Separation method of fibroblast derived from human heart tissue

Abstract

The invention discloses a method for separating fibroblasts from human heart tissue, and belongs to the technical field of cell culture. The method comprises the following steps: standing and digesting heart tissues by using digestive juice containing 0.25 mg / mL collagenase II and collagenase IV; and splitting by using a red blood cell lysis solution, resuspending, centrifuging, and discarding the supernatant to obtain the human heart fibroblasts. According to the invention, heart tissues can be fully dissociated, fibroblasts in the heart tissues can be released, and damage to the heart fibroblasts in the digestion process can be reduced to the greatest extent. According to the method, the problems of low cell activity, low cell yield and the like are solved, and the human heart fibroblasts with full cytoplasm and complete cell nucleuses can be obtained 2-4 days after separation. The method is convenient to operate, short in separation time, high in practicability and high in cell quality, and the human heart fibroblasts obtained through separation have wide application prospects in the aspects of cell culture, screening and testing of new drugs, providing of high-quality cell sources and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture and relates to a method for separating fibroblasts derived from human heart tissue. Background Art

[0002] Fibroblasts are a type of mesenchymal cells found in connective tissue. Fibroblasts can form a highly ordered network structure in the tissue by secreting a variety of extracellular matrix (ECM) components such as collagen, proteoglycans, elastin, fibronectin and laminin, and play an important role in regulating tissue mechanical stress, damage and repair. The heart is rich in fibroblasts, which are mainly distributed in the interstitial spaces between myocardial cells, the epicardium and the endocardium. Among them, fibroblasts originating from the epicardium and endocardial epithelium account for about 60%-70% of the total number of cells in the adult human heart. Cardiac fibroblasts play an important regulatory function under physiological and pathological conditions. Under physiological conditions, cardiac fibroblasts are responsible for forming the extracellular matrix network of the heart, maintaining the integrity of myocardial tissue and heart structure, and maintaining normal electrical conduction and heart rhythm. Under pathological conditions, fibroblasts are activated, proliferated and further differentiated into myofibroblasts under the influence of multiple factors such as mechanical tension and cytokines such as transforming growth factor β (TGF-β) to maintain the integrity of the cardiac structure and ensure the stability of cardiac function. However, excessive activation of fibroblasts can lead to excessive deposition of extracellular matrix and fibrous components, forming fibrotic scars, causing cardiac systolic and diastolic dysfunction, and ultimately causing heart failure or even death. Fibroblasts also play a key role in the repair and regeneration of myocardial tissue. Studies have shown that through the in situ reprogramming technology of myocardial cells, scar-forming fibroblasts can be transdifferentiated into induced cardiomyocytes, thereby effectively regenerating new myocardial tissue, reducing the area of ​​infarction and improving cardiac function.

[0003] High-quality cardiac fibroblasts have the following important significance in basic biological research, regenerative medicine research and clinical applications: 1. Studying the mechanisms of cardiac fibroblast proliferation, differentiation, apoptosis, etc. is crucial for understanding physiological and pathological processes such as cardiac development and fibrosis. 2. By regulating the activity of fibroblasts, cardiac fibrosis can be alleviated, cardiac function can be improved, and it helps to reveal the pathogenesis of heart disease and provide new targets for diagnosis and treatment. 3. By studying the function and regulatory mechanism of fibroblasts, new treatment strategies can be developed to promote the repair and regeneration of damaged tissues, providing a theoretical basis and experimental basis for regenerative medicine.

[0004] Collagen is the main component of the extracellular matrix of cardiac tissue, mainly composed of type I and type III fibrous collagen, as well as type IV, V and VI collagen. Type I and type III collagen are expressed in large quantities in the heart of normal mammals, accounting for 90-95% of cardiac collagen. Different types of collagen have a triple helical domain composed of glycine, proline and hydroxyproline in structure, which gives collagen higher tensile strength and resistance to proteolysis. Due to the dense structure of the extracellular matrix of cardiac tissue, various digestive enzymes are usually required to separate cells from the heart, accompanied by mechanical means such as shaking and vortexing to assist digestion.

