Construction method of human pancreatic cancer tissue organoid model

A construction method and pancreatic cancer technology, applied in the field of biomedicine, can solve the problems of insufficient density of pancreatic cancer organoids, hindering the early development of clinical drug screening, and difficult separation of pancreatic cancer cells, achieving low cost, high success rate, Applicable effect

Inactive Publication Date: 2021-06-25
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, tumor organoids are a new type of drug screening model, although the cost of culture is lower than that of PDX, it is still much higher than that of cell lines
Mainly due to the relatively high cost of Matrigel used in the cultivation process
In addition, compared with other tumors, the amount of surgical specimens for pancreatic cancer is small and the specimens contain more than 90% of stromal components, which makes it difficult to separate pancreatic cancer cells, and the number of effective cancer cells is small, so the success rate of pancreatic cancer organoid culture is higher than that of other tumors. Tumors are lower, and the density of pancreatic cancer organoids often does not meet the standards of high-throughput drug screening in the early stage, hindering the early development of clinical drug screening

Method used

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  • Construction method of human pancreatic cancer tissue organoid model
  • Construction method of human pancreatic cancer tissue organoid model
  • Construction method of human pancreatic cancer tissue organoid model

Examples

Experimental program
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Effect test

Embodiment 1

[0071] A method for constructing a human pancreatic cancer tissue organoid model, comprising the following steps:

[0072] Step S1: Rinse the fresh human pancreatic cancer specimen with washing solution, and shred it mechanically;

[0073] Specifically, transfer the human pancreatic cancer specimens into a 15 mL centrifuge tube, shake and wash with 5 mL of pre-cooled cleaning solution for 30 seconds, discard the supernatant, add 5 mL of pre-cooled cleaning solution for washing again, and repeat 3 times. Then in a pre-cooled 1.5mL EP tube, cut the specimen to 1mm with direct shears that have been autoclaved 3 For left and right tissue fragments, resuspend the tissue fragments with pre-cooled basal medium, pipette the supernatant into a pre-cooled 15mL centrifuge tube, and resuspend 3 times;

[0074] Filter the supernatant obtained three times through a 100 μm filter, and then filter the obtained filtrate through a 20 μm filter to obtain a filter residue, which is resuspended w...

Embodiment 2

[0087] A method for constructing a human pancreatic cancer tissue organoid model differs from Example 1 in that the enzyme digestion solution used in Example 2 contains 2.5 mg / mL collagenase II, 100 μg / mL DNase I and 10.5 μM Y- The basic medium of 27632 was shaken and digested for 30 minutes in step S2, and the rest of the steps and sources of pancreatic cancer specimens were the same as those in Example 1.

Embodiment 3

[0089] A method for constructing a human pancreatic cancer tissue organoid model, which differs from Example 1 in that the pancreatic cancer specimens in Example 3 come from different patients, and the matrix material used in Example 3 is ready-to-use without overnight thawing The matrix material used in Example 3 is made by mixing type I collagen, 10×PBS, NaOH solution; the concentration of type I collagen purchased in this embodiment is C I is 3.17 mg / mL; the volume of the matrix material configured in this example is V=511.5 μL; the protein concentration C of the obtained matrix material is 2.79 mg / mL;

[0090] The volume calculation method of type I collagen, 10×PBS, NaOH solution is:

[0091] Volume V of type I collagen I =V×C / C I ;

[0092] 10 x volume V of PBS PBS =V / 10;

[0093] The volume V of 1M NaOH solution NaOH = V I ×0.023;

[0094] According to the above calculation method, 51.15 μL of 10xPBS and 10.35 μL of 1M NaOH solution are required respectively. I...

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Abstract

The invention relates to the technical field of biological medicine, and concretely relates to a construction method of a human pancreatic cancer tissue organoid model. The method comprises the following steps: S1, washing a fresh human pancreatic cancer specimen with a cleaning solution, and mechanically cutting into pieces; S2, carrying out enzyme digestion on the mechanically cut tissue fragments; S3, carrying out enzyme-free digestion on the tissue fragments subjected to enzyme digestion, collecting supernatant, and centrifuging to obtain pancreatic cancer single cell precipitate; and S4, fully and uniformly mixing the pancreatic cancer single cells obtained in the step S3 with a matrix material, inoculating gel drops, and adding the gel drops into a complete culture medium for culture. The same pancreatic cancer specimen is sequentially treated by adopting mechanical shearing, enzyme digestion and non-enzyme digestion, and single cells and cell masses with excellent organoid yield are separated, so that the utilization rate of the pancreatic cancer specimen is increased, the quantity and activity of inoculated pancreatic cancer cells are ensured, the number of the organoids meeting the requirement of high-throughput clinical drug screening can be obtained within a short culture time, and a solid foundation is laid for clinical transformation of the organoids.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for constructing a human pancreatic cancer tissue organoid model. Background technique [0002] Cancer is an extremely complex disease. In the past few decades, people have made some substantial progress in basic scientific research on some types of cancer, but there are still more or less problems when it is applied to clinical practice. It has also been gradually recognized that the transformation of basic scientific research into clinical practice has become one of the main obstacles to the development of new cancer treatment options. This is mainly because many tumor models only generalize the tumor tissue and cannot maintain the high heterogeneity of tumor cells well, so many drugs that are effective in traditional cancer models cannot be verified in clinical trials. [0003] Traditional two-dimensional (2D) cell culture has played an important role in establis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0677C12N5/0693C12N2509/00C12N2509/10C12N2533/54
Inventor 张太平邱江东陶锦新陈光宇杨刚赵方宇刘悦泽郑连芳
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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