CN112322586A - Tumor tissue digestive juice and method thereof - Google Patents

Abstract

The invention discloses a tumor tissue digestive juice and a method thereof, wherein the digestive juice comprises the following components: collagenase type II 2 mg/ml; collagenase type IV 2 mg/ml; dispase (neutral protease) 0.2 ml/ml; DNase 12 units/ml; the volume fraction of FBS is 10 percent; DMEM 0.8 ml/ml; CaCl₂3 mM. The tumor tissue digestive juice provided by the invention is added with Dispase (neutral protease), the protease can better digest and dissociate protein connection between compact tumor cells, and meanwhile, the content of Dispase enzyme is improved, and the digestion efficiency is obviously improved. In addition, the type IV collagenase is added into the digestive fluid of the tumor tissue, the digestion efficiency can be obviously improved, the digestion time is reduced, and the collagenase IV and II in the digestive fluid of the invention have lower concentration, so the cell injury can be reduced, and the cell survival rate in the cell suspension can be improved.

Description

Tumor tissue digestive juice and method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a tumor tissue digestive juice and a method thereof.

Background

With the development of biomedicine, in order to solve the problem that the genetic high-throughput sequencing technology covers the heterogeneity among cells in the research process of oncology, the single-cell transcriptome sequencing technology can reveal the characteristics and the mutual connection of different cells in tumor tissues on a single-cell level, and in order to research the biological characteristics of single tumor cells, the single-cell transcriptome sequencing technology of tumor cells has become the focus of attention of clinical experts and scientific research workers in the aspect of oncology.

The single cell suspension has wide application in the research direction of tumor pathology and tumor molecular biology, and the preparation method of the tissue single cell suspension of the fresh tumor commonly used at present mainly comprises the following steps: tissue dissociation is performed using mechanical and enzymatic treatment, followed by enzymatic digestion for isolation. The enzyme schemes used at present for digesting different tumor tissues are possibly different, sensitive cell types can be damaged in different cell digestion processes, the tissue content of part of tumor tissues after operation is not high, the digestion of cell suspensions after digestion is insufficient, and the subsequent experiment is influenced.

At present, the problems existing in the process of tumor digestion are mainly as follows: cell death caused by incomplete digestion of tissues, long digestion time, overhigh enzyme concentration and the like causes a series of problems in subsequent experiments.

At present, the tumor cell isolation method is to be commonly obtained by a collagenase type II digestion method, but the method has more defects, mainly comprising the following steps: 1. although type II collagenase is a mild digestive enzyme, it still damages cells, especially when it is over-digested, which is very obvious. 2. Collagenase type II is very easy to lose activity in the using process, the digestion effect is reduced, and the digestion and separation of tumor cells can not be realized seriously. 3. After the digestion by using type II collagenase, the tumor cells are easy to be incompletely digested, and the digestion time is too long, which can cause the subsequent single cell sequencing experiment to be seriously influenced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the tumor tissue digestive juice and the method thereof, which have the advantages of high cell acquisition rate, higher activity of cells, high cell yield, less damage to the cells, low change of transcriptome and capability of subsequent culture or single cell transcriptome sequencing in the tumor tissue digestion.

In order to achieve the purpose, the invention adopts the following technical scheme:

a digestive juice for tumor tissue comprises the following components:

2mg/ml of type II collagenase;

collagenase IV 2 mg/ml;

Dispase 0.2ml/ml；

DNase 12 units/ml;

the volume fraction of FBS is 10 percent;

DMEM 0.8ml/ml；

CaCl₂ 3mmol/L。

the invention also provides a method for preparing the tumor tissue single cell suspension by using the tumor tissue digestive juice, which comprises the following steps:

s1: obtaining tumor tissue;

s2: placing the tumor tissue in a plate, and dripping 2-3 drops of the tumor tissue digestive juice of claim 1 to the tumor tissue by using a liquid-moving machine to prevent the tumor tissue digestive juice from drying;

s3: cutting tumor tissue, adding digestive juice of tumor tissue of claim 1 for digestion; placing on a shaking table at 37 deg.C for 30-40min, and mixing with a pipette every 15 min;

s4: after complete digestion, filtering for 2 times and centrifuging;

s5: adding DPBS (platelet-derived plasma) to the sediment obtained by centrifugation in the step S4 to resuspend cell masses, fully blowing the cell masses, and centrifuging the cell masses at 300 g;

s6: and (4) slowly sucking the supernatant, discarding, and fully and uniformly mixing the lower-layer cell sediment to obtain the cell, namely the tumor cell single-cell suspension with higher activity.

Further, the specific process of step S4 is: filtering with 70um filter screen, filtering with 40um filter screen for 2 times, and centrifuging at 400g for 4 min.