[0005] Commonly used digestive enzymes include trypsin and collagenase. Trypsin is a common general digestive enzyme that mainly hydrolyzes the peptide bond between lysine (Lys) and arginine (Arg) to reduce intercellular connections, thereby achieving the purpose of digesting cells. Trypsin has no substrate specificity and has a strong effect on tissues and cells. In actual use, the dosage and digestion time of trypsin should be closely monitored. If the dosage and digestion time are inappropriate, it will lead to excessive tissue dissociation, causing cell damage or even death. Cardiac fibroblasts are cells that grow on the wall. Incomplete digestion or over-digestion during the separation process will cause changes in cell activity, cell adhesion ability and cell function, thereby affecting the research results. Therefore, how to obtain highly active and high-quality cardiac fibroblasts is the focus and difficulty in the field of cardiac research.

[0006] Collagenase specifically recognizes the Pro-X-Gly-Pro sequence in the substrate and cuts the peptide bond between the neutral amino acid (X) and glycine (Gly) in the sequence, thereby achieving protein cutting and degradation. Collagenase II, the main enzyme substrates include type I collagen, type III collagen, fibronectin, etc. The enzyme is oxidized by reducing agents and Ca 2+ After activation, it shows different substrate specificity from trypsin, and can complement other proteases to further improve the enzyme mixture, which is more conducive to the separation of liver, bone, thyroid, heart and salivary gland tissue cells. Collagenase IV, whose main enzyme substrate is type I collagen, and the enzyme contains low trypsin activity, which can reduce damage to membrane proteins and cell surface receptors.

[0007] At present, there are two main methods reported for isolating mouse cardiac fibroblasts: enzymatic digestion and tissue block adhesion. In the process of isolating mouse cardiac fibroblasts, the invention patent with application publication number CN 110358723 A discloses the specific steps of the enzymatic digestion method, which uses 0.1% type II collagenase in a constant temperature shaker for enzymatic digestion. Using only type II collagenase, the effect is relatively simple and the digestion is insufficient. The invention patent with application publication number CN 114921402 A discloses another enzymatic digestion method, including using 0.5-7.0g / L trypsin, 0.8-10.0g / L type II collagenase, and 0.15-2.0g / L type I deoxyribonuclease (DNase) for shaking digestion. Among them, trypsin cannot specifically hydrolyze collagen, the digestion efficiency is low, and mechanical shear force will be generated during the shaking digestion process to damage the cells. The fibroblasts obtained by these two digestion methods have low vitality and proliferation ability. Another separation method is the tissue block adhesion method, which uses the adhesion properties of cardiac tissue to fix it in the culture dish. Cardiac fibroblasts will migrate out, but the migration cycle is long. After the fibroblasts proliferate and migrate, their original matrix components will change, causing the fibroblasts to transform into myofibroblasts. The resulting cell heterogeneity will have a great impact on subsequent experiments.

[0008] In summary, high-quality human primary cardiac fibroblasts are extremely scarce and urgently needed tool cells in basic scientific research experiments. Different cells have different tolerance to enzymes, and their survival rates vary in enzyme solutions with different ratios. Currently, no effective method for isolating human primary cardiac fibroblasts has been reported. Summary of the invention

[0009] Human-derived primary cardiac fibroblasts are closer to the actual situation in vivo, and the cell source is more clear. Isolating high-quality human primary cardiac fibroblasts has become a rate-limiting step in many cardiac research fields. The purpose of the present invention is to provide a method for isolating fibroblasts derived from human cardiac tissue. The human cardiac fibroblasts isolated by the present invention have broad application prospects in cell culture, screening and testing new drugs, and providing high-quality cell sources.

[0010] The object of the present invention is achieved through the following technical solutions:

[0011] A method for isolating fibroblasts from human cardiac tissue comprises the following steps: digesting cardiac tissue with a digestion solution containing collagenase II and collagenase IV; lysing the digested tissue with a red blood cell lysing solution, resuspending the lysed tissue with a buffer solution, centrifuging, and discarding the supernatant to obtain human cardiac fibroblasts. The concentrations of collagenase II and collagenase IV in the digestion solution are preferably 0.20-0.30 mg / mL, and more preferably 0.25 mg / mL.

