Preparation method and application of heart tissue single-cell suspension
By using complex collagenase and multiple washing centrifugation methods, the cell damage and survival problems in the preparation of single-cell suspension in cardiac tissue were solved, and efficient and stable single-cell suspension preparation was achieved to meet the needs of single-cell sequencing.
Patent Information
- Application Number
- CN202510058477.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-15
AI Technical Summary
The preparation of single-cell suspension in cardiac tissue has problems such as insufficient dissociation, large cell damage, and cell clumps and survival rate and concentration cannot meet single-cell sequencing requirements.
The heart tissue was digested using 0.1 wt.% compound collagenase (the mass ratio of collagenase II and collagenase IV is 1:4), and solid-liquid separation was performed by combining a 70-100-mesh cell filtration screen. The heart cells were gradually separated and digested through multiple washing and centrifugation, and trypsin was avoided.
A single-cell suspension of heart tissue with high cell content and high viability was prepared. The number of cells was 1×106/mL, and the cell viability was >75%, meeting the requirements of single-cell sequencing.
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Figure CN119824070A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of single cell suspensions, and in particular relates to a preparation method of a cardiac tissue single cell suspension and an application thereof. Background Art
[0002] The heterogeneity of cell types during cardiac development makes it difficult to study heart disease using traditional developmental biology techniques. The advent of single-cell RNA sequencing (scRNA-seq) has given cardiac researchers the opportunity to analyze cardiac biology at an unprecedented cellular resolution, revealing the cellular heterogeneity, developmental trajectory, and intercellular communication of the cardiac system, thus making a significant contribution to the study of heart disease. With the development and innovation of scRNA-seq research, single-cell transcriptomics has been increasingly used in the study of heart disease, including sorting cells with specific functions, identifying disease types, discovering new heterogeneous cell subpopulations, accurately identifying diseases at the cellular level, discovering cell populations and genes with specific functions, new therapeutic targets for diseases, and evaluation of drug treatment effects. Therefore, it is necessary to understand heart disease from the perspective of single cells.
[0003] The preparation of single-cell suspension of cardiac tissue is a key step in cardiac scRNA-seq research. However, single-cell sequencing has high requirements for cell suspension: the cell suspension should have a clear background, no large clumps, debris and impurities, and high cell survival rate and concentration. However, the heart is a muscular organ rich in collagen fibers and has a high degree of differentiation. The preparation of single-cell suspension is difficult, and there are still problems such as insufficient dissociation, severe cell damage, cell clumps, and survival rate and concentration that cannot meet sequencing requirements. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a cardiac tissue single-cell suspension and its application, so as to prepare a cardiac tissue single-cell suspension with good dispersion, large quantity, high viability and meeting the requirements of single-cell sequencing.
[0005] The present invention provides a method for preparing a cardiac tissue single cell suspension, comprising the following steps:
[0006] 1) digesting the heart tissue fragments with heart digestion fluid to obtain cell digestion fluid; performing solid-liquid separation on the cell digestion fluid with a 70-100 mesh cell filter to obtain a filtrate and a filter residue;
[0007] The heart digestion solution includes 0.1wt.% composite collagenase and the balance RPMI-1640 culture medium; the composite collagenase includes collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4;
[0008] 2) washing the filter residue in step 1) with the heart digestion solution in step 1) to obtain a cell washing solution; digesting the cell washing solution to obtain a cell digestion solution; performing solid-liquid separation on the cell digestion solution using a 70-100 mesh cell filter screen to obtain a filtrate and a filter residue;
[0009] 3) Repeating the operation of step 2) 1 to 6 times, and in each repetition, using the heart digestion fluid of step 1) to treat the latest obtained filter residue in the manner of step 2) to obtain a filtrate and filter residue obtained by repeated treatment;
[0010] 4) Perform the following S1 or S2 operations:
[0011] S1: centrifuging the filtrate in step 3) respectively, collecting the precipitates and resuspending them respectively to obtain multiple cardiac tissue single cell suspensions;
[0012] S2: Centrifuge the filtrates of step 1) and step 2) and the filtrate obtained by repeated treatment of step 3) respectively, collect the precipitates and resuspend them respectively, combine the resuspended solutions and centrifuge them again, collect the precipitates and resuspend them again to obtain a single cell suspension of cardiac tissue.
