CN116286605A - Reagent, kit and method for preparing fibrotic liver single cell suspension - Google Patents

Abstract

The invention discloses a reagent, kit and method for preparing fibrotic liver single cell suspension. The reagents of the present invention at least include pronase, DNase, type II collagenase, HBSS, NaHCO ₃ , EDTA, FBS and Optiprep separation solution. The present invention utilizes pronase to reduce the viscosity of gelatin and mucin, DNase to degrade DNA and avoid breaking cells to release DNA in the separation process to increase suspension viscosity, good dissociation ability of type II collagenase and the effect of digesting liver, EDTA chelation Calcium ions further avoid cell aggregation; by adding the above components during the separation process, a liver single cell suspension that is fully dissociated from the extracellular matrix components can be efficiently obtained. At the same time, the HBSS solution is used to prepare the buffer throughout the separation process, and the glucose contained in it provides energy supply for living cells, so as to avoid changes in the cell transcriptome caused by the lack of energy substances during the cell separation process to the greatest extent.

Description

Reagent, kit and method for preparing fibrotic liver single cell suspension

technical field

The invention relates to a reagent, kit and method for preparing fibrotic liver single cell suspension, belonging to the technical field of cell separation.

Background technique

With the rise of single-cell omics technology, the isolation of high-quality single cells in various tissues has important scientific significance and application value for accurately understanding the changes of tissues under physiological and pathological conditions. However, compared with the previous flow cytometric detection and sorting, single-cell omics research puts forward higher requirements on the viability of the cells in the obtained cell suspension, whether it is strictly single cells, and the cleaning of cell debris. The cellular composition of the liver is complex and highly heterogeneous. The development of single-cell omics technology is of great significance for in-depth study of the changes of different cells in physiological and pathological states and their impact on the disease process. Single-cell omics research has extremely high requirements for the preparation of high-quality single-cell suspensions, which require living single-cell suspensions with high cell viability, no aggregation and adhesion, and no selection bias during the separation process. The previous method of separating liver single cells, whether collagenase digestion in vitro or liver in situ perfusion digestion, was mainly aimed at healthy liver and liver tumors. However, in the mouse model of liver fibrosis, a large number of collagen fibers in the liver are deposited and intertwined, the microstructure of the liver is changed, and the structure of the lobules is remodeled; the original separation method has low cell yield and more cell clumps or adhesions, which is difficult to obtain. True single cells are obtained, and with prolonged digestion, cell viability is affected and may be biased by inconsistent degrees of dissociation of different cell types.

Contents of the invention

The purpose of the present invention is: the traditional animal liver cell separation method can not well meet the needs of single-cell omics research, especially in the liver of fibrotic mice, the above-mentioned shortcomings are particularly prominent; the present invention provides a preparation Reagents, kits and methods for fibrotic liver single cell suspension; the present invention aims at indigestible liver fibrosis mouse model with a large amount of fiber deposition, adopts liver in situ perfusion and digestion, uses pronase, DNase, type II Collagenase non-biased cell dissociation, using EDTA to further prevent cell aggregation and using Opti-Prep as the medium gradient to centrifuge cells to fully remove impurities and cell debris.

In order to achieve the above object, the present invention provides a reagent for preparing fibrotic liver single cell suspension, said reagent at least including pronase, DNase, type II collagenase, HBSS (Hank's balanced salt solution), NaHCO ₃ , EDTA, Ca ^{2 +} , Fetal Bovine Serum (FBS) and Optiprep Separation Medium (Optiprep ^™ Density Gradient Medium).

Preferably, the reagent further includes at least one of erythrocyte lysate, phenol red and DMEM high glucose medium.

The present invention also provides a kit for preparing a single-cell suspension of fibrotic liver, the kit includes the above-mentioned reagents and instructions for preparing a single-cell suspension of fibrotic liver, and the instructions describe the preparation method and use of the reagent method.

