Kit for efficiently preparing retina single-cell suspension and method for dissociating hard retina tissue

Abstract

The invention relates to the technical field of biology, in particular to a kit for efficiently preparing retina single-cell suspension and a method for dissociating hard retina tissue, a mixed enzyme of collagenase II and collagenase IV, a dissociation buffer solution, a washing buffer solution, a stop solution and a resuspension solution are included, the mass ratio of the collagenase II to the collagenase IV is (1.0-1.8): 1, and the total concentration is 1.2-2.2 mg / mL. By using the kit and the dissociation method provided by the invention, the hard retinal tissue can be effectively dissociated, the protruding link between cells can be fully digested, the sufficient digestion of substances in the protruding gap can be realized, and the single-cell dissociation of the retinal tissue can be well realized and the high cell activity can be maintained through the verification of multiple experiments.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for efficiently preparing a retinal single cell suspension and a method for dissociating hard retinal tissue. Background Art

[0002] Single-cell sequencing technology has played an important role in biological research in recent years. It can analyze the genome, transcriptome, and epigenome at the single-cell level, revealing the heterogeneity and complexity between cells. This technology has great potential in revealing the diversity of cells in organisms, understanding cell fate decisions, and exploring disease mechanisms. Especially in retinal research, single-cell sequencing technology can analyze the gene expression profiles of various cells in the retina in detail, revealing the changes of different cell types in development, function, and disease states, thereby providing important information for understanding the biological functions of the retina and related diseases.

[0003] However, there are significant technical difficulties in the preparation of single-cell suspensions of retinal tissue. The retinal tissue is hard in texture, with a wide variety of cell types and tight connections between cells. Most of them are photosensitive functional neurons, which are large in size and intertwined with each other. These characteristics make it difficult to obtain single, complete and active cells during the dissociation process of retinal tissue. In addition, traditional dissociation methods may damage neurons, reduce cell activity and purity, and thus affect the quality of subsequent single-cell sequencing. These difficulties lead to information loss in retinal single-cell studies, restricting in-depth research on retinal biology and disease mechanisms.

[0004] It is of great significance to solve the technical difficulties in preparing retinal single-cell suspensions and optimize the dissociation method. The process of preparing single-cell suspensions needs to be highly repeatable and stable to ensure the consistency of results between different experiments. Efficient and gentle dissociation methods can ensure the acquisition of high-quality retinal single-cell suspensions, improve the success rate and data quality of single-cell sequencing, thereby providing more accurate and rich information for retinal research and promoting the progress of retinal biology and disease research. Summary of the invention

[0005] One of the purposes of the present invention is to provide a kit for efficiently preparing retinal single cell suspensions, which can efficiently dissociate retinal tissues and obtain single cell suspensions with high viability and low impurities through optimized enzymatic hydrolysis combinations and processing steps.

[0006] The second object of the present invention is to provide a method for dissociating hard retinal tissue, which is suitable for single-cell sequencing, gene expression analysis, drug screening and cell function research, etc. The method is simple and convenient, and can obtain highly active and highly dispersed single-cell suspensions, and has broad application prospects.

[0007] The scheme adopted by the present invention to achieve one of the purposes is: a kit for efficiently preparing a retinal single cell suspension, comprising a mixed enzyme of collagenase II and collagenase IV, a dissociation buffer, a washing buffer, a stop solution and a resuspension solution, wherein the mass ratio of collagenase II to collagenase IV is 1.0 to 1.8:1, and the total concentration is 1.2 to 2.2 mg / mL.

[0008] In the present invention, the mixed enzyme of collagenase II and collagenase IV is used to dissociate the extracellular matrix and basement membrane in the retinal tissue to help separate single cells.

[0009] Preferably, the main components of the dissociation buffer include 15-20 mM HEPES buffer, 100-120 mM NaCl, 3-5 mM KCl, 1.5-2.0 mM CaCl2, and 1.0-2.0 mM MgCl2.

[0010] Specifically, HEPES buffer: 15-20 mM, used to maintain the pH stability of the solution.

[0011] NaCl (sodium chloride): 100-120 mM, used to maintain the osmotic pressure of cells.

[0012] KCl (potassium chloride): 3-5 mM, helps maintain the osmotic pressure of cells.