[0013] Preferably, in step 1), the ratio of the cardiac digestive fluid to cardiac tissue is 5 mL: (0.2-2.2) g.
[0014] Preferably, the volume ratio of the heart digestive solution used for digestion treatment in step 1) and the heart digestive solution used for washing in step 2) is 1:1.
[0015] Preferably, the temperatures of the centrifugation in S1, the centrifugation in S2, and the centrifugation again in S2 are 4° C., the rotation speeds are 1500 rpm, and the times are 5 min, respectively.
[0016] Preferably, the solutions used for resuspension in S1, resuspension in S2, and resuspension in S2 respectively include 5% v / v dual antibody and the remainder PBS buffer; the dual antibody includes penicillin and streptomycin.
[0017] Preferably, the temperatures of the digestion treatment in step 1) and the digestion treatment in step 2) are 37° C., the time is 5 min, and the rotation speed is 60 rpm.
[0018] Preferably, the heart tissue fragments include rhesus monkey heart tissue fragments.
[0019] Preferably, the size of the heart tissue fragments is 0.5 to 1 mm. 3 .
[0020] Preferably, the method for preparing the rhesus monkey heart tissue fragments comprises: soaking the rhesus monkey heart tissue in 75% v / v ethanol, taking it out and placing it in a pre-cooled PBS buffer containing 5% double antibody and chopping it to obtain the rhesus monkey heart tissue fragments; the double antibody comprises penicillin and streptomycin.
[0021] The present invention also provides the use of the cardiac tissue single cell suspension prepared by the preparation method described in the above technical solution in the preparation and research and development of heart disease products.
[0022] Beneficial effects:
[0023] The present invention provides a method for preparing a cardiac tissue single cell suspension, comprising the following steps: 1) digesting cardiac tissue fragments with cardiac digestion fluid to obtain a cell digestion fluid; performing solid-liquid separation on the cell digestion fluid with a 70-100 mesh cell filter screen to obtain a filtrate and a filter residue; the cardiac digestion fluid comprises 0.1wt.% composite collagenase and the remainder of RPMI-1640 culture medium; the composite collagenase comprises collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4; 2) washing the filter residue with the cardiac digestion fluid in step 1) to obtain a cell washing fluid; digesting the cell washing fluid to obtain a cell digestion fluid; using a 70-100 mesh cell filter screen to separate the solid from the liquid to obtain a filtrate and a filter residue; the cardiac digestion fluid comprises 0.1wt.% composite collagenase and the remainder of RPMI-1640 culture medium; the composite collagenase comprises collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4; 3) washing the filter residue with the cardiac digestion fluid in step 1) to obtain a cell washing fluid; performing solid-liquid separation on the cell washing fluid to obtain a cell digestion fluid; using a 70-100 mesh cell filter screen to separate the solid from the liquid to obtain a filtrate and a filter residue; 1) using a cell filter sieve with meshes of 100 to 200 to separate the cell digestion solution into solid and liquid to obtain a filtrate and a filter residue; 2) repeating the operation of step 2) for 1 to 6 times, and in each repetition, using the heart digestion solution of step 1) to treat the latest filter residue in the manner of step 2) to obtain the filtrate and filter residue obtained by repeated treatment; 3) performing the following S1 or S2 operation: S1: centrifuging the filtrate of step 3) respectively, collecting the precipitate and resuspending it respectively to obtain multiple heart tissue single cell suspensions; S2: centrifuging the filtrate of step 1) and step 2) and the filtrate obtained by repeated treatment of step 3) respectively, collecting the precipitate and resuspending it respectively, combining the resuspended solution and centrifuging it again, collecting the precipitate and resuspending it again to obtain a heart tissue single cell suspension. The method of the present invention is simple, and by limiting the combination and concentration of the heart digestion solution, trypsin is not used, and the damage to cells can be reduced; and by stepwise separation and digestion, the heart cells can be stably and reliably digested, and the obtained heart tissue single cell suspension has a high cell content and high viability, and the number of cells is 1×10 6 / mL, cell viability> 75%, and the cardiac tissue cell suspension can be obtained efficiently. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0025] Figure 1Schematic diagram of the process for preparing single-cell suspension of rhesus monkey heart tissue. DETAILED DESCRIPTION