[0012] In some embodiments, the digestion solution is a cell culture medium containing 0.25 mg / mL collagenase II and 0.25 mg / mL collagenase IV, and the cell culture medium is preferably RPMI-1640 medium. Furthermore, the digestion solution may also contain penicillin-streptomycin, DNase, and β-mercaptoethanol, and the concentrations of each are preferably 100 U / mL penicillin-streptomycin, 1 mg / mL DNase, and 0.05 mM β-mercaptoethanol.

[0013] In some embodiments, 1 g of heart tissue is preferably digested with 3 mL of digestion solution for 2-4 hours, wherein the heart tissue is cut into a paste with sterile surgical scissors and sterile blades.

[0014] In some embodiments, the composition of the red blood cell lysis solution is preferably: 0.15M ammonium chloride, 0.1M disodium ethylenediaminetetraacetic acid, 10mM potassium bicarbonate; the lysis condition is preferably lysis on ice for 0.5-1 minute.

[0015] In some embodiments, the FACS buffer is 1× Dulbecco's phosphate buffered saline (DPBS) containing 2% (v / v) fetal bovine serum and 2.5 mM ethylenediaminetetraacetic acid (EDTA).

[0016] Furthermore, the method for isolating fibroblasts derived from human heart tissue comprises the following steps:

[0017] (1) Under a sterile environment, use sterile surgical scissors and forceps to cut away excess fat, blood vessels and other tissues from the heart tissue, cut it into small pieces, and rinse the heart several times with pre-cooled washing solution.

[0018] The washing solution is preferably 1×DPBS containing 100 U / mL penicillin-streptomycin.

[0019] (2) Use sterile surgical scissors to cut the heart tissue into pieces, and then use a sterile blade to cut it into a paste. Transfer 1 g / well of the minced tissue pieces to a 6-well plate.

[0020] (3) Add 3 mL of digestion solution to each well of a 6-well plate to cover the tissue pieces and gently blow them apart.

[0021] (4) Place the 6-well plate in a 37°C, 5% CO 2 Digest in a constant temperature incubator for 2-4 hours until complete digestion.

[0022] (5) After the deionized tissue is completely blown away, it is filtered through a cell sieve into a centrifuge tube.

[0023] (6) The tissue mixture obtained by filtration is centrifuged and the supernatant is discarded. Red blood cell lysis buffer is added to lyse the mixture at low temperature in the dark and then FACS buffer is added to resuspend the cell pellet. The centrifugation condition is preferably 600g (rcf) centrifugation for 10 minutes.

[0024] (7) Centrifuge the tissue mixture obtained in step (6) and discard the supernatant. The obtained cell precipitate is the isolated human cardiac fibroblasts. The centrifugation condition is preferably 600g (rcf) for 5 minutes.

[0025] Furthermore, the method for isolating fibroblasts derived from human cardiac tissue also includes the following steps: resuspending the isolated human cardiac fibroblasts in a human cardiac fibroblast culture medium to obtain primary human cardiac fibroblasts that can be cultured adherently.

[0026] The human cardiac fibroblast culture medium is preferably Dulbecco's Modified Eagle Medium (DMEM) containing 20% ​​(v / v) fetal bovine serum and 100 U / mL penicillin-streptomycin.