[0013] CaCl2 (calcium chloride): 1.5-2.0 mM, an essential ion for enzyme activity.

[0014] MgCl2 (magnesium chloride): 1.0~2.0mM, as an auxiliary ion for enzyme activity.

[0015] The pH was adjusted to 7.4 to maintain the normal state of cells at physiological pH.

[0016] Preferably, the main components of the washing buffer include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

[0017] DPBS (Phosphate Buffered Saline): 1× (1x) DPBS is used to wash cells to remove residual enzymes and cell debris.

[0018] BSA (bovine serum albumin): 0.04%, used to protect cells from mechanical damage during washing.

[0019] Preferably, the main component of the stop solution is a complete culture medium containing 10%-15% fetal bovine serum by mass.

[0020] Complete culture medium: usually contains 10%-15% (mass percentage) fetal bovine serum (FBS), which is used to terminate the enzymatic reaction.

[0021] FBS (fetal bovine serum): 10%-15% by mass, provides nutrients required for cell growth and protects cells.

[0022] Preferably, the main components of the resuspension include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

[0023] DPBS (Phosphate Buffered Saline): 1× (1x) DPBS is used for the final resuspension of cells.

[0024] BSA (bovine serum albumin): 0.04%, used to stabilize the cell suspension and prevent cell aggregation and adhesion.

[0025] Preferably, the mass ratio of collagenase II to collagenase IV is 1:1, and the total concentration is 2 mg / mL.

[0026] The solution adopted by the present invention to achieve the second purpose is: a method for dissociating hard retinal tissue, using the kit, comprising the following steps:

[0027] Step 1: Obtain retinal tissue sample;

[0028] Step 2: Wash the tissue several times with wash buffer to remove blood and other nonspecific attachments;

[0029] Step 3: Cut the retinal tissue into small pieces of appropriate size;

[0030] Step 4: Transfer the tissue pieces to the dissociation buffer containing a mixture of collagenase II and collagenase IV and incubate until dissociation is complete;

[0031] Step 5: After the enzymatic hydrolysis is completed, remove the tissue and add it to the stop solution to terminate the enzymatic hydrolysis reaction;

[0032] Step 6: After filtering the tissue, centrifuge the filtrate to collect the cell precipitate;

[0033] Step 7: Resuspend the cell pellet with a resuspension solution to prepare a single cell suspension.

[0034] Preferably, in step 4, the specific incubation operation is to incubate at 37° C. with gentle shaking for 25 to 35 minutes.

[0035] Incubate at 37°C with gentle shaking for 25 to 35 minutes. During this period, the test tube can be gently shaken several times to promote enzymatic hydrolysis. The dissociation time can be adjusted appropriately according to the hardness and condition of the tissue. For harder and older sample tissues, the dissociation time should be appropriately increased, and for younger and softer tissues, the dissociation time can be appropriately reduced. The control of the dissociation time is crucial.

[0036] The present invention effectively reduces the damage to the cell membrane and significantly improves the activity and purity of single cells by optimizing the combination and dissociation time of collagenase II and collagenase IV.

[0037] Preferably, in step 5, DNase I may also be added to the suspension.

[0038] DNase I is added to degrade the released DNA and prevent cell aggregation. An additional wash step can be added to wash the cell pellet with DPBS (containing 0.04% BSA) to further remove residual enzymes and cell debris.

[0039] Preferably, in step 6, filtration is performed using a 60-80 μm cell filter, the centrifugal force is 300-500 g, and the centrifugation is performed for 4 to 6 minutes, wherein steps 1 to 7 are all performed at 4 to 6° C.

[0040] During the operation after the enzymatic hydrolysis is terminated, the operation temperature should be kept low (4-6°C) to reduce cell stress and death. During each centrifugation step (300-500g, 4°C centrifugation for 4 to 6 minutes) and resuspension, the cells should be handled gently to avoid severe mechanical stress. During all washing processes, an appropriate amount of DNase I (0.1 mg / mL) can be added to degrade the released DNA and prevent cell aggregation.

[0041] The cell suspension prepared by the dissociation method of the present invention is used to detect the viability of the cell suspension using the trypan blue staining method, and the cell viability is above 95%.