Preferably, the preparation method of the reagent includes selecting at least two of the above-mentioned reagents to be prepared into lavage solution A, lavage solution B, pronase buffer, DNase buffer, type II collagenase buffer, FBS-containing At least one of buffer, diluted Optiprep separation solution and mixed enzyme solution:

The lavage solution A is prepared from Ca ²⁺ -free HBSS (Hank's Balanced Salt Solution), NaHCO ₃ and EDTA;

The lavage solution B is prepared from HBSS (Hank's Balanced Salt Solution) containing Ca ²⁺ , NaHCO ₃ , EDTA and Ca ²⁺ ;

The pronase buffer is prepared from pronase and the lavage solution B;

The DNase buffer is prepared from DNase and the lavage solution B;

The type II collagenase buffer is prepared from type II collagenase and the lavage solution B;

The FBS-containing buffer is prepared from the lavage solution A and fetal bovine serum FBS.

The diluted Optiprep separation solution is prepared from 60% w/v Opti-Prep separation solution and the FBS-containing buffer;

The mixed enzyme liquid is prepared from the type II collagenase buffer, pronase and DNase.

More preferably, the lavage solution A is formulated in a ratio of 50ml: 500 μl: 65 μl according to HBSS (Hank's Balanced Salt Solution) without Ca ²⁺ , 7.5wt% NaHCO ₃ solution and 160 mg/ml EDTA solution ; Wherein, NaHCO _solution and EDTA solution can be replaced by other concentrations, and the dosage can be adjusted accordingly;

And/or, the lavage solution B is based on Ca2 ⁺ containing HBSS (Hank's balanced salt solution), 7.5wt% _NaHCO3 solution, 160mg/ml EDTA solution and 2.5M _CaCl2 solution with 50ml: 500 μl: Prepared at a ratio of 100 μl; among them, NaHCO ₃ solution, EDTA solution and CaCl ₂ solution can be replaced by other concentrations, and the dosage can be adjusted accordingly;

And/or, the pronase buffer solution is prepared from pronase and the lavage solution B at a concentration of 1 mg/ml;

And/or, the DNase buffer solution is prepared by DNase and the lavage solution B at a concentration of 0.1 mg/ml;

And/or, the type II collagenase buffer solution is prepared from type II collagenase and the lavage solution B at a concentration of 0.01-0.03 g/ml;

And/or, the diluted Optiprep separation solution is prepared from 60% w/v OptiPrep separation solution and the FBS-containing buffer solution at a concentration of 10-40% w/v;

And/or, the mixed enzyme solution is prepared from the type II collagenase buffer solution, pronase and DNase in a ratio of 10ml:4mg:1mg.

The present invention also provides the above reagent for preparing fibrotic liver single cell suspension, or the application of the above kit for preparing fibrotic liver single cell suspension in the preparation of fibrotic liver single cell suspension, characterized in that the preparation The single cell suspension of fibrotic liver is prepared after dissociation of fibrotic liver tissue from sacrificed animals or directly from fresh animal fibrotic liver tissue isolated from the body.

The present invention also provides a method for preparing fibrotic liver single cell suspension, comprising the following steps:

Step 1: Cut the isolated fresh animal fibrotic liver tissue into pieces, add a mixture of pronase, DNase and type II collagenase to obtain a tissue suspension, pass the tissue suspension through a cell sieve and put it in a water bath Digestion;

Step 2: After full digestion, add DMEM+10% FBS to stop the digestion, blow off the cells with a pipette gun and centrifuge to obtain the cell pellet and supernatant containing primary stem cells, and then centrifuge again to collect the cell pellet;

Step 3: Add red blood cell lysate to resuspend the cell pellet, incubate on ice, add buffer to stop lysis, obtain cell suspension, pass through a cell sieve and centrifuge, collect cell pellet and resuspend in buffer, then add 40% OptiPrep separation medium , blowing evenly with a pipette gun, injecting 10% OptiPrep separation solution and buffer solution containing phenol red chromogen in sequence to obtain cell fluid divided into upper, middle and lower layers; wherein, the buffer solution is composed of Ca-free ²⁺ HBSS, NaHCO ₃ , EDTA and fetal bovine serum FBS;

Step 4: Centrifuge the cell liquid obtained in step 3, avoid destroying the stratification by adjusting the speed up and down of the centrifuge, collect the upper and middle layer cells after centrifugation, add DMEM+2% FBS to resuspend and centrifuge again to collect the cells precipitation;

Step 5: According to the application and requirements of the analytical instrument, select the medium to resuspend the cell pellet obtained in step 4, and dilute to the target concentration to obtain the fibrotic liver single cell suspension.