[0027] The present invention is the first to isolate primary cardiac fibroblasts from human origin, and has the following advantages and beneficial effects: 1. It is the first to establish a human cardiac fibroblast digestion scheme: it determines the optimal fixed ratio of type IV collagenase and type II collagenase, and utilizes the enzymatic digestion activity and substrate specificity of different enzymes to improve tissue digestion efficiency. 2. In terms of digestion conditions, the static digestion method is used for the first time to avoid mechanical damage to cells by vortex and oscillation digestion methods. 3. It is the first to provide a ratio of digestive fluid to tissue and a fixed digestion container area, with one hole (9.62cm) in each 6-well plate. 2) is the spatial basis, and 3mL digestion solution is used for every 1g tissue, and the optimal digestion time is 2-4 hours. In short, such a digestion method weakens the damage of physical mechanical force to the original state of the cell, and through the combination and proportion of multiple types of collagenase, the heart tissue is fully digested, and highly active, adherent growth and passaged human primary cardiac fibroblasts are successfully separated. After 2-4 days of adherent culture of the separated product, human cardiac fibroblasts with full cytoplasm and complete nuclei can be obtained, which avoids the problems of low cell activity, poor cell morphology, low cell yield, slow cell proliferation growth, and difficulty in cell culture caused by excessive digestion of the tissue. The method provided by the present invention is convenient to operate, short separation time, strong practicality, high cell yield, and high cell quality. The obtained primary cardiac fibroblasts of human origin have high purity, complete structural function, and good cell state. The present invention helps to obtain primary cardiac fibroblasts from the human heart, helps to construct related cardiovascular disease models in vitro, studies the mechanism of cardiac fibrosis, and provides a good cell source and research basis for screening and identifying small molecule targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The morphological diagram of cardiac fibroblasts obtained from 3 human heart tissues isolated from the present invention and cultured for 1, 3, and 5 days.

[0029] Figure 2 This is an immunofluorescence identification diagram of vimentin isolated from human cardiac fibroblasts of the present invention.

[0030] Figure 3 This is a bar graph showing the cell number growth from Day 1 to Day 8 of human cardiac fibroblasts isolated and recovered from cryopreservation.

[0031] Figure 4 These are morphological images of cardiac fibroblasts obtained after isolating human cardiac tissue samples according to six different schemes in Example 3 and culturing them for 1, 2, 4, and 8 days, wherein AF are cell morphological images of schemes 1 to 6, respectively. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific implementation modes, but the protection scope of the present invention is not limited to the described contents.

[0033] Example 1

[0034] Human heart samples 1, 2 and 3 were selected, and the method for isolating fibroblasts from human heart tissue provided by the present invention was used, which specifically comprises the following steps:

[0035] (1) Move the retrieved heart tissue sample into the clean bench, wash it 2-3 times with detergent, use sterilized forceps in the left hand and sterilized surgical scissors in the right hand to cut off excess fat, blood vessels, etc. on the surface of the heart tissue, then wash the heart tissue repeatedly with detergent 3-4 times, and use sterilized surgical scissors to cut the heart tissue into pieces of about 0.5-1 cm 3 The small pieces were weighed with an electronic balance sterilized with UV light in advance, and about 1g / piece was temporarily stored in the washing solution for the next step. The composition of the washing solution used in this step was: 100U / mL penicillin-streptomycin (Gibco, 15140), 1×DPBS (Biosharp, BL310A).

[0036] (2) Place 1 g of weighed heart tissue on the lid of a 10 cm culture dish without culture medium. Use an aspirator to remove the residual washing solution around it, being careful not to suck up the tissue. Then use sterile surgical scissors to cut the heart tissue into approximately 1 mm thick pieces. 3 Use a sterile blade and sterile forceps to carefully transfer the tissue to a hole (9.62cm) in a 6-well plate. 2 )middle.

[0037] (3) Add 3 mL of digestion solution to the wells of a 6-well plate to cover the tissue blocks, then use a pipette to gently peel off the paste-like tissue blocks and continue to gently blow them apart with a sterile Pasteur pipette to mix them. The digestion solution used in this step is RPMI-1640 medium (Gibco, C11875500BT) containing 0.25 mg / mL collagenase IV (BioFRoXX, 2091GR001), 0.25 mg / mL collagenase II (Worthington, LS004177), 100 U / mL penicillin-streptomycin (Gibco, 15140), 1 mg / mL DNase (Sigma, DN25) and 0.05 mM β-mercaptoethanol (Thermofisher).

[0038] (4) Place the 6-well plate containing tissue and digestion solution in a 37°C, 5% CO 2 The culture medium was placed in an incubator at 4 °C for 2-4 hours until the tissue was completely digested. During the digestion period, the digestion fluid containing the tissue changed from slightly pink to turbid yellow.