[0042] The present invention has the following advantages and beneficial effects:

[0043] The use of the kit and dissociation method of the present invention can effectively dissociate retinal tissue with a harder texture, fully digest the protrusion links between cells, and achieve relatively sufficient digestion of the protrusion gap material. After multiple experimental verifications, it can better achieve single-cell dissociation of retinal tissue and maintain high cell activity.

[0044] The kit of the present invention can rapidly and efficiently dissociate retinal tissue, shorten the dissociation time, and improve the preparation efficiency of single cell suspension.

[0045] The preparation method of the present invention is not only applicable to mouse retinal tissue, but also applicable to human retinal tissue, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of AOPI staining and counting of single cell suspension obtained from normal mouse retinal tissue in Example 2 of the present invention;

[0047] Figure 2This is a schematic diagram of AOPI staining and counting of single cell suspensions obtained from retinal tissues of retinal ischemia-reperfusion model mice in Example 3 of the present invention;

[0048] Figure 3 This is a schematic diagram of AOPI staining and counting of single cell suspensions obtained from retinal tissues of gene knockout mice in Example 4 of the present invention;

[0049] Figure 4 Annotate cell types for data obtained from single-cell transcriptome sequencing;

[0050] Figure 5 The average gene expression per cell and the proportion of mitochondrial gene expression;

[0051] Figure 6 Comparison of the proportions of different cell types among samples. DETAILED DESCRIPTION

[0052] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0053] Example 1

[0054] A kit for efficiently preparing a retinal single cell suspension, comprising a mixed enzyme of collagenase II and collagenase IV, a dissociation buffer, a washing buffer, a stop solution and a resuspension solution, wherein the mass ratio of collagenase II to collagenase IV is 1.0 to 1.8:1, and the total concentration is 1.2 to 2.2 mg / mL. In this embodiment, the specific mass ratio of collagenase II to collagenase IV is 1:1, and the total concentration is 2 mg / mL.

[0055] The main components of the dissociation buffer include 15-20 mM HEPES buffer, 100-120 mM NaCl, 3-5 mM KCl, 1.5-2.0 mM CaCl2, and 1.0-2.0 mM MgCl2.

[0056] Specifically, the main component of the dissociation buffer in this embodiment is HEPES buffer: 20 mM, which is used to maintain the pH stability of the solution.

[0057] NaCl (sodium chloride): 120 mM, used to maintain the osmotic pressure of cells.

[0058] KCl (potassium chloride): 5 mM, helps maintain the osmotic pressure of cells.

[0059] CaCl2 (Calcium chloride): 2 mM, an essential ion for enzyme activity.

[0060] MgCl2 (magnesium chloride): 1 mM, as a co-ion for enzyme activity.

[0061] The pH was adjusted to 7.4 to maintain the normal state of cells at physiological pH.

[0062] The main components of the washing buffer solution include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

[0063] The main component of the stop solution is a complete culture medium containing 10%-15% fetal bovine serum by mass. The main component of the stop solution in this embodiment is a complete culture medium containing 10% fetal bovine serum by mass.

[0064] The main components of the resuspension solution include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

[0065] Example 2

[0066] A method for dissociating retinal tissue, using the kit of Example 1 to dissociate retinal tissue, comprising the following steps:

[0067] Step 1: Tissue sampling

[0068] Samples were taken from fresh mouse eyeballs and immediately placed in pre-cooled DPBS (containing 0.04% BSA).

[0069] Carefully separate the retinal tissue using a dissecting microscope and surgical instruments in a clean bench. (Note: Try to minimize contact between the retina and the instrument, do not separate from DPBS during the peeling process, and keep the operation on ice. Keep the retina from being pulled to avoid causing a large number of cell death).

[0070] Step 2: Tissue washing

[0071] Wash the tissue several times with wash buffer to remove blood and other nonspecific attachments.

[0072] Step 3: Cut the tissue into pieces

[0073] Cut the retinal tissue into approximately 1 mm pieces using a scalpel or scissors on ice. 3 of small pieces.

[0074] Step 4: Enzymatic treatment

[0075] The cut retinal tissue was transferred into a dissociation buffer containing a mixture of collagenase II and collagenase IV (mass ratio 1:1, total concentration 2 mg / mL).

