CN111088222A - Preparation method of single cell suspension of adipose tissues - Google Patents
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Abstract
The invention relates to the technical field of cells, in particular to a preparation method of a single cell suspension of adipose tissues. The invention combines six enzymes of neutral protease, collagenase I, collagenase II, collagenase IV, trypsin and DNAse to carry out enzymolysis on adipose tissues for preparing single cell suspension. The method has simple steps, and the obtained cells have high number and high cell survival rate, wherein the total number of the viable cells is 9.13 × 105～1.12×107The cell/g adipose tissue has the cell survival rate of 80-88%, and the cell number and the cell survival rate are obviously superior to those of the comparison method. Meanwhile, experiments show that the single cell suspension prepared by the invention is suitable for sequencing 10xGenomics single cell transcriptome, and can also be used for primary culture of tissue cells and cell analysis.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a preparation method of a single cell suspension of adipose tissues.
Background
With the rapid development and popularization of high-throughput Sequencing technologies, researchers at the front of life sciences are almost talking about the significance and value of Single-Cell Sequencing for the whole life sciences research. A very important research direction for sequencing of the single cell transcriptome is to redefine cell types under the single cell resolution and mine new cell subsets and marker genes.
Based on the latest chromosome Single Cell 3' system of a 10x Genomics platform, the method can rapidly mark, sequence and analyze the Single Cell, efficiently obtain the Single Cell level digital gene expression profile of as many as 10000 cells, realize the deep analysis of complex Cell groups, perfectly overcome the problems of rare sequencing samples, Cell heterogeneity and the like, and is widely applied. The main application fields of single cell sequencing are the research fields of human medicine, neurobiology, genetic reproduction, stem cells and the like, and the types of the related main samples are very wide, but the single cell sequencing can be divided into the following categories according to the sources: 1) tumor tissues such as brain tumor, liver cancer, gastric cancer, breast cancer, etc.; 2) primary isolated cells from solid tissues, such as adipocytes, brain neurons, epithelium and dry cells from intestinal tissues, etc.; 3) certain subpopulations of cells of blood origin (which can be FACS sorted, typically by different biorarkers), such as by a population of 5-color labeled hematopoietic stem cells; 4) stem cells induce differentiation/reprogramming related cell samples.
Adipose tissue is the energy reservoir in the body and is also a very important endocrine organ of human and animal body, and fat metabolism is the basic energy metabolism mode of the life body. Abnormal differentiation and proliferation of adipocytes can cause excessive accumulation of adipose tissue, which easily leads to obesity and insulin resistance. The adipose-derived stem cells have the functions of identifying damaged parts, regulating immunity and repairing damage, and have wide clinical research and application prospects. The development of adipocytes involves the proliferation and differentiation of Adipocyte Stem Cells (ASCs) and the close interaction with host immune cells. The development mechanism of different parts is different, and the heterogeneity of the adipose stromal cells cannot be analyzed by the past research means. Researchers identify the types of stromal cells and explore the differentiation tracks of the adipose-derived stem cells by using a single-cell transcriptome sequencing technology, and the clustering result shows that the adipose-derived stem cell population can be clustered into eight sub-cell populations including four adipose-derived stem cell subsets, which indicates that the adipose-derived stem cells have heterogeneity. Researchers have also analyzed different subsets of mouse subcutaneous adipose Stromal Vascular Fraction (SVF) adipose stem cells and precursor cells, identifying a subset of cells that not only cannot differentiate into adipocytes, but also can inhibit Adipogenesis in a paracrine fashion in vivo/in vitro, named Adipogenesis modulating cells (Aregs). This study points to a potentially critical role for Aregs: regulating plasticity of adipose tissue, and improving the ability to control obesity and insulin resistance, thereby treating metabolic diseases including type 2 diabetes.