Preferably, the isolated fresh animal fibrotic liver tissue in step 1 is prepared by the following steps:

Step S1: Fix the limbs of the sacrificed mice, disinfect the abdominal skin of the mice with 75% alcohol, quickly open the abdominal cavity, and fully expose the liver and portal vein;

Step S2: Use a syringe to draw the lavage solution A preheated at 37°C, and connect it to a 24G intravenous indwelling needle. After exhausting the gas in the indwelling needle, perform hepatic portal vein puncture and intubation, withdraw the needle tip into the flexible tube, and use an arterial clamp Fixing the indwelling needle; wherein, the lavage solution A is prepared from Ca ²⁺ -free HBSS (Hank's Balanced Salt Solution), NaHCO ₃ and EDTA;

Step S3: Gently push the syringe, see that the liver is slightly swollen and the color becomes lighter, cut the inferior vena cava, continue perfusion, see that the liver swells and becomes shallow again, then pause for 3-5 seconds, and repeat the perfusion operation after the residual blood flows out;

Step S4: until the liver as a whole is earthy white and retracts without residual blood flowing out, replace the pronase buffer and perfuse 3-5ml; wherein, the pronase buffer is composed of pronase, HBSS containing Ca ²⁺ (Hank's Balanced Salt Solution), NaHCO ₃ , EDTA and Ca ²⁺ ;

Step S5: Perfuse the liver with type II collagenase buffer until the liver is soft and completely digested. Use ophthalmic forceps to clamp the inferior vena cava and perfuse type II collagenase buffer until the liver expands slightly. Release the tweezers after internal digestion for 5-10 seconds, and repeat the process 2-3 times; wherein, the type II collagenase buffer is composed of type II collagenase, HBSS (Hank's balanced salt solution) containing Ca ²⁺ , NaHCO ₃ , EDTA and Ca ²⁺ ;

Step S6: Gently press the liver with a cotton swab. When the liver is soft and fully digested and easy to dissociate, remove the whole liver and put it into a centrifuge tube.

Principle of the present invention:

The present invention utilizes the effect of pronase to reduce the viscosity of gelatin and mucin, DNA enzyme degrades DNA and avoids breaking cells to release DNA in the separation process to increase suspension viscosity, and type II collagenase can degrade triple-helix natural collagen fibers of connective tissue, And relatively mild, with good dissociation ability under physiological temperature and pH value conditions, no need for mechanical stirring or special equipment, and type II collagenase is more suitable for digesting the liver than other types of collagenase; EDTA chelates calcium ions to further avoid cell aggregation ; By adding the above components during the separation process, the liver single cell suspension fully dissociated from the extracellular matrix components can be obtained efficiently. At the same time, during the separation process, the HBSS solution is used to prepare the buffer throughout the whole process. The glucose contained in the HBSS solution provides energy supply for living cells, and avoids changes in the cell transcriptome caused by the lack of energy substances during the cell separation process to the greatest extent.

Compared with prior art, the beneficial effect of the present invention is:

(1) The single-cell suspension prepared by the preparation method of the present invention has a high cell yield: 2× ¹⁰⁷ cells can be obtained from a single 25g mouse, counted under a microscope after staining with trypan blue, and the percentage of surviving cells is 90-98% between;

(2) Impurities and cell debris are few in the cell suspension prepared by the present invention: among the present invention, cell debris and cell residues have removed more than 99.9% of the cell debris by the Opti-Prep medium gradient centrifugation method; a small amount of cell suspension After staining under the microscope, if cell debris and residues can still be seen, a second gradient centrifugation can be performed to obtain a high-purity living cell suspension;

(3) Does not affect the detection of intracellular RNA and protein: adopt the single cell suspension separated by the method of the present invention, add glucose in the separation buffer, avoid the change of cell transcriptomics caused by energy shortage in the separation process to the greatest extent; The digestion is thorough, and the various cells in the liver are fully digested and separated from the extracellular matrix, which avoids the interference of the adhered extracellular matrix on the results of single-cell proteomics in single-cell proteomics monitoring;

(4) The method of the present invention obtains a true single-cell suspension, which effectively avoids cell adhesion and aggregation.

Detailed ways

In order to make the present invention more comprehensible, preferred embodiments are described in detail as follows.

The suppliers and article numbers of some reagents used in the following examples are as follows:

7.5% NaHCO ₃ : Biyuntian C0220;

2.5M CaCl ₂ solution: Sangong A600506-0100;

EDTA (0.5M, pH8.0): Biyuntian ST066.

