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Kit for separating single cells and applications thereof

A kit and single-cell technology, applied in the field of enzyme engineering, 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: 2019-04-30
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kit for separating single cells and its application in order to overcome the problems of low single cell yield and cell viability in the prior art

Method used

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  • Kit for separating single cells and applications thereof
  • Kit for separating single cells and applications thereof
  • Kit for separating single cells and applications thereof

Examples

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Embodiment approach

[0036] In addition, the kit of the present invention can be used to separate single cells from pancreatic tumor samples, thereby facilitating the analysis of pancreatic tumor samples at the cellular level. Therefore, according to a preferred embodiment of the present invention, the pancreatic sample is a pancreatic tumor sample (pancreatic tumor tissue).

[0037]The kit of the present invention can also be used to separate single cells from non-tumor pancreas samples (such as normal pancreas at the site of the tumor), thereby facilitating the analysis of the influence of tumors on the pancreas at the cellular level. Therefore, according to another aspect of the present invention A preferred embodiment, the pancreas sample is the normal pancreas (normal pancreas tissue) where the tumor is located.

[0038] That is, preferably, the pancreatic sample is normal pancreatic tissue and / or pancreatic tumor tissue.

[0039] The present invention will be described in detail below by wa...

Embodiment 1

[0045] This example is used to illustrate the method for isolating single cells from normal pancreatic tissue (normal pancreas at the tumor site) using the kit for isolating single cells of the present invention, wherein the tissue preservation solution, digestion solution, and lysate used are and washing liquid are as shown in Table 1, the enzyme activity unit value and concentration of each component in every liter of digestive juice are as shown in Table 2, and the preparation method of digestive juice is: mix type VIII collagenase, Dispase II and trypsin inhibitor , use a solvent to dissolve the mixed powder, then add DNaseI and mix well.

[0046] The samples were divided into five groups for experimentation (see Table 3), and the specific operation steps were as follows:

[0047] 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...

Embodiment 2

[0069] This example is used to illustrate the method for isolating single cells from pancreatic-derived tumor tissue using the kit for isolating single cells 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 the concentration of each component in every liter of digestive juice (preparation method is the same as embodiment 1) are as shown in I-1 of table 2.

[0070] 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.

[0071] 2) Wash 3 times with sterile PBS.

[0072] 3) Put the sample into a 5 mL sterile EP tube (eppendorf), and add about 500 μl of digestion solution.

[0073] 4) Use sterile ophthalmic scissors to cut the tissue into debris (approximat...

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Abstract

The invention relates to the field of enzyme engineering, and discloses a kit for separating single cells and applications thereof. The kit comprises VIII type collagenase, neutral protease, a trypsininhibitor, and deoxyribonuclease; and the enzyme active unit ratio of VIII type collagenase: neutral protease: a trypsin inhibitor: deoxyribonuclease is 50-90: 0.5-1: 2000-5000: 1. The invention alsodiscloses an application of the kit on separating single cells from a pancreas sample. The provided kit can extract single cells (in particular from pancreas tissues), the yield is high, high activity of single cells is maintained, and the problems that the yield of single cells extracted from pancreas tissues is low and the activity of the single cells is low in the prior art are solved.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to a kit for separating single cells and its application. 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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Patent Type & Authority Applications(China)
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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