[0039] (5) After static digestion, remove the 6-well plate from the incubator and move it to the clean bench. Use a 1 mL pipette to gently blow the tissue 10-15 times. You can see that the tissue is completely dispersed, the tissue digestion fluid is slightly viscous, and there are no obvious large pieces of tissue. Use a pipette to gently aspirate the digested tissue fluid, first filter it with a 100 μm cell sieve, and wash the remaining tissue digestion fluid in the 6-well plate with 3 mL RPMI-1640 culture medium. After all the filtration, use a sterile 10 mL syringe rubber head to grind the remaining tissue fragments on the sieve, then wash the sieve with 20 mL RPMI-1640 culture medium, and then add more culture medium to the centrifuge tube until the liquid in the 50 mL tube is no longer viscous. Then use a 70 μm cell sieve to filter the liquid into a new 50 mL centrifuge tube.

[0040] (6) Centrifuge at 600g (rcf) for 10 minutes. Aspirate the supernatant, protect from light, add 1 mL of red blood cell lysis buffer to the cell pellet on ice, mix gently, lyse on ice for 0.5-1 minute in the dark, and then add 10 mL of FACS Buffer to resuspend the cell pellet. The composition of the red blood cell lysis buffer used in this step is: 0.15 M ammonium chloride (NH 4 Cl), 0.1 M ethylenediaminetetraacetic acid disodium dihydrate (EDTANa 2 ·2H 2 O), 10 mM potassium bicarbonate (KHCO 3 ); FACS buffer composition: 2% (v / v) fetal bovine serum (NEWZERUM, FBS-S500), 97.5% (v / v) 1×DPBS (Biosharp, BL310A), 0.5% (v / v) 0.5M EDTA.

[0041] (7) Continue centrifugation at 600 g (rcf) for 5 minutes, discard the supernatant, and the resulting cell pellet is the isolated fibroblasts.

[0042] (8) Resuspend the fibroblasts obtained in step (7) with 10 mL of human cardiac fibroblast culture medium, and take 10 μL for counting. Mix the remaining cell suspension and add it to a 10 cm dish that has been pre-plated with 0.2% (v / v) Gelatin for 30 minutes, and place it at 37°C and 5% CO. 2Cultured overnight in an incubator, the cell status was observed the next day. The human cardiac fibroblast culture medium used in this step was DMEM (Gibco, C11995500BT) containing 20% ​​(v / v) fetal bovine serum (NEWZERUM, FBS-S500), 100U / mL penicillin-streptomycin (Gibco, 15140); 0.2% (v / v) Gelatin was composed of: 50mL 2% Gelatin (G7041-100G), 450mL 1×DPBS (Biosharp, BL310A).

[0043] (9) The next day, the isolated human cardiac fibroblasts were observed under a microscope, washed 2-3 times with detergent to remove dead cells and tissue fragments remaining on the cell surface, replaced with fresh human cardiac fibroblast culture medium, and placed at 37°C and 5% CO. 2 Continue culturing in the incubator.

[0044] (10) After the cells have grown for about 3-5 days, they can be subcultured. The fibroblasts have the following morphology: Figure 1 As shown, the fibroblasts isolated from samples 1, 2, and 3 all had intact nuclei but less cytoplasm on the first day; on the third day, the cytoplasm gradually became full and the cell state gradually recovered; after the fifth day, a large number of human fibroblasts in good condition with intact cytoplasm and a flat multi-protrusion shape could be obtained.

[0045] The human cardiac fibroblasts isolated from sample 1 were identified using human cardiac fibroblast marker vimentin and nuclear fluorescent dye 4',6-diamidino-2-phenylindole (DAPI). The specific method is as follows:

[0046] (1) Discard the cell culture supernatant, wash the cell surface with 1×DPBS to remove the residual culture medium, fix the cells with 4% paraformaldehyde for 15-20 minutes, and wash the cells three times with 1×DPBS again to remove the residual fixative.

[0047] (2) Add 0.1% triton X-100 to permeabilize the cell membrane at room temperature for 15 minutes, and wash the cells three times with 1× DPBS to remove residual triton X-100.

[0048] (3) Add 5% bovine serum albumin (BSA) and block at room temperature for 1 hour. After blocking, remove 5% BSA and add primary antibody (rabbit polyclonal anti-vimentin antibody) solution prepared with 1% BSA as solvent and incubate at 4°C overnight.

