CN110452874B - Method for obtaining neuron - Google Patents

Method for obtaining neuron Download PDF

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CN110452874B
CN110452874B CN201910839160.8A CN201910839160A CN110452874B CN 110452874 B CN110452874 B CN 110452874B CN 201910839160 A CN201910839160 A CN 201910839160A CN 110452874 B CN110452874 B CN 110452874B
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neurons
lysate
mixture
enzymolysis
papain
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CN110452874A (en
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郑瑞茂
王炳蔚
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Peking University
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Peking University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Abstract

The invention provides a method for obtaining neurons, which belongs to the technical field of biological medicines and comprises the following steps: 1) mixing the nerve nucleus and papain, carrying out enzymolysis and blow beating on the obtained mixture to obtain filtrate; the enzyme activity unit of the papain in the mixture is 18-22U/ml; 2) mixing the filtrate obtained in the step 1) with erythrocyte lysate, and then cracking to obtain a lysate; 3) centrifuging the lysate obtained in the step 2) to obtain a precipitate as neurons. The nerve nucleus is subjected to enzymolysis through papain to release neurons, the obtained filtrate contains red blood cells besides the neurons, and the red blood cells are cracked by red blood cell cracking liquid and then centrifuged to remove cell fragments, so that the neurons are obtained. The average cell body diameter of the neuron is 10 μm, the activity is high, the clustering rate is low, and the method is suitable for 10 x single cell sequencing.

Description

Method for obtaining neuron
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for obtaining neurons.
Background
Single cell sequencing refers to a new technology for performing high-throughput sequencing analysis on genomes, transcriptomes, appearance groups and the like on a single cell level, is used for revealing the gene structure and gene expression state of a single cell, reflects the heterogeneity among cells, and is listed as the leader of six fields which are most worthy of attention in 2013 by Science. The single cell sequencing can be applied to the research of nervous system cells, developmental biology and the like. The nervous system of the brain is composed of thousands of different types of nerve cells, which have a high diversity in cell morphology, synaptic connections and physiological functions. The genome, proteome, chemical molecular composition and metabolism of different types of nerve cells are also very different. The sequencing of the single cell transcriptome analyzes the gene expression condition in each cell by sequencing the RNA of a single neuron, and performs classification comparison on the cells, thereby providing a new direction for neural development, neural regulation and metabolism, drug effect prediction, new drug research and development and the like.
In order to label RNA in each cell individually for data resolution, the cells before labeling must be single suspended and intact living cells, so single cell sequencing requires high requirements on the sample, including cell suspension state, concentration, activity, etc. The neuron further provides a challenge for single cell extraction due to its vulnerability, so that it is necessary to develop a new sample processing method to more conveniently obtain a suitable sample for single cell transcriptome sequencing.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for obtaining neurons, wherein the diameter of the cell body of the neurons obtained by the method is 10 μm on average, the activity is high, the aggregation rate is low, and the method is suitable for 10 × single cell sequencing.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for obtaining neurons, which comprises the following steps:
1) mixing the nerve nucleus and papain, carrying out enzymolysis and blow beating on the obtained mixture to obtain filtrate; the enzyme activity unit of the papain in the mixture is 18-22U/ml;
2) mixing the filtrate obtained in the step 1) with erythrocyte lysate, and then cracking to obtain a lysate;
3) centrifuging the lysate obtained in the step 2) to obtain a precipitate as neurons.
Preferably, the composition of the erythrocyte lysate comprises: 0.1 to 0.2M NH4CL、8～12mM KHCO3、0.05～0.15mM Na2EDTA, 0.03 to 0.08mM DL-AP5 and 0.03 to 0.08mM kynurenine.
Preferably, the volume ratio of the filtrate to the erythrocyte lysate is (0.5-1.5) to (2.5-3.5).
Preferably, the cracking time in the step 2) is 8-12 min.