The rest of the unspecified reagents are conventional commercially available reagents.

Example

This embodiment provides a preparation method of fibrotic liver single cell suspension:

1. Reagent preparation:

Lavage solution A: 50ml HBSS (without Ca ²⁺ )+500μl 7.5wt% NaHCO ₃ solution+65μl EDTA (160mg/ml) or 71μl 0.5M EDTA;

Lavage solution B: 50ml HBSS (containing Ca ²⁺ )+500μl 7.5wt% NaHCO ₃ solution+100μl 2.5MCaCl ₂ solution;

Buffer C: lavage solution A+2% (v/v) FBS, 98ml lavage solution A+2ml FBS per 100ml;

Pronase buffer solution: 10mg pronase dry powder dissolved in 10ml lavage solution B;

DNase buffer: 1mg DNase dry powder dissolved in 10ml lavage solution B;

Type II collagenase buffer: add type II collagenase dry powder to the lavage solution B until the color turns light brown (approximately 0.15g type II collagenase dry powder is added to each 10ml lavage solution B);

10% (w/v) and 40% (w/v) Opti-Prep separation solution: both are prepared with 60% (w/v) Opti-Prep and buffer C; the specific preparation is as follows:

12ml 40% (w/v) Opti-Prep: 8ml 60% Opti-Prep+4ml Buffer C;

12ml 60% (w/v) Opti-Prep: 2ml 60% Opti-Prep+10ml Buffer C;

Mixed enzyme solution: 10ml of the above type II collagenase buffer + 4mg pronase + 1mg DNase;

Others: red blood cell lysate; phenol red; high-glucose DMEM medium (containing 4.5g/L D-glucose, L-glutamine, phenol red, 0.11g/L sodium pyruvate; without HEPES); fetal bovine serum ( FBS) etc.

2. Item preparation:

24G intravenous indwelling needle; 10ml syringe; arterial clip; ophthalmic forceps; ophthalmic scissors; 75% alcohol; infrared heating lamp; 15ml/50ml centrifuge tube; 30μm/100μm cell sieve, etc.

3. Experimental steps:

3.1 Liver isolation

(1) Fix the limbs of the sacrificed mice, disinfect the abdominal skin of the mice with 75% alcohol, and quickly open the abdominal cavity to fully expose the liver and portal vein.

(2) Use a 10ml syringe to draw a certain volume of lavage solution A preheated at 37°C, and connect it to a 24G intravenous indwelling needle. After the gas in the indwelling needle is exhausted, puncture the hepatic portal vein and insert the needle tip into the flexible tube. Secure the catheter with an arterial clip.

(3) Gently push the syringe and see that the liver swells slightly and becomes lighter in color. Cut the inferior vena cava and continue perfusion. See the liver swells and becomes shallow again, then pause for 3-5 seconds, and repeat the perfusion operation after the residual blood flows out.

(4) When the whole liver is earthy white and retracts without residual blood flowing out, replace the pronase buffer and perfuse 3-5ml.

(5) Perfuse the liver with type II collagenase buffer until the liver is soft and completely digested. Clamp the inferior vena cava with ophthalmic tweezers and perfuse type II collagenase buffer until the liver swells slightly, then stop the perfusion, release the tweezers after the collagenase has been digested in the liver for 5-10 seconds, and repeat this process 2-3 times.

(6) Gently press the liver with a cotton swab. When the liver is soft and fully digested and easy to dissociate, remove the whole liver and place it in a 50ml centrifuge tube.

3.2 Preparation of single cell suspension

(1) Slightly chop the fresh liver tissue obtained in 3.1, add 10ml of pronase (4mg/10ml), DNase (1mg/10ml) and type II collagenase mixed enzyme solution, and pass the suspension through 100μm cell sieve.

(2) The filtered cell suspension was continuously shaken and digested in a water bath at 37°C (10 min for normal control mouse model, 20 min for liver fibrosis mouse model).

(3) After fully digested, add 40ml DMEM+10% (v/v) FBS to stop the digestion, and fully blow off the cells with a pipette gun.

(4) Centrifuge the cell suspension at 50 g for 3 min at 4°C, the precipitates are mainly primary hepatocytes, which can be collected as needed.