[0049] (4) The next day, the primary antibody was discarded, the cells were washed three times with 1×DPBS to remove the unbound primary antibody, and a secondary antibody solution (anti-rabbit IgG labeled with Alexa Fluor 488) prepared with 1% BSA as solvent was added and incubated in the dark for 1 hour.

[0050] (5) Discard the secondary antibody, wash the cells three times with 1×DPBS to remove the unbound secondary antibody, and use DAPI dye to stain the cell nucleus. After staining, the sample is imaged and analyzed, and the cells are photographed under a fluorescence microscope. Figure 2 As shown, the human cardiac fibroblasts separated by the method of the present invention have normal cell morphology, intact cell nuclei, and all cells highly express the fibroblast marker vimentin. Therefore, the present invention can obtain high-quality human cardiac fibroblasts.

[0051] Example 2

[0052] Human heart sample 4 was selected, and human cardiac fibroblasts were isolated according to the method in Example 1. After culturing for 7 days, the obtained cells were cryopreserved and the cryopreserved cells were resuscitated.

[0053] 1. Cell cryopreservation:

[0054] (1) Preheat trypsin and human cardiac fibroblast culture medium in a 37°C water bath.

[0055] (2) Open the lid of the cell culture dish in the biosafety cabinet, tilt the dish, and quickly aspirate the waste liquid using an aspirator.

[0056] (3) Use a pipette to slowly add 4-5 mL of 1× DPBS along the wall of the culture dish, gently shake the culture dish, and quickly aspirate and discard the 1× DPBS using an aspirator.

[0057] (4) Add 3 mL of trypsin to the culture dish, gently shake the dish to allow the trypsin to cover the bottom of the dish, and quickly place the cells in a 37°C incubator to digest for 2-3 minutes.

[0058] (5) Take the digested cells out of the incubator, gently tap the edge of the culture dish, and observe under a microscope to see whether the cells are completely digested.

[0059] (6) After the cells are completely digested, add 3 mL of serum-containing neutralization medium to the culture dish to terminate the digestion. Slowly pipette the cells to remove all cells from the culture dish.

[0060] (7) Collect the cell suspension into a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the waste liquid.

[0061] (8) While the cells are being centrifuged, a cell freezing solution is prepared. The formula of the cell freezing solution in this process is: 90% (v / v) fetal bovine serum + 10% (v / v) dimethyl sulfoxide (DMSO). DMSO is added to the fetal bovine serum and quickly pipetted to mix.

[0062] (9) According to the freezing ratio, add an appropriate amount of cell freezing solution to the cell pellet, pipette to mix well, and add 1 mL of freezing solution to each cryopreservation tube.

[0063] (10) Place the cryotube in a programmed cooling box, place it in a -80°C refrigerator to cool for 24 hours, and then transfer it to a cryobox for storage.

[0064] 2. Cell recovery:

[0065] (1) In a biosafety cabinet, take 8 mL of human cardiac fibroblast culture medium and preheat it in a 37°C water bath.

[0066] (2) Quickly take out the frozen cells from the -80℃ freezer and quickly thaw them in a 37℃ water bath. When a small amount of ice crystals remain in the cryotube, take out the cells and slowly add the frozen cells to the preheated culture medium. After slowly pipetting to mix, centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0067] (3) Add 8 mL of culture medium to the cell pellet and mix well using a pipette. Add the mixed cell suspension to a 10 cm dish for adherent culture.

[0068] 3. Cell digestion and counting:

[0069] (1) Preheat trypsin in a 37°C water bath.

[0070] (2) In a biosafety cabinet, half-open a 6-well cell culture plate, tilt it, and quickly aspirate and discard the waste liquid using a pipette.

[0071] (3) Use a pipette to quickly draw 1 mL of 1× DPBS and slowly add it along the wall of the culture dish. Shake gently and then use a pipette to discard the 1× DPBS.

[0072] (4) Add 500 μL of trypsin to the 6-well plate, gently shake to allow the trypsin to cover the 6-well plate, and quickly place the cell plate in a 37°C incubator for digestion for 2 minutes.