10XGenomics single cell transcriptome sequencing is a microfluid platform established on GemCode technology, and gel beads with bar codes and primers and single cells are wrapped in oil drops; next within each oil droplet, the gel beads are lysed and the cells lysed to release mRNA, which by reverse transcription generates barcoded cDNA for sequencing; after the liquid oil layer is damaged, cDNA is constructed subsequently, and then an Illumina sequencing platform is used for sequencing and detecting the library, so that a large amount of gene expression data of single cells can be obtained at one time, and the purpose of performing expression sequencing at the single cell level is realized. Selecting proper single cell preparation method to obtain optimal single cell suspension of adipose tissue, ensuring that the cell survival rate reaches more than 80 percent, the cell loss is less, the suspension is clean and has no fragments, and the method is the first factor of success and failure of the technology!
Although the prior adipose tissue primary cell separation method is mature, the prior adipose tissue single cell separation method for single cell sequencing is still lack of research, and a new method is needed to improve the cell yield, the cell viability, the cell debris and the erythrocyte residue.
Disclosure of Invention
In view of the above, the present invention is directed to a method for preparing a single cell suspension of adipose tissue, which can obtain a single cell suspension with high cell viability, large number of cells and good state.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method of preparing a single cell suspension of adipose tissue comprising:
step 1: taking adipose tissues, and carrying out enzymolysis by using a complex enzymolysis liquid;
step 2: filtering and centrifuging the product after enzymolysis, taking cell sediment, and adding erythrocyte lysate for cracking;
and step 3: centrifuging the cracked product, taking cell sediment, cleaning and resuspending to obtain a single cell suspension;
the compound enzymolysis solution contains neutral protease, collagenase I, collagenase II, collagenase IV, trypsin and DNAse.
The invention adopts the compound enzyme composed of neutral protease, collagenase I, collagenase II, collagenase IV, trypsin and DNAse to carry out enzymolysis on adipose tissues for preparing the single cell suspension, and experiments show that the single cell suspension prepared by the invention has the advantages of large cell number, high activity rate and good state, and can be used for sequencing 10XGenomics single cell transcriptome.
In step 1 of the invention, adipose tissues are pretreated before enzymolysis, wherein the pretreatment is specifically to rinse the adipose tissues for three times by using a DMEM (DMEM) culture medium, remove blood vessels and surface membrane structures in the tissues and then cut the tissues into 1-3mm3Tissue mass of size.
In the step 1 of the invention, in the complex enzyme, the working concentration of the collagenase I is 200-400u/mL or 1.0-2.0 mg/mL; the working concentration of the collagenase II is 100-200u/mL or 0.5-1.0 mg/mL; the working concentration of the collagenase IV is 50-200u/mL or 0.25-1.0 mg/mL; the working concentration of the neutral protease is 0.25-0.5 mg/mL; the working concentration of the trypsin is 0.25-0.5 mg/mL; the working concentration of DNAse is 5-20u/mL or 0.01-0.02 mg/mL.
In some specific embodiments, in the complex enzyme, the working concentrations of collagenase I, collagenase II, collagenase iv, neutral protease, trypsin and DNAse are 1.0mg/ml, 0.5mg/ml, 0.25mg/ml and 0.01mg/ml in sequence, that is, the mass ratio is 100:50:25: 25:1.
In step 1 of the invention, the solvent of the composite enzymolysis solution is a basal culture medium or an enzyme buffer solution.
In some embodiments, the complex enzymatic hydrolysate consists of the complex enzyme and an enzyme buffer; preferably, the concentration of the complex enzyme is 0.5-5 mg/ml. In some specific embodiments, the complex enzymatic hydrolysate consists of the complex enzyme and PBS buffer solution, and the concentration of the complex enzyme in the complex enzymatic hydrolysate is 4.52 mg/ml.
In some embodiments, the complex enzymatic hydrolysate consists of the complex enzyme and a basal medium; preferably, the concentration of the complex enzyme in the complex enzymolysis liquid is 0.5-5 mg/ml, and more preferably 2.26 mg/ml.
In some embodiments, the volume ratio of the adipose tissues to the composite enzymolysis liquid in the step 1 is 1: 5-10.
In some embodiments, in the step 1, the temperature of the enzymolysis is 35-38 ℃, and the time of the enzymolysis is 20-40 min; the enzymatic hydrolysis was terminated with complete medium containing serum.
