer 1 for 5 or 6 times, each time for 4-6 minutes; Wash in Washing Buffer 2 for 2-4 minutes; wash the tissue washed in Washing Buffer 2 in Washing Buffer 3 for 2-4 minutes; wash the tissue washed in Washing Buffer 3 in Washing Buffer 4 at 4 Soaking for 28-32 minutes under the condition of ℃; and washing the tissue soaked in the washing buffer 4 in the washing buffer 5 for 5 or 6 times, each time for 4-6 minutes.

[0046] 2) Excluding the samples after the first cleaning treatment, so as to remove fat, blood, necrosis and tissues with a content of interstitium greater than 20%;

[0047] 3) Perform the first cleaning treatment on the removed tissue. Wash the tissue samples in washi...

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Abstract

The invention proposes a tumor sample pretreatment method. The method comprises: performing a first cleaning treatment on a fresh tumor sample in a washing buffer solution; removing the sample after the first cleaning treatment, so as to remove fat, blood, necrosis and interstitial mass fraction greater than 18-22%. tissue; and performing a second cleaning process on the removed tissue. This method effectively reduces the probability of microbial contamination after tumor sample culture, and improves the success rate of tumor sample culture. In the method, fat, blood, necrosis, and tissue with a mass fraction greater than 18-22% are selected. Strictly limited, on the one hand, it can remove some pollution, on the other hand, it makes the second cleaning more thorough and effective, and also greatly improves the tissue parts with more tumor cell content, improves the subsequent enzymatic hydrolysis efficiency and increases the subsequent tumor cell culture. Number of successful clones.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a pretreatment method of tumor samples. Background technique [0002] In the existing tumor organoid culture methods, the main focus is on sample enzymatic hydrolysis and medium formulation, and there are few detailed plans for sample pretreatment, but the sample pretreatment process is one of the main reasons for culture failure. [0003] For example, in the existing tumor organoid culture technology, the activity of the sample is likely to decrease during the transfer process, and the reduction in the number of viable cells will lead to culture failure; One of the main reasons for the failure; after the sample is obtained, the existing technology lacks the standard for selective elimination, and often uses tissue parts with less tumor cell content for culture, resulting in a small number of successfully cultured clones. [0004] The above reasons are the mai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09A01N1/02
CPCA01N1/0215C12N5/0693C12N2509/00
Inventor 曹志鹏卓朗陈璞
Owner NANOPEPTIDE DEPOT INC
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