(5) Transfer the supernatant and centrifuge at 500 g at 4°C for 7 min, retain the cell pellet, and discard the supernatant.

(6) Add 2ml of erythrocyte lysate to resuspend the cell pellet, and incubate on ice for 2min.

(7) Add 10ml buffer C to stop lysis.

(8) Pass the cell suspension through a 30 μm cell sieve.

(9) Centrifuge at 500g for 3min at 4°C and discard the supernatant.

(10) Resuspend the cell pellet with 3ml buffer C, then add 3ml 40% w/v Opti-Prep, and blow evenly with a pipette gun.

(11) Inject 3ml of 10% w/v Opti-Prep containing a small amount of phenol red for color development close to the inner wall of the centrifuge tube, pay attention to gentle operation, and try to avoid disturbing the layered interface.

(12) Slowly inject 3ml of buffer C into the top of the inner wall of the tube. It can be seen that the liquid in the tube is divided into upper, middle and lower layers, and the layered interface is clear.

(13) Centrifuge at 1500g at 4°C for 30 minutes, and adjust the speed up of the centrifuge to 6 and the speed down to 0 to reduce the disturbance of the stratification by external factors.

(14) After centrifugation, mixed hepatocytes and dead cells can be seen at the bottom of the tube. Take the upper and middle layer cells and transfer them to a new centrifuge tube.

(15) After adding DMEM+2% (v/v) FBS to resuspend, centrifuge at 500g for 7min at 4°C and discard the supernatant.

(16) Repeat step 15 to remove residual EDTA.

(17) According to the further use (such as single-cell RNA sequencing or single-cell proteome detection), resuspend the cell pellet directly in the solution medium required by the corresponding instrument, dilute to the target concentration, count the cells, and stain with trypan blue After calculating the ratio of viable cells, the next step of detection and analysis was performed.

In this embodiment, select 1ml DMEM+2% (v/v) FBS to resuspend the cells obtained in step (16), take a small amount of cell suspension trypan blue staining under the microscope, count the cells under the microscope, calculate the proportion of living cells, and the result Display: 2×10 ⁷ cells can be obtained from a single 25g mouse, the percentage of viable cells is between 90-98%, and no cell fragments and residues are seen in the stained pictures, indicating that this method has obtained high-purity living cells Suspension.

The foregoing embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form and in essence. It should be pointed out that those of ordinary skill in the art can also make Several improvements and supplements should also be considered as the protection scope of the present invention.

Claims (7)

1. A reagent for preparing a single-cell suspension of a fibrotic liver is characterized by at least comprising pronase, DNase, type II collagenase, HBSS (Hank's balanced salt solution) and NaHCO ₃ 、EDTA、Ca ²⁺ Fetal Bovine Serum (FBS) and Optiprep isolates (Optiprep) ^TM Density gradient centrifugation).

2. The reagent for preparing a single cell suspension of fibrotic liver of claim 1, further comprising at least one of red blood cell lysate, phenol red and DMEM high sugar medium.

3. A kit for preparing a single-cell suspension of a fibrotic liver, comprising the reagent for preparing a single-cell suspension of a fibrotic liver according to claim 1 and instructions describing a preparation method and a use method of the reagent.

4. A kit for preparing a single cell suspension of a fibrotic liver as claimed in claim 3, wherein the method of preparation of the reagent comprises selecting at least two of the reagents of claim 1 to be formulated as at least one of lavage solution a, lavage solution B, pronase buffer, dnase buffer, collagenase buffer type II, FBS containing buffer, diluted Optiprep separation solution and mixed enzyme solution:

the lavage liquid A is prepared from the materials without Ca ²⁺ HBSS (Hank's balanced salt solution), naHCO ₃ And EDTA;

the lavage liquid B contains Ca ²⁺ HBSS (Hank's balanced salt solution), naHCO ₃ EDTA and Ca ²⁺ Is prepared by the steps of preparing;

the pronase buffer solution is prepared from pronase and the lavage liquid B;

the DNase buffer solution is prepared from DNase and lavage liquid B;

the type II collagenase buffer solution is prepared from type II collagenase and lavage fluid B;

the buffer containing FBS is prepared from the lavage liquid A and fetal bovine serum FBS.