In some embodiments, in step 2, the filtration is 70 μm cell sieve filtration; the centrifugation is 300g to 400g for 5 min; the cleaning is to take cell sediment for re-suspending by DMEM culture solution, centrifuge for 10-20min by Percoll discontinuous density gradient centrifugate 3000g, remove cell debris and supernatant, re-suspend cells by PBS buffer solution, centrifuge for 5min by 500g, and discard supernatant. Wherein, the density of the Percoll discontinuous density gradient centrifugate is preferably 1.19-1.31 mg/ml.
Centrifuging the cracked product, taking cell sediment, cleaning and resuspending to obtain a single cell suspension; wherein the centrifugation is 300 g-400 g for 5 min; the washing is to take the cell sediment to be resuspended by PBS buffer solution, centrifuge for 5min at 200g, remove the residual erythrocyte lysate and debris, and collect the bottom cells.
Wherein the resuspension is resuspended in PBS buffer containing 0.5% BSA, the number of resuspensions is two, and a step of filtering with a 40 μm cell sieve is further included between the two resuspensions.
In some embodiments, step 3 is specifically: centrifuging 300-400 g of the cracking product for 5 min; removing supernatant, taking cell sediment, and washing the cell sediment by using PBS buffer solution, wherein the washing steps are as follows: adding PBS buffer solution to resuspend the cells, centrifuging for 5min at 200g and 150-. The washed cells were resuspended in PBS buffer containing 0.5% BSA, filtered using a 40 μm cell sieve, and then resuspended in PBS buffer (0.5% BSA) to obtain a single cell resuspension.
The invention adopts the compound enzyme composed of neutral protease, collagenase I, collagenase II, collagenase IV, trypsin and DNAse to carry out enzymolysis on adipose tissues for preparing single cell suspension, the method has simple steps, the obtained cells have a large quantity, and the total number of the living cells is 1.06～1.12×107The cell has high cell viability rate which is 78-88%, and the cell number and the cell viability rate are obviously superior to those of the cell prepared by the comparative method. Meanwhile, experiments show that the single cell suspension prepared by the invention is suitable for sequencing 10xGenomics single cell transcriptome, and can also be used for primary culture of tissue cells and cell analysis.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the cell viability of single cell suspensions, wherein FIG. 1A is a single cell suspension prepared in example 1, FIG. 1B is a single cell suspension prepared in comparative example 1, and FIG. 1C is a single cell suspension prepared in comparative example 2;
FIG. 2 is a schematic diagram showing the labeling of adipose tissue single cells and library construction;
FIG. 3 is a schematic diagram showing quality inspection of a single cell library of adipose tissue, in which FIG. 3A shows the analysis result of a cDNA library constructed using a single cell suspension of example 1, and FIG. 3B shows the analysis result of a standard DNA library;
FIG. 4 is a schematic diagram showing the analysis of the sequencing data of the adipose tissue single Cell library, wherein FIG. 4A is the QC result of data sequencing, and FIGS. 4B to 4C are the results of Cell Range analysis of single Cell data.
Detailed Description
The invention discloses a preparation method of a single cell suspension of adipose tissues, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of Single cell suspension of adipose tissue
Preparing a composite enzymolysis liquid: the compound enzyme is prepared from a compound enzyme and a basic culture medium, wherein the concentration of the compound enzyme is about 2.26mg/ml, and the mass ratio of collagenase I, collagenase II, collagenase IV, neutral protease, trypsin and DNAse in the compound enzyme is 100:50:25:25:25:1(1.0mg/ml, 0.5mg/ml, 0.25mg/ml and 0.01 mg/ml).