[0026] The present invention provides a method for preparing a cardiac tissue single cell suspension, comprising the following steps:
[0027] 1) digesting the heart tissue fragments with heart digestion fluid to obtain cell digestion fluid; performing solid-liquid separation on the cell digestion fluid with a 70-100 mesh cell filter to obtain a filtrate and a filter residue;
[0028] The heart digestion solution includes 0.1wt.% composite collagenase and the balance RPMI-1640 culture medium; the composite collagenase includes collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4;
[0029] 2) washing the filter residue in step 1) with the heart digestion solution in step 1) to obtain a cell washing solution; digesting the cell washing solution to obtain a cell digestion solution; performing solid-liquid separation on the cell digestion solution using a 70-100 mesh cell filter screen to obtain a filtrate and a filter residue;
[0030] 3) Repeating the operation of step 2) 1 to 6 times, and in each repetition, using the heart digestion fluid of step 1) to treat the latest obtained filter residue in the manner of step 2) to obtain a filtrate and filter residue obtained by repeated treatment;
[0031] 4) Perform the following S1 or S2 operations:
[0032] S1: centrifuging the filtrate in step 3) respectively, collecting the precipitates and resuspending them respectively to obtain multiple cardiac tissue single cell suspensions;
[0033] S2: Centrifuge the filtrates of step 1) and step 2) and the filtrate obtained by repeated treatment of step 3) respectively, collect the precipitates and resuspend them respectively, combine the resuspended solutions and centrifuge them again, collect the precipitates and resuspend them again to obtain a single cell suspension of cardiac tissue.
[0034] As an embodiment, the present invention prepares heart tissue fragments. As an embodiment, the size of the heart tissue fragments of the present invention is 0.5-1 mm 3 As another embodiment, the size of the heart tissue fragments of the present invention is 0.6 to 0.8 mm 3. As an embodiment, the heart tissue fragments of the present invention include rhesus monkey heart tissue fragments. As an embodiment, the rhesus monkey heart tissue fragments of the present invention include fragments of the apical part of the rhesus monkey heart tissue. As an embodiment, the present invention uses 75% v / v ethanol to soak the rhesus monkey heart tissue, takes it out and places it in a pre-cooled PBS buffer containing 5% double antibody and cuts it into pieces to obtain the rhesus monkey heart tissue fragments. As an embodiment, the double antibody of the present invention includes penicillin and streptomycin; the penicillin and streptomycin. As an embodiment, the ratio of penicillin and streptomycin in the double antibody of the present invention is 500U:0.5mg. As an embodiment, the soaking time of the present invention is 3 to 5min. The present invention uses 75% v / v ethanol to soak the rhesus monkey heart tissue to remove the membrane and connective tissue.
[0035] After obtaining the heart tissue fragments, the present invention uses heart digestion fluid to perform a first digestion treatment on the heart tissue fragments to obtain a first cell digestion fluid. The heart digestion fluid of the present invention includes 0.1wt.% composite collagenase and the remainder of RPMI-1640 culture medium; the composite collagenase includes collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4. As an embodiment, the temperature of the first digestion treatment of the present invention is 37°C, the time is 5min, and the rotation speed is 60rpm. As an embodiment, the present invention uses 1640 full culture fluid to terminate the first digestion treatment. As an embodiment, the 1640 full culture fluid of the present invention includes 10% v / v FBS, 1% v / v PS and the remainder of RPMI-1640 culture medium.
[0036] As one embodiment, the dosage ratio of the heart digestive fluid and heart tissue of the present invention is (0.2-2.2) g:5 mL; as another embodiment, the dosage ratio of the heart digestive fluid and heart tissue of the present invention is (0.3-0.5) g:5 mL.