[0076] Incubate in a 37°C water bath for 30 min, stirring gently every 5 min to promote enzymatic hydrolysis.

[0077] Step 5: Stop enzymatic digestion

[0078] Immediately add the enzymatically hydrolyzed tissue block into the pre-cooled (4-6°C) stop solution to terminate the enzymatic reaction.

[0079] Step 6: Collect the cell pellet

[0080] Filter the tissue through a 70 μm cell strainer to remove undigested tissue chunks.

[0081] The cells were centrifuged at 300 g for 4-6 minutes at 4-6°C to collect the cell pellet.

[0082] Step 7: Washing

[0083] Resuspend the cell pellet in resuspension buffer, centrifuge at 400 g for 5 min, and remove the supernatant.

[0084] An appropriate amount of DNase I (0.1 mg / mL) was added to degrade the released DNA and prevent cell aggregation.

[0085] Repeat the washing step 3 times to remove residual enzymes and cell debris.

[0086] Step 8: Resuspension

[0087] Resuspend the cell pellet in resuspension buffer to obtain a single cell suspension.

[0088] Step 9: Cell viability assay

[0089] The cell activity was detected by trypan blue staining, and the cell survival rate was above 95%.

[0090] The temperature should be kept low (4°C) throughout the operation to reduce cell stress and death.

[0091] During each centrifugation and resuspension step, handle cells gently and avoid severe mechanical stress.

[0092] Verification

[0093] A live cell counter (such as AOPI staining) was used to confirm the activity and concentration of the single cell suspension. The specific activity test results were overall cell viability: 95.57%, total cell concentration: 1.07E+06 / ml, and clumping rate: 12.4%. Figure 1 Shown is a schematic diagram of AOPI staining counting of single cell suspension obtained for normal mouse retinal tissue in this example, BR: bright field counting image of Countstar cell counter; FL1: green fluorescence counting image of Countstar cell counter (living cells); FL2: red fluorescence counting image of Countstar cell counter (dead cells); fusion image: green and red fluorescence counting images of Countstar cell counter; lower figure: diameter distribution, green fluorescence and red fluorescence distribution statistics of counted cells.

[0094] Example 3

[0095] The difference between this embodiment and embodiment 2 is that the experimental object is the retinal tissue of retinal ischemia-reperfusion model mice.

[0096] A live cell counter (such as AOPI staining) was used to confirm the activity and concentration of the single cell suspension. The specific activity test results were overall cell viability: 95.63%, total cell concentration: 9.92E+05 / ml, and clumping rate: 12.6%. Figure 2 Shown is a schematic diagram of AOPI staining counting of single cell suspension obtained from retinal tissue of retinal ischemia-reperfusion model mice in this embodiment, BR: bright field counting image of Countstar cell counter; FL1: green fluorescence counting image of Countstar cell counter (living cells); FL2: red fluorescence counting image of Countstar cell counter (dead cells); fusion image: green and red fluorescence counting images of Countstar cell counter; bottom image: diameter distribution, green fluorescence and red fluorescence distribution statistics of counted cells.

[0097] Example 4

[0098] The difference between this embodiment and embodiment 2 is that the experimental object is the retinal tissue of gene knockout mice.

[0099] A live cell counter (such as AOPI staining) was used to confirm the activity and concentration of the single cell suspension. The specific activity test results were overall cell viability: 96.02%, total cell concentration: 9.15E+05 / ml, and clumping rate: 10.16%. Figure 3 Shown is a schematic diagram of AOPI staining counting of single cell suspension obtained from retinal tissue of gene knockout mice in this embodiment, BR: bright field counting image of Countstar cell counter; FL1: green fluorescence counting image of Countstar cell counter (living cells); FL2: red fluorescence counting image of Countstar cell counter (dead cells); fusion image: green and red fluorescence counting images of Countstar cell counter; bottom image: diameter distribution, green fluorescence and red fluorescence distribution statistics of counted cells.

