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Digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells

A technology for digestive juice and pancreatic cancer, applied in the field of single cell analysis and cell culture, can solve the problems of expensive equipment and reagent consumables, time-consuming, unable to be completed in ordinary laboratories, etc., to achieve strong practicability, reduce costs, and operate The effect of simple process

Inactive Publication Date: 2019-04-02
SINGLERON NANJING BIOTECHNOLOGIES LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the time-consuming technical method currently used to disperse human fresh pancreatic cancer tissue into single living cells; the viability and purity of the obtained single cells are not high; some methods require expensive equipment and reagent consumables, and cannot be completed in ordinary laboratories , the present invention provides a method for obtaining single cancer cells with high purity and high activity in a short period of time, providing materials for further single cell analysis and cell culture

Method used

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  • Digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells
  • Digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells
  • Digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells

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Embodiment 1

[0036] The method for preparing highly active human pancreatic cancer single cells in a short time by adopting the present invention is as follows:

[0037] (1) Prepare digestion solution, mix well according to 120U / ml Collagenase Type IV, 0.8U / ml Dispase, 15U / ml DNase I, and set aside.

[0038] (2) Mechanical separation: take 100mg of fresh pancreatic cancer tissue into a 1.5ml sterile centrifuge tube, put it in an ice water bath, use ophthalmic scissors to cut the tissue thoroughly to 0.1-2mm3, add 1ml of the prepared digestive solution, transfer Transfer to a 15ml centrifuge tube, and repeatedly grind the tumor tissue suspension 8 times with a Pasteur pipette.

[0039] (3) Enzyme digestion: shake and incubate at 37°C for 15 minutes for digestion, during which time every 5 minutes pipette with a 5ml pipette 10 times, and vortex intermittently at the maximum speed for 1 minute.

[0040](4) Termination of digestion: after the incubation, pipette fully with a 5ml pipette, cent...

Embodiment 2

[0045] (1) Prepare digestion solution, mix well according to 50U / ml Collagenase Type IV, 0.1U / ml Dispase, 2U / ml DNase I, set aside.

[0046] (2) Mechanical separation: Take 10 mg of fresh pancreatic cancer tissue into a 1.5 ml sterile centrifuge tube, place it in an ice water bath, and use ophthalmic scissors to cut the tissue thoroughly to 0.1-2 mm 3 , add 1ml of the prepared digestive solution, transfer to a 15ml centrifuge tube, and repeatedly grind the tumor tissue suspension 10 times with a Pasteur pipette.

[0047] (3) Enzyme digestion: shake and incubate at 37°C for 15 minutes for digestion, during which time every 5 minutes pipette with a 5ml pipette 10 times, and vortex intermittently at the maximum speed for 1 minute.

[0048] (4) Termination of digestion: after incubation, pipette fully with a 5ml pipette, centrifuge at 800rpm at 4°C for 5min, discard the supernatant, wash twice with PBS, filter with a 40um filter, collect the single cell suspension and place it on ...

Embodiment 3

[0053] (1) Prepare digestion solution, mix well according to 300U / ml Collagenase Type IV, 5U / ml Dispase, 30U / ml DNase I, set aside.

[0054] (2) Mechanical separation: Take 1000mg of fresh pancreatic cancer tissue into a 1.5ml sterile centrifuge tube, place it in an ice water bath, and use ophthalmic scissors to cut the tissue thoroughly to 0.1-2mm 3 , add 1ml of the prepared digestive solution, transfer to a 15ml centrifuge tube, and repeatedly grind the tumor tissue suspension 10 times with a Pasteur pipette.

[0055] (3) Enzyme digestion: shake and incubate at 37°C for 30 minutes for digestion, during which time every 15 minutes pipette with a 5ml pipette 10 times, and vortex intermittently at the maximum speed for 1 minute.

[0056] (4) Termination of digestion: after incubation, pipette fully with a 5ml pipette, centrifuge at 800rpm at 4°C for 5min, discard the supernatant, wash twice with PBS, filter with a 70um filter, collect the single cell suspension and place it on ...

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Abstract

The invention relates to digestive juice and method for dispersing pancreatic cancer tissue of human into single living cells, and belongs to the field of unicellular analysis and cell culture. The digestive juice prepared by mixing three enzymes of Collagenase Type IV, Dispase and DNase I is mainly used for treating pancreatic cancer tissue, and then operations of performing centrifugation, removing dead cells and the like are performed, so that single pancreatic cancer cells high in purity and high in activity can be obtained within a short time. The used reagent is a frequently-used molecule cell biology reagent. Used consumptive materials are also common cell culture consumptive materials, are low in price and are easy to obtain, so that the cost is effectively reduced. The entire operation process is simple, special instruments and equipment are not needed, the operation process can also be performed in a general purpose laboratory, and the method has high practicality.

Description

technical field [0001] The invention belongs to the field of single cell analysis and cell culture, in particular to a reagent and method for dispersing pancreatic cancer tissue into single living cells. Background technique [0002] Pancreatic cancer is a common malignant tumor of the digestive tract with a high degree of malignancy and a median overall survival rate of 6-12 months. It is difficult to diagnose and treat, and the prognosis is poor. The 5-year survival rate is less than 7%. One of the worst malignancies. In recent years, its morbidity and mortality have increased significantly. It is estimated that by 2030, pancreatic cancer may become the second cause of cancer death. In order to study the tumor biological characteristics of pancreatic cancer, scientists often use surgery or biopsy tissue as the starting material for research. On the one hand, fresh tissue can be directly used for related research, and on the other hand, fresh cancer tissue can be dispersed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2509/00
Inventor 訾晓渊陈芳
Owner SINGLERON NANJING BIOTECHNOLOGIES LTD
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