[0073] (5) Take the digested cell plate out of the incubator, tap it gently, and observe whether the cells are completely digested under a microscope. After confirming that the cells are completely digested, add 500 μL of 2% (v / v) fetal bovine serum to the culture dish to neutralize it. Use a pipette to slowly blow the cells, try to make all cells fall off the culture dish and become a single cell suspension. Add the blown cell suspension to a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the waste liquid.

[0074] (6) Add 1 mL of culture medium to the cell pellet and resuspend by pipetting. Take 10 μL of the cell solution and add an equal amount of trypan blue dye to count the cells.

[0075] (7) Take the above adherent cultured cells, digest three wells every 24 hours, and count them using a hemocytometer to obtain a statistical chart of the number of cells from Day 1 to Day 8. Figure 3 As shown in the figure, the human fibroblasts isolated in the early stage were revived and cultured for 1 to 8 days for counting. After 8 days of culture, the number of human cardiac fibroblasts increased from the initial 2.75×10 4 The number of cells increased to 1.57×10 5 cells.

[0076] Example 3

[0077] This example developed different digestion schemes based on the currently reported methods for isolating cardiac fibroblasts from different species, and compared the isolation efficiency of human cardiac fibroblasts under different digestion schemes. Human heart sample 4 was selected for digestion test.

[0078] Solution 1: Using the method of the present invention, the core digestive enzymes include: 0.25 mg / mL collagenase IV, 0.25 mg / mL collagenase II.

[0079] Solution 2: The core digestive enzyme was changed to 10 mg / mL collagenase II, and the other digestion conditions and methods were consistent with the method of the present invention.

[0080] Solution 3: The core digestion enzymes were changed to: 0.6 mg / mL collagenase II, 0.2 mg / mL collagenase IV, 0.6 mg / mL BSA, and the other digestion conditions and methods were consistent with the method of the present invention.

[0081] Scheme 4: The core digestive enzymes were changed to: 0.5 mg / mL collagenase IV, 0.5 mg / mL collagenase II, and the other digestion conditions and methods were consistent with the method of the present invention.

[0082] Plan 5: The specific operation steps include: moving the retrieved heart into the clean bench, cutting off excess fat and other tissues, and repeatedly washing the heart with pre-cooled 1×DPBS for 3-4 times. Cut the heart into pieces, and then cut it into 1mm pieces with a blade. 3, transfer to a 50mL centrifuge tube, add 10mL trypsin, and digest at 37°C for 15 minutes. After digestion, add about 0.5mg / mL collagenase II, disperse the tissue mass, vortex for 1 minute, bath at 37°C for 8 minutes, vortex for 1 minute, and filter the digested cell supernatant into the neutralization medium using a 70μm mesh. Repeat this step 6-8 times. Centrifuge the filtered supernatant at 600g (rcf) for 5 minutes and discard the supernatant.

[0083] Plan 6: The specific operation steps include: moving the retrieved heart into the clean bench, cutting off excess fat and other tissues, and repeatedly washing the heart 3-4 times with pre-cooled 1×DPBS. Cut the heart into pieces, and further chop the heart into a paste with a sterile blade, transfer it to a new 10cm culture dish, and add 3mL of human cardiac fibroblast culture medium: 80% (v / v) DMEM, 20% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin. After culturing for 2 hours, add human cardiac fibroblast culture medium to 8mL.

[0084] The morphology of fibroblasts isolated by different methods was photographed under bright field. Figure 4 As shown, AF are the results of Scheme 1 to Scheme 6, respectively. On the first day after separation, no obvious cell morphology appeared in the visual field of the six digestion schemes; on the second day after separation, fibroblasts appeared in the visual field of Scheme 1, and several flat multi-protruding fibroblasts were visible, and no obvious cell morphology appeared in the other schemes; on the fourth day after separation, the number of fibroblasts in the visual field of Scheme 1 gradually increased, and the cell state gradually recovered, and no obvious cell morphology appeared in the other schemes. On the eighth day after separation, the number of fibroblasts in the visual field of Scheme 1 increased significantly, the cell state was good, the cytoplasm was intact, and the cell extension was more obvious, and no obvious cell morphology appeared in the other schemes. In this embodiment, only Scheme 1, that is, the method of the present invention, can successfully obtain human cardiac fibroblasts derived from heart sample 4, and the cell morphology is intact and in good condition, and subsequent subculture can be carried out normally. Human cardiac fibroblasts derived from sample 4 were not obtained in other schemes. At present, there is no reported method for obtaining primary human cardiac fibroblasts. The present invention establishes an optimal method for obtaining human cardiac fibroblasts by static digestion of a fixed ratio of collagenase II and collagenase IV.