[0100] The cell suspension was further analyzed by single-cell sequencing and other techniques. The results are as follows:

[0101] Figure 4The cell type annotations of the data obtained by single-cell transcriptome sequencing are shown. Single-cell sequencing was performed on the three types of case samples prepared in Examples 2-4, and the sequencing results were used for cell clustering, showing that the clustering had good clustering, clear clustering categories, and no scattered clustering. From the results of use, it can be inferred that the patent of the present invention can achieve a relatively sufficient preparation of retinal single-cell suspensions, and the preparation survival rate is high, which can establish a good foundation for subsequent single-cell sequencing experiments.

[0102] Figure 5 The figure shows the average gene expression per cell and the proportion of mitochondrial gene expression. After the sample transcriptome sequencing prepared in Example 2, the number of probes per cell, the number of molecules and the proportion of mitochondrial genes were statistically analyzed. It can be seen that the number of molecules (features) is mainly in the range of 500-5000, the total number of probes is within 20000, and the proportion of mitochondrial genes is within 20%. The above analysis reflects that the dissociation of retinal single cells is relatively successful, and the preparation of high-viability single-cell suspension is achieved.

[0103] Figure 6 The comparison of the proportion of different cell types between samples. After dividing the samples tested by the patent of this invention into subgroups, the proportion of the number of cells in each subgroup is counted and a bar chart is displayed. The main points are: the proportion of cells in each sample is reasonable, and the subgroup categories of each sample are balanced, which conforms to the scientific and objective results, reflecting that the patent of this invention can fully dissociate retinal cells and maintain a high viability, and each type of cell is less damaged.

[0104] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A kit for efficiently preparing retinal single cell suspension, characterized in that: The method comprises a mixed enzyme of collagenase II and collagenase IV, a dissociation buffer, a washing buffer, a stop solution and a resuspension solution, wherein the mass ratio of collagenase II to collagenase IV is 1.0-1.8:1, and the total concentration is 1.2-2.2 mg / mL.

2. The kit for efficiently preparing retinal single cell suspension according to claim 1, characterized in that: The main components of the dissociation buffer include 15-20 mM HEPES buffer, 100-120 mM NaCl, 3-5 mM KCl, 1.5-2.0 mM CaCl2, and 1.0-2.0 mM MgCl2.

3. The kit for efficiently preparing retinal single cell suspension according to claim 1, characterized in that: The main components of the washing buffer solution include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

4. The kit for efficiently preparing retinal single cell suspension according to claim 1, characterized in that: The main component of the stop solution is a complete culture medium containing 10%-15% fetal bovine serum by mass.

5. The kit for efficiently preparing retinal single cell suspension according to claim 1, characterized in that: The main components of the resuspension solution include phosphate buffered saline solution and bovine serum albumin with a mass concentration of 0.04%.

6. The kit for efficiently preparing retinal single cell suspension according to claim 1, characterized in that: The mass ratio of collagenase II to collagenase IV was 1:1, and the total concentration was 2 mg / mL.

7. A method for dissociating hard retinal tissue, characterized in that: The kit according to any one of claims 1 to 6 comprises the following steps: Step 1: Obtain retinal tissue sample; Step 2: Wash the tissue several times with wash buffer to remove blood and other nonspecific attachments; Step 3: Cut the retinal tissue into small pieces of appropriate size; Step 4: Transfer the tissue pieces to the dissociation buffer containing a mixture of collagenase II and collagenase IV and incubate until dissociation is complete; Step 5: After the enzymatic hydrolysis is completed, remove the tissue and add it to the stop solution to terminate the enzymatic hydrolysis reaction; Step 6: After filtering the tissue, centrifuge the filtrate to collect the cell precipitate; Step 7: Resuspend the cell pellet with a resuspension solution to prepare a single cell suspension.

8. The method for dissociating hard retinal tissue according to claim 7, characterized in that: In step 4, the specific incubation operation is to incubate at 37° C. with gentle shaking for 25 to 35 minutes.

9. The method for dissociating hard retinal tissue according to claim 7, characterized in that: In step 5, DNase I may also be added to the suspension.

10. The method for dissociating hard retinal tissue according to claim 7, characterized in that: In step 6, a 60-80 μm cell filter is used for filtration, the centrifugal force is 300-500 g, and the centrifugation is performed for 4-6 minutes, wherein steps 1-7 are all performed at 4-6° C.

Images