(1) Obtaining and preprocessing adipose tissues: adipose tissues were taken from adipose tissue waste of a professional hospital or beauty salon. Rinsing adipose tissue with DMEM medium for three times, removing blood vessel and surface membrane structure in tissue, weighing 1g tissue, and cutting into 1mm with scissors or surgical blade3Size;
(2) adding complex enzymolysis solution 5-6 times of adipose tissue volume for digestion treatment, and dissociating for 20-30min in combination with a Meitian and whirlpool single cell preparation instrument;
(3) adding 5ml of complete culture medium containing serum, stopping, filtering by a 70-micron cell sieve, centrifuging for 5min by 300-400 g, and then separating the cell suspension into 3 layers: grease, digestive juice and sediment;
(4) carefully remove the lipid layer and the digestive layer to obtain cells, and resuspend the cells using 2ml DMEM;
(5) observing under a microscope, and focusing on the quantity of red blood cells and fragments and counting cells (an automatic cell counter);
(6) centrifuging for 5min at 300-400 g to obtain cell sediment, re-suspending cells by using 1-2ml of PBS, adding 2-4ml of density gradient centrifugate according to the proportion of 1:2, gently mixing, then adding 3-4ml of PBS to cover, performing density gradient centrifugation to remove cell debris, centrifuging 15ml of centrifuge tube at 3000g for 10-20min, wherein the debris is at the interface, the cells are at the bottom, and the debris is remained on the cell;
(7) removing debris and supernatant, resuspending with PBS, centrifuging at 500g (2000rpm) for 5min, and removing supernatant;
(8) adding 1x erythrocyte lysate for removing erythrocytes, processing for 2-3min in a dark place, and centrifuging for 5min at 300-400 g;
(9) removing supernatant, adding PBS to resuspend cells, centrifuging at 200g for 5min, and removing residual erythrocyte lysate and debris;
(10) PBS buffer (0.5% BSA) resuspended cells, filtered using a 40 μm cell sieve;
(11) PBS buffer (0.5% BSA) resuspended cells to obtain a single cell resuspension.
Example 2 preparation of a Single cell suspension of adipose tissue
Preparing a composite enzymolysis liquid: the compound enzyme is prepared from compound enzyme and PBS, the concentration of the compound enzyme is about 4.52mg/ml, wherein in the compound enzyme, the mass ratio of collagenase I, collagenase II, collagenase IV, neutral protease, trypsin and DNAse is 100:50:25:25: 1(2.0mg/ml, 1.0mg/ml, 0.5mg/ml, 0.02 mg/ml).
(1) Obtaining and preprocessing adipose tissues: adipose tissues were taken from adipose tissue waste of a professional hospital or beauty salon. Rinsing adipose tissue with DMEM medium for three times, removing blood vessel and surface membrane structure in tissue, weighing 1.5g tissue, and cutting into 1mm with scissors or surgical blade3Size;
(2) adding 6 times of complex enzymolysis solution of adipose tissue volume for digestion treatment, and dissociating for 30min by combining with a Meitian and whirly single cell preparation instrument;
(3) adding 5ml of complete culture medium containing serum, stopping, filtering by a 70-micron cell sieve, centrifuging for 5min by 300-400 g, and then separating the cell suspension into 3 layers: grease, digestive juice and sediment;
(4) carefully remove the lipid layer and the digestive layer to obtain cells, and resuspend the cells using 2ml DMEM;
(5) observing under a microscope, and focusing on the quantity of red blood cells and fragments and counting cells (an automatic cell counter);
(6) after 1-1.5ml PBS is used for resuspending cells, adding 2-3ml density gradient centrifugate according to the proportion of 1:2, gently mixing, then adding 2-3ml PBS for covering, performing density gradient centrifugation to remove cell fragments, carrying out 15ml centrifuge tube, 3000g, 10-20min, wherein the fragments are at the interface, the cells are at the bottom, and the fragments are remained on the surface;
(7) removing debris and supernatant, resuspending with PBS, centrifuging at 500g (2000rpm) for 5min, and removing supernatant;
(8) adding 1x erythrocyte lysate for removing erythrocytes, processing for 2-3min in a dark place, and centrifuging for 5min at 300 g-400 g;
(9) removing supernatant, adding PBS to resuspend cells, centrifuging at 200g for 5min, and removing residual erythrocyte lysate and debris;
(10) PBS buffer (0.5% BSA) resuspended cells, filtered using a 40 μm cell sieve;
(11) PBS buffer (0.5% BSA) resuspended cells to obtain a single cell resuspension.
Example 3
Preparing a composite enzymolysis liquid: the enzyme composition is prepared from complex enzyme and DMEM, wherein the concentration of the complex enzyme is about 4.52mg/ml, and the mass ratio of collagenase I, collagenase II, collagenase IV, neutral protease, trypsin and DNAse in the complex enzyme is 100:50:25:25:25:1(2.0mg/ml, 1.0mg/ml, 0.5mg/ml and 0.02 mg/ml).