The invention has the beneficial effects that: the tumor tissue digestive juice provided by the invention is added with Dispase (neutral protease), the protease can better digest and dissociate protein connection between compact tumor cells, and meanwhile, the content of Dispase enzyme is improved, and the digestion efficiency is obviously improved. In addition, the type IV collagenase is added into the digestive fluid of the tumor tissue, the digestion efficiency can be obviously improved, the digestion time is reduced, and the collagenase IV and II in the digestive fluid of the invention have lower concentration, have less damage to cells and are beneficial to improving the cell survival rate in cell suspension.

Drawings

FIG. 1 is a graph showing the cell viability in the case of the method of example 2 of the present invention;

FIG. 2 is a graph showing the cell viability in the case of the conventional digestion method.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.

Example 1

This example provides a tumor tissue digestive juice, comprising the following components:

type II collagenase 2mg/ml

Collagenase IV 2mg/ml

Dispase (neutral protease) 0.8ml/ml

DNase 12units/ml

The volume fraction of FBS is 10 percent

DMEM 0.8ml/ml

CaCl₂ 3mmol/L

Example 2

This example provides a method for preparing single cell suspension of tumor tissue by using the digestive juice of tumor tissue described in example 1, comprising the following steps:

s1: obtaining tumor tissue;

s2: placing the tumor tissue obtained in the step S1 in a plate, and dropwise adding 2-3 drops of the tumor tissue digestive juice described in example 1 to the tumor tissue by using a liquid-moving machine to prevent drying;

s3: mincing 0.5-2g of tumor tissue, and adding 5ml of the digestive juice of tumor tissue described in example 1 into the tumor tissue for digestion; placing on a shaking table at 37 deg.C for 30-40min, and mixing with a pipette every 15 min;

s4: after digestion was complete, filtration was performed 2 times and centrifugation was performed.

S5: 5ml of DPBS was added to the pellet centrifuged in step S4 to resuspend the cell pellet, and the pellet was centrifuged at 300g after being sufficiently blown off.

S6: slowly sucking 3.5ml of supernatant and discarding, and fully and uniformly mixing the lower layer cell sediment by using a conventional caliber gun head to obtain the cell, namely the tumor cell single cell suspension with higher activity.

In this embodiment, the specific process of step S4 is: filtering with 70um filter screen, filtering with 40um filter screen for 2 times, and centrifuging at 400g for 4 min.

The results of the tumor cell suspensions digested by the conventional digestion method and the tumor cell suspensions digested by the method of example 1 are shown in Table 1.

TABLE 1

The cell viability map of the method of example 2 is shown in FIG. 1, and the cell viability map of the conventional digestion method is shown in FIG. 2. By AO/PI staining, it can be seen that the number of live cells (white, large, brighter spots in FIG. 1, and green in the original) is much greater in the example 2 method than in the dead cells (smaller, darker spots in FIG. 1, and red in the original) in the conventional digestion method (white, larger, brighter spots in FIG. 2 are live cells, green in the original, smaller, darker spots are dead cells, and red in the original).

It should be noted that the conventional digestion method comprises the following steps: taking a tumor tissue sample, cleaning DPBS, shearing the tumor tissue sample into fine fragments by ophthalmic scissors, adding 0.25% (m/v) pancreatin to digest the tumor tissue sample for 30min at 37 ℃, then sucking and removing supernatant, adding 1ml of 0.02% (v/v) type II collagenase to digest the tumor tissue sample overnight, filtering cell suspension by using a 100um cell filter screen after digestion, adding 10% (v/v) FBS high-sugar DMEM culture medium to stop digestion, and centrifuging the cell suspension at 800rpm for 5min to obtain cell sediment.

Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.

Claims (3)

1. The tumor tissue digestive juice is characterized by comprising the following components:

collagenase type II 2 mg/ml;

collagenase type IV 2 mg/ml;

Dispase 0.2ml/ml；

DNase 12 units/ml;

the volume fraction of FBS is 10 percent;

DMEM 0.8ml/ml；

CaCl₂ 3mmol/L。

2. a method for preparing single cell suspension of tumor tissue by using the digestive juice of tumor tissue as claimed in claim 1, comprising the following steps:

s1: obtaining tumor tissue;

s2: placing the tumor tissue in a plate, and dripping 2-3 drops of the tumor tissue digestive juice of claim 1 to the tumor tissue by using a liquid-moving machine to prevent the tumor tissue digestive juice from drying;

s3: cutting tumor tissue, adding digestive juice of tumor tissue of claim 1 for digestion; placing on a shaking table at 37 deg.C for 30-40min, and mixing with a pipette every 15 min;

s4: after complete digestion, filtering for 2 times and centrifuging;

s5: adding DPBS (platelet-derived plasma) to the sediment obtained by centrifugation in the step S4 to resuspend cell masses, fully blowing the cell masses, and centrifuging the cell masses at 300 g;

s6: and (4) slowly sucking the supernatant, discarding, and fully and uniformly mixing the lower-layer cell sediment to obtain the cell, namely the tumor cell single-cell suspension with higher activity.

3. The method according to claim 2, wherein the step S4 is specifically performed by: filtering with 70um filter screen, filtering with 40um filter screen for 2 times, and centrifuging at 400g for 4 min.

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