Preferably, the nerve core group obtained in the step 1) is mixed with papain and also mixed with L-cysteine and Na2Mixing EDTA and DNase-I to obtain a mixture; the concentration of L-cysteine in the mixture is 0.8-1.2M, and Na in the mixture2The concentration of EDTA is 0.4-0.6 mM, and the unit of enzyme activity of DNase-I in the mixture is 90-110U/ml.
Preferably, the conditions for enzymolysis in step 1) include: the temperature of the enzymolysis is 35-42 ℃, the time of the enzymolysis is 50-70 min, the enzymolysis is carried out under oscillation, and the rotation speed of the oscillation is 90-110 rpm.
Preferably, the tool used in the blowing in step 1) is a fire polished glass pasteur pipette.
Preferably, in the step 3), the lysate is placed above a centrifugate and then is centrifuged, wherein the centrifugation temperature is 4-6 min, and the centrifugation force is 280-320 g.
Preferably, the components of the centrifugate comprise: 8-12 mg/ML ovomucin inhibitor and 8-12 mg/ML bovine serum albumin.
Preferably, the obtained neurons are maintained in a cell maintenance fluid comprising the following components: 1XEBSS, 0.04-0.06 mM DL-AP5, 0.04-0.06 mM KA, and 140-160 mM trehalose.
The invention provides a method for obtaining neurons, which comprises the following steps: 1) mixing the nerve nucleus and papain, carrying out enzymolysis and blow beating on the obtained mixture to obtain filtrate; the enzyme activity unit of the papain in the mixture is 18-22U/ml; 2) mixing the filtrate obtained in the step 1) with erythrocyte lysate, and then cracking to obtain a lysate; 3) centrifuging the lysate obtained in the step 2) to obtain a precipitate as neurons. The nerve nucleus is subjected to enzymolysis through papain to release neurons, the obtained filtrate contains red blood cells besides the neurons, and the red blood cells are cracked by red blood cell cracking liquid and then centrifuged to remove cell fragments, so that the neurons are obtained. The average cell body diameter of the neuron is 10 μm, the activity is high, the clustering rate is low, and the method is suitable for 10 x single cell sequencing.
Drawings
FIG. 1 is a test of neurons obtained in example 1;
FIG. 2 is a test of neurons obtained in comparative example 1.
Detailed Description
The invention provides a method for obtaining neurons, which comprises the following steps:
1) mixing the nerve nucleus and papain, performing enzymolysis, beating, and filtering to obtain filtrate; the enzyme activity unit of the papain in the mixture is 18-22U/ml;
2) mixing the filtrate obtained in the step 1) with erythrocyte lysate, and then cracking to obtain a lysate;
3) centrifuging the lysate obtained in the step 2) to obtain a precipitate as neurons.
Mixing a nerve nucleus and papain, carrying out enzymolysis, beating and filtering on the obtained mixture to obtain a filtrate; the unit of the enzyme activity of the papain in the mixture is 18-22U/ml.
In the present invention, the nucleus pulposus is preferably obtained from brain tissue, the brain tissue is preferably from a mouse, and the brain tissue is preferably minced by forceps of the ophthalmology department and then is in a minced state.
In the invention, the unit of the enzyme activity of the papain in the mixture is 18-22U/ml, and preferably 20U/ml. In the invention, the papain can carry out enzymolysis on intercellular protein skeletons in brain tissues and release neurons. In the present invention, when the nerve core group is mixed with papain, L-cysteine and Na are preferably added2Mixing EDTA and DNase-I to obtain a mixture; the concentration of the L-cysteine in the mixture is preferably 0.8-1.2M, and more preferably 1M; na in the mixture2The concentration of EDTA is preferably 0.4-0.6 mM, more preferably 0.5M; the enzyme activity unit of DNase-I in the mixture is preferably 90-110U/ml, and more preferably 100U/ml. In the present invention, the L-cysteine functions to protect neurons. In the present invention, the Na is2EDTA as a metal chelating agent, chelating Ca2+And the like, and after chelating with the ions, the separation of the cells and the culture dish is accelerated, and the enzymolysis is promoted. In the present invention, the DNase-I functions to reduce cell clumping.