Other conditions were the same as in example 1.
Comparative examples 1 to 4
Comparative example 1: preparing single cell suspension by adopting a kit made in China;
comparative example 2: the German brand MeitianNi kit-adipose tissue dissociation kit is adopted to prepare the single cell suspension (product goods number: 130-
Comparative example 3: preparing a composite enzymolysis liquid: the compound enzyme is prepared from compound enzyme and DMEM, wherein the concentration of the compound enzyme is about 3.52mg/ml, and the mass ratio of collagenase I, collagenase II, collagenase IV and DNAse in the compound enzyme is 100:50:25:1(2.0mg/ml, 1.0mg/ml, 0.5mg/ml and 0.02 mg/ml). Other conditions were the same as in example 1.
Comparative example 4: preparing a composite enzymolysis liquid: the enzyme composition is prepared from a compound enzyme and DMEM, wherein the concentration of the compound enzyme is about 3.52mg/ml, and the mass ratio of collagenase I, collagenase II, trypsin and DNAse in the compound enzyme is 100:50:25:1(2.0mg/ml, 1.0mg/ml, 0.5mg/ml and 0.02 mg/ml). Other conditions were the same as in example 1.
The adipose tissues adopted in comparative examples 1-4 have the same source and the same material taking method as in example 1.
Example 4
Trypan blue staining, microscopic observation, cell counting and viability detection are carried out on the single cell suspensions of examples 1-3 and comparative examples 1-4, the number of cells obtained per 1g of adipose tissue is counted, and the cell number and viability detection results of different methods are shown in tables 1-2 and FIG. 1. Wherein, samples 1-3 are from three different adipose tissues respectively.
TABLE 1 comparison of cell viability rates
TABLE 2 comparison of cell numbers
As can be seen from the results in tables 1 to 2, the number of cells and the cell viability rate obtained by the method provided by the invention are significantly higher than those of the comparative method and the comparative kit (P < 0.05).
Example 510 XGenomics adipose tissue Single cell labeling and library construction
The adipose tissue Single Cell suspension prepared in the example 1 is coated with the glue beads with the Cell tag sequence and the cells in the liquid drops by utilizing a microfluidic chip, the liquid drops coated with the cells are collected, the cells are cracked in the liquid drops, mRNA in the cells is connected with Cell Barcode on the bead to form Single Cell GEMs, RT reaction is carried out in the liquid drops, then emulsion breaking is carried out, and cDNA library construction is carried out. The cell Barcode on the library sequence can distinguish which cell the target sequence comes from, and the sample the target sequence comes from can distinguish by sample Index on the sequence. The GEM is formed by the cells and the reaction reagent in one channel on the microfluidic chip and the glue beads in the other channel. Reverse transcription was performed independently in each GEM, after which the tagged cdnas were mixed, amplified and library constructed. A schematic diagram of 10XGenomics adipose tissue single cell labeling and library construction is shown in FIG. 2.
Example 610 XGenomics adipose tissue Single cell library quality inspection
The library constructed in example 5 was subjected to quality inspection, and the results are shown in FIG. 3: (1) the Qubit 4.0 quantifies the library concentration, and the mass concentration is 28.6 ng/ul; (2) agilent 2100 detects the integrity of the library DNA fragment and the size of the inserted fragment, and the average fragment size is 426 bp; (3) the Q-PCR method accurately quantifies the effective concentration of the library, and the concentration is 82.8 nM. The results show that the library prepared from the single cell suspension prepared in example 1 of the present invention meets the sequencing requirements (the single cell library can be sequenced under the conditions that the size of the inserted fragment is qualified, the peak type is single, no hybrid peak, no linker and no primer dimer are generated, and the Q-PCR concentration is more than or equal to 4 nM.).
Example 710 XGenomics adipose tissue single cell library sequencing and data analysis
Sequencing is carried out by using Illumina Novaseq 6000 platform gallery, so that 108.98G is co-produced in total Raw data, and 107.08G is co-produced in total filtered Clean data. According to the unique library structure of 10x scRNA-Seq, the Barcode and UMI insert parts of reads are split, then the insert parts are aligned to a reference genome, then the proportion of each region is counted, and expression quantity statistics is carried out.