[0037] The heart digestion solution of the present invention utilizes collagenase complex enzymes instead of trypsin which may cause great damage to cells. The preparation operation is simple, the price is low, the cost is low, and the heart tissue can be stably and reliably digested to improve the cell content and survival rate.
[0038] After obtaining the first cell digestion liquid, the present invention uses a 70-mesh to 100-mesh cell filter screen to perform a first solid-liquid separation on the first cell digestion liquid to obtain a first filtrate and a first filter residue. As an embodiment, the present invention uses a 70-mesh cell filter screen to perform a first solid-liquid separation on the first cell digestion liquid to obtain a first filtrate and a first filter residue. As an embodiment, the cell filter screen of the present invention is purchased from Corning.
[0039] After obtaining the first filtrate and the first filter residue, the present invention uses the heart digestion solution described in the above technical solution to wash the first filter residue to obtain a cell washing solution; the cell washing solution is subjected to a second digestion treatment to obtain a second cell digestion solution; the second cell digestion solution is subjected to a second solid-liquid separation using a 70-mesh to 100-mesh cell filter screen to obtain a second filtrate and a second filter residue. The temperature of the second digestion treatment of the present invention is 37°C, the time is 5min, and the speed is 60rpm. As an embodiment, the volume ratio of the heart digestion solution used for the first digestion treatment of the present invention and the heart digestion solution used for washing is 1:1. As an embodiment, the present invention uses a 70-mesh cell filter screen to perform a second solid-liquid separation on the second cell digestion solution to obtain a second filtrate and a second filter residue. As an embodiment, the 1640 full culture solution of the present invention includes 10% v / v FBS, 1% v / v PS and the remainder of RPMI-1640 culture medium.
[0040] After obtaining the second filtrate and the second filter residue, the present invention repeats the steps of preparing the second filtrate and the second filter residue 1 to 6 times, and in each repetition, the newly obtained filter residue is treated with the heart digestive fluid in the manner of preparing the second filtrate and the second filter residue to obtain the filtrate and filter residue obtained by repeated treatment. As an embodiment, the present invention repeats the steps of preparing the second filtrate and the second filter residue 2 to 6 times; as another embodiment, the present invention repeats the steps of preparing the second filtrate and the second filter residue 3 to 6 times; as another embodiment, the present invention repeats the steps of preparing the second filtrate and the second filter residue 5 to 6 times.
[0041] After obtaining the filtrate obtained by the repeated treatment, the present invention performs the following S1 or S2 operation: S1: centrifuging the filtrate obtained by the repeated treatment respectively, collecting the precipitates and resuspending them respectively to obtain multiple cardiac tissue single cell suspensions; S2: centrifuging the first filtrate, the second filtrate and the filtrate obtained by the repeated treatment respectively, collecting the precipitates and resuspending them respectively, combining the resuspensions and centrifuging again, collecting the precipitates and resuspending them again to obtain a cardiac tissue single cell suspension.
[0042] The present invention centrifuges the filtrate obtained by the repeated treatment, collects the precipitate and resuspends them respectively to obtain multiple cardiac tissue single cell suspensions. As an embodiment, the centrifugation conditions of the present invention are: 4°C, 1500rpm, 5min. As an embodiment, the solution used for resuspension of the present invention includes 5% v / v double antibody and the remaining PBS buffer. As an embodiment, the double antibody of the present invention includes penicillin and streptomycin; the penicillin and streptomycin. As an embodiment, the ratio of penicillin and streptomycin in the double antibody of the present invention is 500U: 0.5mg. As an embodiment, the PBS buffer of the present invention includes NaCl 137mmol / L, KCl 2.7mmol / L, Na2HPO410mmol / L, KH2PO42mmol / L, and pH value 7.2-7.4. The present invention centrifuges the filtrate obtained by the repeated treatment, collects the precipitate and resuspends them respectively, and can obtain cardiac tissue single cell suspensions with higher cell viability. The results of the embodiment show that the cell viability of cardiac tissue single cell suspensions obtained by different centrifuges is greater than 75%.