In the present invention, the conditions for the enzymatic hydrolysis preferably include: the enzymolysis temperature is preferably 35-42 ℃, and more preferably 37 ℃; the enzymolysis time is preferably 50-70 min, and more preferably 60 min; the enzymolysis is preferably carried out under the oscillation, and the rotation speed of the oscillation is preferably 90-110 rpm, and more preferably 100 rpm. The present invention preferably uses a fire polished glass pasteur pipette to perform the gentle pipetting. In the present invention, the pore size of the filter used for the filtration is preferably 20 μm. In the present invention, the filtrate contains red blood cells in addition to neurons.
The obtained filtrate is mixed with erythrocyte lysate and then cracked to obtain lysate.
In the invention, the volume ratio of the filtrate to the erythrocyte lysate is preferably (0.5-1.5): (2.5-3.5)More preferably 1: 3. In the present invention, the components of the erythrocyte lysate preferably include: 0.1 to 0.2M NH4CL、8～12mMKHCO3、0.05～0.15mM Na2EDTA, 0.03 to 0.08mM DL-AP5 and 0.03 to 0.08mM kynurenine; more preferably, it comprises: 0.15M NH4CL、10mM KHCO3、0.1mM Na2EDTA, 0.05mM DL-AP5 and 0.05mM kynurenine. In the invention, the cracking time is preferably 8-12 min, and more preferably 10 min. In the present invention, the filtrate and the erythrocyte lysate are mixed and then left on ice for lysis. In the present invention, the lysis lyses red blood cells into cell debris.
The present invention centrifuges the obtained lysate to obtain the precipitate as neuron.
According to the invention, the lysate is preferably placed above the centrifugate and then centrifuged, wherein the centrifugation temperature and time are preferably 4-6 min, and more preferably 5 min; the centrifugal force of the centrifugation is preferably 280-320 g. More preferably 300 g. In the present invention, the centrifugation removes the cell debris and the resulting pellet is a neuron.
The neuron obtained by the invention has the average cell body diameter of 10 mu m, high activity and low clustering rate, and is suitable for 10 multiplied by single cell sequencing.
In the present invention, the components of the centrifugate preferably comprise: 8-12 mg/ML ovomucin inhibitor and 8-12 mg/ML bovine serum albumin, more preferably 10mg/ML ovomucin inhibitor and 10mg/ML bovine serum albumin.
The present invention preferably maintains the obtained neurons in a cell maintenance solution, the cell maintenance solution preferably comprising: 1X EBSS, 0.04-0.06 mM DL-AP5, 0.04-0.06 mM KA and 140-160 mM trehalose; more preferably 1XEBSS, 0.05mM DL-AP5, 0.05mM KA, and 150mM trehalose.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Isolation of C57BL/6J neurons
Experimental animals: 8 weeks of C57BL/6J 5
(I) preparation of reagents
1. The neuron digestive juice comprises 20U/ML papain, 1mM L-cysteine and 0.5mM Na2EDTA，100U/MLDNase-I。
2. Erythrocyte lysate: 0.15M NH4CL，10mM KHCO3，0.1mM Na2EDTA, 0.05mM DL-AP5, 0.05mM kynurenine (Kynurenic acid, KA).
3. Gradient centrifugate: the gradient centrifugation solution was 10mg/ML ovomucin inhibitor and 10mg/ML bovine serum albumin.
4. Cell maintenance solution: 1X EBSS, 0.05mM DL-AP5, 0.05mM KA, 150mM trehalose.
(II) neuronal isolation
1.37 ℃ preheating: the neuron digestive juice is preheated in a carbon dioxide constant temperature incubator.
2. A plastic petri dish, 1.5ml sterile centrifuge tube was placed on ice for pre-cooling.
3. Taking out the nerve nucleus, and cutting to less than 1mm with surgical blade3The pieces were quickly placed into a pre-sterilized 1.5ml sterile centrifuge tube.