The library prepared in example 5 was sequenced by the above method, and the result is shown in fig. 4, and mRNA expression profile information of a total of 5332 single cells was obtained, the average cell basis factor was 3992, the sequencing depth was about 70k Reads/cell, and the expected standard was met.
The above experiments were carried out on the single cell suspensions prepared in examples 2-3, and the experimental results were similar to those of example 1, and were different (P > 0.05).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for preparing a single cell suspension of adipose tissue, comprising:
step 1: taking adipose tissues, and carrying out enzymolysis by using a complex enzymolysis liquid;
step 2: filtering and centrifuging the product after enzymolysis, taking cell sediment, and adding erythrocyte lysate for cracking;
and step 3: centrifuging the cracked product, taking cell sediment, cleaning and resuspending to obtain a single cell suspension;
the compound enzymolysis solution contains neutral protease, collagenase I, collagenase II, collagenase IV, trypsin and DNAse.
2. The method as claimed in claim 1, wherein in step 1, the working concentration of collagenase I in the complex enzyme is 200-; the working concentration of the collagenase II is 100-200u/mL or 0.5-1.0 mg/mL; the working concentration of the collagenase IV is 50-200u/mL or 0.25-1.0 mg/mL; the working concentration of the neutral protease is 0.25-0.5 mg/mL; the working concentration of the trypsin is 0.25-0.5 mg/mL; the working concentration of DNAse is 5-20u/mL or 0.01-0.02 mg/mL.
3. The method according to claim 1, wherein in step 1, the solvent of the complex enzymatic hydrolysate is a basal medium or a PBS buffer.
4. The preparation method of claim 1, wherein in the step 1, the concentration of the complex enzyme in the complex enzymolysis solution is 0.5-5 mg/ml.
5. The method according to claim 1, wherein in step 1, the volume ratio of the adipose tissue to the composite enzymatic hydrolysate is 1: 5-10.
6. The preparation method according to claim 1, wherein in the step 1, the temperature of the enzymolysis is 35-38 ℃, and the time of the enzymolysis is 20-40 min; the enzymatic hydrolysis was terminated with complete medium containing serum.
7. The method according to claim 1, wherein in step 2, the filtration is 70 μm cell sieve filtration; the centrifugation is 300g to 400g for 5 min; the cleaning is to take cell sediment for re-suspending by DMEM culture solution, centrifuge for 10-20min by Percoll discontinuous density gradient centrifugate 3000g, remove cell debris and supernatant, re-suspend cells by PBS buffer solution, centrifuge for 5min by 500g, and discard supernatant.
8. The method according to claim 1, wherein in step 3, the centrifugation is performed for 5min at 300-400 g; the washing is to take cell sediment for resuspension by PBS buffer solution, centrifuge for 5-10min at 200g, remove residual erythrocyte lysate and debris, and collect bottom cells.
9. The method according to claim 1, wherein the resuspension is performed in step 3 by resuspending in PBS buffer containing 0.5% BSA twice, and a filtration step with a 40 μm cell sieve is further included between the resuspensions.
10. The method according to any one of claims 1 to 9, wherein the enzyme is used in the production of the enzymeBefore the solution, the method also comprises the step of pretreating the adipose tissues, wherein the pretreatment comprises the steps of rinsing the adipose tissues for three times by using a DMEM (DMEM) culture medium, removing blood vessels and surface membrane structures in the tissues, and then shearing the tissues into 1-3mm3Tissue mass of size.
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| CN112226406A (en) * | 2020-10-19 | 2021-01-15 | 中国医学科学院北京协和医院 | Preparation method of human perivascular adipose tissue single cell suspension |
| CN113604539A (en) * | 2021-08-06 | 2021-11-05 | 上海欧易生物医学科技有限公司 | Low-temperature dissociation kit suitable for single cell sequencing and application thereof |
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| CN113604539A (en) * | 2021-08-06 | 2021-11-05 | 上海欧易生物医学科技有限公司 | Low-temperature dissociation kit suitable for single cell sequencing and application thereof |
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