[0043] The present invention centrifuges the first filtrate, the second filtrate and the filtrate obtained by repeated treatment, collects the precipitate and resuspends them respectively, combines the resuspended liquid and centrifuges again, collects the precipitate and resuspends them again to obtain a single cell suspension of cardiac tissue. As an embodiment, the temperature of the centrifugation and re-centrifugation of the present invention is 4°C, the speed is 1500rpm, and the time is 5min. As an embodiment, the solution used for resuspension and re-resuspending of the present invention includes 5% v / v double antibody and the remaining PBS buffer. As an embodiment, the double antibody of the present invention includes penicillin and streptomycin; the penicillin and streptomycin. As an embodiment, the ratio of penicillin and streptomycin in the double antibody of the present invention is 500U: 0.5mg. As an embodiment, the PBS buffer of the present invention includes NaCl 137mmol / L, KCl2.7mmol / L, Na2HPO410mmol / L, KH2PO42mmol / L, and pH value 7.2-7.4. The present invention combines different resuspensions after centrifugation and resuspending of different filtrates, and then centrifuges and resuspends again, which can improve the cell viability in the cardiac tissue single cell suspension. The results of the embodiment show that the viability of the obtained cardiac tissue single cell suspension is greater than 83%.
[0044] The present invention also provides the use of the cardiac tissue single cell suspension prepared by the preparation method described in the above technical solution in the preparation of products for studying cardiac diseases.
[0045] In order to further illustrate the present invention, a preparation method of a cardiac tissue single cell suspension and its application provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0046] Example 1
[0047] according to Figure 1 Procedure: Preparation of single cell suspension of rhesus monkey heart tissue, the specific steps are as follows:
[0048] 1. Preparation of composite collagenase: Take 80 mL RPMI-1640 medium (purchased from Hyclone), add 0.2 g collagenase II (purchased from Gibco) and 0.8 g collagenase IV (purchased from Gibco), fully dissolve and mix, add RPMI-1640 medium to make up to 100 mL, filter with 0.22 μm filter membrane, package into 25 mL / tube, store in a -20 ° C refrigerator, and restore to 37 ° C before use.
[0049] Preparation of 2.5% double antibody buffer: Take 495mL PBS buffer (NaCl 137mmol / L, KCl2.7mmol / L, Na2HPO410mmol / L, KH2PO42mmol / L, pH 7.2-7.4, ensure high pressure sterilization before the experiment), add 25mL 100× penicillin-streptomycin double antibody to form a 5% double antibody buffer, the concentration of penicillin and streptomycin is 500U / mL and 0.5mg / mL respectively, and store it in 4℃ precooling for later use;
[0050] 3. Preparation of 1640 full culture medium: Take 445mL RPMI-1640 culture medium, add 50mL fetal bovine serum (FBS, purchased from Gibco), 5mL 100× double antibody buffer (PS, purchased from Institute of Medical Biology, Chinese Academy of Medical Sciences), and prepare 10% FBS-1% PS (Penicillin-Streptomycin)-1640; the whole preparation process is carried out in a clean bench;
[0051] 4. Obtaining Rhesus Monkey Cardiomyocytes:
[0052] After the monkeys were euthanized, they were immediately disinfected and dissected to obtain 0.2-2.2 g of the apical part of the heart tissue, which was placed in pre-cooled PBS and quickly transported to the laboratory in an ice box. The heart tissue was then placed in a culture dish filled with 75% v / v ethanol and soaked and rinsed for 3-5 minutes. The temperature was kept low (placed on ice) throughout the process to ensure the viability of the cells. Sterile ophthalmic forceps were used during the soaking and rinsing process, and gentle movements were ensured throughout the process.