1ml of neuron digestive juice is added into a 4.1.5ml centrifuge tube, a sealing membrane is sealed, and the neuron digestive juice is digested for 60 minutes in a constant temperature shaking water bath (100 r/min) at 37 ℃.
5. The cell suspension was formed by gently pipetting with a fire polished glass pasteur pipette.
6. The cell suspension was filtered through a 20 μm screen into a fresh sterile 1.5ml centrifuge tube.
(II) lysis of erythrocytes
Mixing the filtered cell suspension and the cell lysate according to the volume ratio of 1:3, turning upside down and uniformly mixing, standing on ice for 10 minutes, and slightly mixing once again in the middle to ensure that the red blood cells are fully lysed.
(III) gradient centrifugation to remove cell debris
The gradient centrifugation solution is firstly placed in a 15ml centrifuge tube, and then the cell suspension after the red blood cells are cracked is added above the gradient centrifugation solution. The addition amount is the total volume of the lysed cells: the solution was centrifuged at 300 × g for 5min at 1:5 gradient.
(III) neuronal resuspension and cell viability assay
1. The supernatant was carefully removed, leaving only the centrifuged cell pellet.
2. The neurons were resuspended in Cell maintenance fluid, Cell counter for Cell counting. The results are shown in FIG. 1.
As can be seen from FIG. 1, the cell activity was 89.8%, the average cell diameter was 10.2 μm, and the single cell content was 94.0%.
Comparative example 1
Erythrocyte lysate: 0.15M NH4CL，10mM KHCO3，0.1mM Na2EDTA。
1X EBSS。
The other conditions were the same as in example 2, and the results are shown in FIG. 2.
As can be seen from FIG. 2, the cell activity was 66.1%, the average cell diameter was 9.1 μm, and the single cell content was 90.0%.
The embodiment can show that the neuron obtained by the method provided by the invention has high activity and low clustering rate, and is suitable for 10 x single cell sequencing.
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 (2)

1. A method of obtaining neurons, comprising the steps of:
1) mixing the nerve nucleus and papain, performing enzymolysis, beating, and filtering to obtain filtrate; the enzyme activity unit of the papain in the mixture is 18-22U/ml;
2) mixing the filtrate obtained in the step 1) with erythrocyte lysate, and then cracking to obtain a lysate;
3) centrifuging the lysate obtained in the step 2) to obtain a precipitate as neurons;
when the nerve nucleus is mixed with papain, the mixture is also mixed with L-cysteine and Na2Mixing EDTA and DNase-I to obtain a mixture; the concentration of L-cysteine in the mixture is 0.8-1.2M, and Na in the mixture2The concentration of EDTA is 0.4-0.6 mM, and the unit of enzyme activity of DNase-I in the mixture is 90-110U/ml;
placing the lysate above a centrifugate and then centrifuging, wherein the temperature time of centrifugation is 4-6 min, and the centrifugal force of centrifugation is 280-320 g;
the components of the centrifugate comprise: 8-12 mg/ML ovomucin inhibitor and 8-12 mg/ML bovine serum albumin;
the erythrocyte lysate comprises the following components: 0.1 to 0.2M NH4CL、8～12mM KHCO3、0.05～0.15MmNa2EDTA, 0.03 to 0.08mM DL-AP5 and 0.03 to 0.08mM kynurenine;
the volume ratio of the filtrate to the erythrocyte lysate is 0.5-1.5: 2.5-3.5;
the cracking time in the step 2) is 8-12 min;
the enzymolysis condition in the step 1) comprises the following steps: the temperature of the enzymolysis is 35-42 ℃, the time of the enzymolysis is 50-70 min, the enzymolysis is carried out under oscillation, and the rotation speed of the oscillation is 90-110 rpm;
maintaining the obtained neurons in a cell maintenance solution, wherein the cell maintenance solution comprises the following components: 1X EBSS, 0.04-0.06 mM DL-AP5, 0.04-0.06 mM KA, and 140-160 mM trehalose.
2. The method of claim 1, wherein the tool used in the step 1) of whipping is a fire polished glass pasteur pipette.
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