Method for separating single cells from sample

A single-cell and sample technology, applied in the field of single-cell separation, can solve the problems of low single-cell yield and cell viability, and achieve the effect of promoting further research, high-yield and high-viability extraction

Pending Publication Date: 2019-04-23
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a new method for separating single cells from

Method used

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  • Method for separating single cells from sample
  • Method for separating single cells from sample
  • Method for separating single cells from sample

Examples

Experimental program
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Embodiment approach

[0034] According to a specific embodiment of the present invention, the method includes the following steps:

[0035] 1) In the tissue preservation solution, use sterile ophthalmic scissors to trim off the fiber, fat and necrotic tissue on the normal tissue sample;

[0036] 2) washing with sterile PBS;

[0037] 3) Put the sample into a sterile centrifuge tube, add the first part of the digestion solution (relative to the volume of 1cm 3 For normal tissue samples, the amount of the first part of the digestive solution can be 400-1000μL);

[0038] 4) Use sterile ophthalmic scissors to cut the tissue into debris (approximately 2×2×1mm 3 ), operated on ice, and the shearing time was not more than 5 minutes;

[0039] 5) Transfer the tissue debris to a sterile petri dish, add the second part of the digestive solution (relative to the volume of 1cm 3 For normal tissue samples, the amount of the second part of the digestive solution can be 5-20mL);

[0040] 6) Use a sterile Buchn...

Embodiment 1

[0057] This example is used to illustrate the method for isolating single cells from normal pancreatic tissue (normal pancreas at the tumor site) of the present invention, wherein the tissue preservation solution, digestion solution, lysate and washing solution used are shown in Table 1, The enzyme activity unit value and concentration of each component in each liter of digestive juice are shown in Table 2, and the preparation method of the digestive juice is: mix type VIII collagenase, Dispase II and trypsin inhibitor, use solvent to dissolve the mixed powder, and then add DNaseI and mix well.

[0058] The samples were divided into five groups for experimentation (see Table 3), and the specific operation steps were as follows:

[0059] 1) In the tissue preservation solution, use sterile ophthalmic scissors to trim off the fiber, fat and necrotic tissue on the specimen (the normal pancreatic tissue size is about 1.5×1.5×0.5cm 3 , yellow in color, medium to soft in texture, no...

Embodiment 2

[0081] This example is used to illustrate the method of the present invention for isolating single cells from pancreatic-derived tumor tissue, wherein the tissue preservation solution, digestion solution, lysate and washing solution used are as shown in Table 1, and each liter of digestion solution (preparation method The enzyme activity unit value and concentration of each component in Example 1) are as shown in I-1 of Table 2.

[0082] 1) In the tissue preservation solution, use sterile ophthalmic scissors to trim off the pancreatic tumor sample (1.5×1.5×0.5cm 3 , Obtained by surgical resection, the patient has signed the informed consent) on the fiber, fat and necrotic tissue, etc.

[0083] 2) Wash 3 times with sterile PBS.

[0084] 3) Put the sample into a 5 mL sterile EP tube (eppendorf), and add about 500 μl of digestion solution.

[0085] 4) Use sterile ophthalmic scissors to cut the tissue into debris (approximately 2×2×1mm 3 ), operated on ice, and the shearing tim...

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Abstract

The invention relates to the field of single cell separation, and discloses a method for separating single cells from a sample. The method comprises the steps that the normal tissue sample is mixed with digestive juice for digestion, wherein the digestive juice contains VIII-type collagenase, neutral protease, a trypsin inhibitor and deoxyribonuclease; the ratio between enzyme activity units of the VIII-type collagenase, the neutral protease, the trypsin inhibitor and the deoxyribonuclease is (100-312.5):(1-5):(5000-25000):1. The method can achieve high-yield and high-activity extraction of the single cells under the condition that the total digestion time is short, especially pancreatic tissue, and the problem that existing pancreatic tissue single cells are relatively low in yield and activity is solved.

Description

technical field [0001] The invention relates to the field of single cell separation, in particular to a method for separating single cells from samples. Background technique [0002] The pancreas is an organ with both endocrine and exocrine functions in the human body. Histologically, it contains multiple cellular components including islet cells, acinar cells, ductal cells, and interstitial cells. The pancreatic juice secreted by the pancreas contains various digestive enzymes such as trypsin, pancreatic lipase, and pancreatic amylase. With the rise of single-cell analysis technology in recent years, people's research on human tissues and organs has gradually deepened from the analysis of the overall tissue where different components mix and interfere with each other to the study of individual cells that make up tissues and organs. Its importance is self-evident. . However, in the field of pancreas research, because the pancreas organ is generally soft and fragile, its h...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0676C12N5/0693C12N2509/00
Inventor 吴文铭彭俊雅陈澔黄丹刘路路洪夏飞丛林李冬晶赵玉沛
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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