[0053] 5. Digestion of cardiac tissue: Take out the sterilized and cleaned cardiac tissue and place it in a 2 mL EP tube containing 1 mL pre-cooled PBS. Use sterile ophthalmic surgical scissors to cut the cardiac tissue into pieces of about 0.5-1 mm. 3After that, centrifuge the tube twice at 4°C, 1500 rpm, 5 min, remove the supernatant, add it to a 50 mL centrifuge tube containing 5 mL of 37°C preheated composite collagenase in step 1, and gently invert to mix to resuspend the rhesus monkey myocardial tissue fragments.
[0054] 6. Preparation of cardiac tissue single cells: After resuspending the myocardial tissue fragments, place the above centrifuge tube in a 37°C shaker at 60 rpm for 5 minutes, and then terminate the digestion with 5 mL of 1640 full culture to obtain cell mixture 1. Filter the cell mixture 1 through a 70-mesh cell filter and collect filtrate 1. Rinse the remaining myocardial tissue fragments on the above cell filter with 5 mL of step 1 composite collagenase. During the rinsing process, gently grind with the back of a 1 mL gun tip to obtain more cardiac single cells that have been digested well on the tissue surface but have not yet detached; harvest the obtained tissue cell suspension and place it in a 37°C shaker at 60 rpm for 5 minutes, and then terminate the digestion with 1640 full culture to obtain cell mixture 2. Filter the cell mixture 2 through a 70-mesh cell filter and collect filtrate 2. Repeat the above steps to obtain filtrate 3, filtrate 4, filtrate 5, filtrate 6, filtrate 7 and filtrate 8.
[0055] 7. Collect cardiac single cells by centrifugation and prepare single cell suspension: centrifuge filtrates 1 to 8 to remove supernatant, resuspend in PBS, blow and disperse cells to obtain multiple cardiac tissue single cell suspensions, mark them from 5min-1 to 5min-8 in sequence for counting.
[0056] 8. Combine multiple cardiac tissue single cell suspensions in step 7, centrifuge and remove the supernatant, resuspend in PBS, blow and disperse the cells to obtain the final rhesus monkey cardiac tissue single cell suspension.
[0057] 9. Microscopic examination of viable cell yield and cell viability: dilute the multiple cardiac tissue single-cell suspensions in step 7 and the final rhesus monkey cardiac tissue single-cell suspension to appropriate concentrations, aspirate 10 μL of each, add an equal volume of trypan blue dye, mix thoroughly, aspirate 10 μL and add to a blood cell counting plate, count under a microscope, measure the viable cell ratio, repeat the test 4 times (RM1-4), the results are shown in Table 1.
[0058] Table 1 Viability of single cell suspension prepared from rhesus monkey heart tissue (%)
[0059] Single cell suspension RM1 Mean RM2 Mean RM3 Mean RM4 Mean 5min-1**** 37.74 46.73 47.54 42.83 49.12 46.57 45.00 40.70
[0060]
[0061] Note: In the table, * indicates p≤0.05, **** indicates p≤0.0001.
[0062] It can be seen from Table 1 that the technical solution provided by the present invention can stably and reliably digest rhesus monkey heart tissue, and the obtained cell viability is >75%. The method for preparing a single cell suspension of heart tissue of the present invention is simple to operate, low in price, low in cost, and can efficiently obtain a single cell suspension of heart tissue.
[0063] Comparative Example 1
[0064] The same as Example 1, the only difference is that in step 5, 0.25% pancreatic enzyme (provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences, containing phenol red) is used to digest the heart tissue, and the viability of the final single cell suspension preparation of rhesus monkey heart tissue is shown in Table 2:
[0065] Table 2 Viability of single cell suspension prepared from rhesus monkey heart tissue (%)
[0066]
[0067]
[0068] Note: **** in the table means p≤0.0001.
[0069] According to Table 2, the cell viability of digestion with 0.25% pancreatin is significantly lower than that in Example 1. The data of the first 1 to 8 times of 5 min were statistically analyzed by T test, and it was found that the cell viability of the single cell suspension of heart tissue prepared in the first 4 times was statistically different.
[0070] Comparative Example 2
[0071] The same as Example 1, the only difference is that in step 5, the heart tissue is digested with collagenase II, and the viability of the final single-cell suspension preparation of rhesus monkey heart tissue is shown in Table 3.