The diluted Optiprep separating liquid is prepared from 60% of Optiprep separating liquid and buffer liquid containing FBS;

the mixed enzyme solution is prepared from the type II collagenase buffer solution, pronase and DNase.

5. The use of a reagent for preparing a fibrotic liver single cell suspension according to claim 1, or a kit for preparing a fibrotic liver single cell suspension according to any one of claims 2 to 4, in preparing a fibrotic liver single cell suspension from a sacrificed animal after dissociation of the fibrotic liver tissue or directly from an isolated fresh animal fibrotic liver tissue.

6. A method of preparing a suspension of single cells of a fibrotic liver, comprising the steps of:

step 1: cutting fresh animal fibrotic liver tissue in vitro, adding mixed enzyme solution of pronase, DNase and type II collagenase to obtain tissue suspension, sieving the tissue suspension with a cell sieve, and digesting in a water bath;

step 2: adding DMEM and 10% FBS after full digestion to stop digestion, fully blowing off cells by a pipetting gun, centrifuging to obtain cell sediment and supernatant containing primary stem cells, centrifuging again, and collecting the cell sediment;

step 3: adding erythrocyte lysate to resuspend cell sediment, incubating on ice, adding buffer solution to stop lysis to obtain cell suspension, sieving with cell sieve, centrifuging, collecting cell sediment, resuspending in buffer solution, adding 40% OptiPrep separating solution, blowing with a pipette, sequentially injecting 10% OptiPrep separating solution containing phenol red color developing agent and buffer solutionObtaining cell fluid divided into an upper layer, a middle layer and a lower layer; wherein the buffer solution is prepared from a solution containing no Ca ²⁺ HBSS, naHCO ₃ EDTA and fetal bovine serum FBS;

step 4: centrifuging the cell sap obtained in the step 3, avoiding damaging layering by adjusting the speed up and speed down of the centrifuge, collecting cells at the upper layer and the middle layer after centrifugation, adding DMEM+2% FBS for re-suspension, centrifuging again, and collecting cell sediment;

step 5: and (3) selecting a medium according to the requirements of the application and an analytical instrument, re-suspending the cell sediment obtained in the step (4), and diluting to a target concentration to obtain the single-cell suspension of the fibrotic liver.

7. The method of preparing a single cell suspension of fibrotic liver of claim 6, wherein the isolated fresh animal fibrotic liver tissue of step 1 is prepared by:

step S1: fixing the four limbs of the sacrificed mice, sterilizing the abdominal skin of the mice by 75% alcohol, rapidly opening the abdominal cavity, and fully exposing the liver and portal vein;

step S2: sucking the lavage liquid A preheated at 37 ℃ by using a syringe, connecting the lavage liquid A with a 24G specification venous indwelling needle, discharging gas in the indwelling needle, performing portal vein puncture intubation, retracting a needle point into a hose, and fixing the indwelling needle by using an arterial clamp; wherein the lavage liquid A is prepared from a lavage liquid containing no Ca ²⁺ HBSS (Hank's balanced salt solution), naHCO ₃ And EDTA;

step S3: pushing the injector lightly until the liver is slightly swelled and the color becomes light, cutting off the inferior vena cava, continuing to perfuse until the liver is swelled again and becomes light, stopping for 3-5 seconds, and repeating the perfusion operation after the residual blood flows out;

step S4: changing the pronase buffer solution to perfuse 3-5ml until the whole liver is earth white and no residual blood flows out after retraction; wherein the pronase buffer solution consists of pronase and Ca-containing buffer solution ²⁺ HBSS (Hank's balanced salt solution), naHCO ₃ EDTA and Ca ²⁺ Is prepared by the steps of preparing;

step S5: perfusing the liver with a type II collagenase buffer until the liver is softSoft and completely digested, filling type II collagenase buffer solution after clamping the inferior vena cava with ophthalmic forceps until the liver slightly swells, stopping filling, loosening forceps after collagenase is digested in the liver for 5-10 seconds, and repeating the process for 2-3 times; wherein the type II collagenase buffer solution consists of type II collagenase and Ca-containing buffer solution ²⁺ HBSS (Hank's balanced salt solution), naHCO ₃ EDTA and Ca ²⁺ Is prepared by the steps of preparing;

step S6: the liver is gently pressed by a cotton swab, and when the liver is soft and is fully digested and easy to dissociate, the whole liver is picked up and put into a centrifuge tube.