[0085] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for isolating fibroblasts from human heart tissue, Features: The following steps are involved: The heart tissue is statically digested with a digestion solution containing collagenase II and collagenase IV; the digested tissue is lysed with a red blood cell lysing solution, the lysed tissue is resuspended with a buffer solution, centrifuged, and the supernatant is discarded to obtain human heart fibroblasts; the concentrations of collagenase II and collagenase IV in the digestion solution are both 0.20-0.30 mg / mL.

2. The method for isolating fibroblasts derived from human heart tissue according to claim 1, Features: The digestive juice also contains penicillin-streptomycin, DNase and beta-mercaptoethanol.

3. The method for isolating fibroblasts derived from human heart tissue according to claim 2, Features: The digestion solution is a cell culture medium containing 0.25 mg / mL collagenase II, 0.25 mg / mL collagenase IV, 100 U / mL penicillin-streptomycin, 1 mg / mL DNase, and 0.05 mM β-mercaptoethanol.

4. The method for isolating fibroblasts derived from human heart tissue according to claim 1, Features: Use 3 mL of digestion solution for 1 g of heart tissue and allow to digest for 2-4 hours.

5. The method for isolating fibroblasts derived from human heart tissue according to claim 4, Features: The heart tissue was minced into a paste using sterile surgical scissors and a sterile blade.

6. The method for isolating fibroblasts derived from human heart tissue according to claim 1, Features: The composition of the red blood cell lysis solution is: 0.15M ammonium chloride, 0.1M disodium ethylenediaminetetraacetate, and 10mM potassium bicarbonate; the lysis condition is lysis on ice for 0.5-1 minute.

7. The method for isolating fibroblasts derived from human heart tissue according to claim 1, Features: The FACS buffer is 1×DPBS containing 2% fetal bovine serum and 2.5 mM EDTA.

8. The method for isolating fibroblasts derived from human heart tissue according to claim 1, Features: The following steps are involved: (1) Under sterile conditions, use sterile surgical scissors and forceps to cut away excess fat and other tissue from the heart tissue, cut it into small pieces, and rinse the heart several times with pre-cooled washing solution; (2) Cut the heart tissue into pieces with sterile surgical scissors, and then cut it into a paste with a sterile blade. Transfer 1 g / well of the minced tissue pieces to a 6-well plate; (3) Add 3 mL of digestion solution to each well of a 6-well plate to cover the tissue pieces and gently blow them apart; (4) Place the 6-well plate in a 37°C, 5% CO 2 Digest in a constant temperature incubator for 2-4 hours; (5) After the deionized tissue is completely blown away, it is filtered into a centrifuge tube using a cell sieve; (6) After centrifugation of the filtered tissue mixture, the supernatant was discarded, red blood cell lysis buffer was added to lyse the cells at low temperature in the dark, and FACS buffer was added to resuspend the cell pellet; (7) Centrifuging the tissue mixture obtained in step (6) and discarding the supernatant. The resulting cell precipitate is the isolated human cardiac fibroblasts.

9. The method for isolating fibroblasts derived from human heart tissue according to claim 8, Features: The centrifugation conditions described in steps (6) and (7) are 600g centrifugation for 5-10 minutes.

10. The method for isolating fibroblasts derived from human heart tissue according to any one of claims 1 to 9, Features: The following steps are also included: The separated human cardiac fibroblasts are resuspended in human cardiac fibroblast culture medium to obtain primary human cardiac fibroblasts that can be cultured adherently; the human cardiac fibroblast culture medium is DMEM containing 20% ​​fetal bovine serum and 100U / mL penicillin-streptomycin.

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