[0072] Table 3 Viability of single cell suspension prepared from rhesus monkey heart tissue (%)
[0073]
[0074]
[0075] Note: In the table, * indicates p≤0.05, **** indicates p≤0.0001.
[0076] According to Table 3, the cell viability of collagenase II digestion was significantly lower than that of Example 1. The data of the first 1 to 8 times of 5 min were statistically analyzed by T test, and it was found that the cell viability of the single cell suspension of heart tissue prepared in the first 4 times was statistically different.
[0077] It can be seen from the above content that the technical solution provided by the present invention can stably and reliably digest heart cells, and the obtained heart tissue single cell suspension has a high cell content and high viability.
[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for preparing a cardiac tissue single cell suspension, characterized in that: The steps include: 1) digesting the heart tissue fragments with heart digestion fluid to obtain cell digestion fluid; performing solid-liquid separation on the cell digestion fluid with a 70-100 mesh cell filter to obtain a filtrate and a filter residue; The heart digestion solution includes 0.1wt.% composite collagenase and the balance RPMI-1640 culture medium; the composite collagenase includes collagenase II and collagenase IV; the mass ratio of collagenase II to collagenase IV is 1:4; 2) washing the filter residue in step 1) with the heart digestion solution in step 1) to obtain a cell washing solution; digesting the cell washing solution to obtain a cell digestion solution; performing solid-liquid separation on the cell digestion solution using a 70-100 mesh cell filter screen to obtain a filtrate and a filter residue; 3) Repeating the operation of step 2) 1 to 6 times, and in each repetition, using the heart digestion fluid of step 1) to treat the latest obtained filter residue in the manner of step 2) to obtain a filtrate and filter residue obtained by repeated treatment; 4) Perform the following S1 or S2 operations: S1: centrifuging the filtrate in step 3) respectively, collecting the precipitates and resuspending them respectively to obtain multiple cardiac tissue single cell suspensions; S2: Centrifuge the filtrates of step 1) and step 2) and the filtrate obtained by repeated treatment of step 3) respectively, collect the precipitates and resuspend them respectively, combine the resuspensions and centrifuge again, collect the precipitates and resuspend them again to obtain a single cell suspension of cardiac tissue.
2. The preparation method according to claim 1, characterized in that: In step 1), the ratio of the cardiac digestive fluid to the cardiac tissue is 5 mL: (0.2-2.2) g.
3. The preparation method according to claim 1, characterized in that: The volume ratio of the heart digestion solution used in the digestion treatment of step 1) and the heart digestion solution used in the washing of step 2) is 1:
1.
4. The preparation method according to claim 1, characterized in that: The temperatures of the centrifugation in S1, the centrifugation in S2, and the centrifugation again in S2 are 4° C., the rotation speeds are 1500 rpm, and the times are 5 min.
5. The preparation method according to claim 1, characterized in that: The solutions used for resuspending in S1, resuspending in S2, and resuspending in S2 respectively include 5% v / v dual antibody and the remainder PBS buffer; the dual antibody includes penicillin and streptomycin.
6. The preparation method according to claim 1, characterized in that: The temperature of the digestion treatment in step 1) and the digestion treatment in step 2) are 37° C., the time is 5 min, and the rotation speed is 60 rpm.
7. The preparation method according to any one of claims 1 to 6, characterized in that: The heart tissue fragments include rhesus monkey heart tissue fragments.
8. The preparation method according to claim 7, characterized in that: The size of the heart tissue fragments is 0.5 to 1 mm 3 .
9. The preparation method according to claim 7, characterized in that: The preparation method of the rhesus monkey heart tissue fragments comprises: soaking the rhesus monkey heart tissue in 75% v / v ethanol, taking it out and placing it in a pre-cooled PBS buffer containing 5% double antibody and chopping it to obtain the rhesus monkey heart tissue fragments; the double antibody comprises penicillin and streptomycin.
10. Use of the cardiac tissue single cell suspension prepared by the preparation method according to any one of claims 1 to 9 in the preparation and development of cardiac disease products.