Method for extracting, purifying and identifying fibroblasts and application of fibroblasts

Abstract

The invention provides a method for extracting, purifying and identifying fibroblasts, which comprises the following steps of: cutting meninx or spinal membrane tissues into pieces, digesting, and centrifuging to obtain a precipitate; re-suspending the obtained precipitate by using a fibroblast culture solution, spreading the precipitate in a polylysine pre-coated culture flask, culturing until the cell confluence degree is 90%, shaking, and discarding the supernatant liquid to obtain purified fibroblasts; and carrying out passage on the obtained purified fibroblasts, carrying out immobilization and permeabilization, sealing with goat serum, sequentially adding a primary antibody solution and a fluorescent secondary antibody solution, dyeing with DAPI, and observing and identifying the fibroblasts under a fluorescence microscope to complete the extraction, purification and identification of the fibroblasts. The invention also provides an application of the method. The method is used for extracting, purifying and identifying meningeal fibroblasts or spinal fibroblasts. The fibroblasts extracted by the method are high in purity, short in culture period and simple to operate.

Description

Technical Field

[0001] This invention belongs to the field of cell extraction and purification technology, specifically relating to a method and application for the extraction, purification and identification of fibroblasts. Background Technology

[0002] Spinal cord injury repair remains an unsolved international challenge, and the formation of fibrous scars after spinal cord injury is one of the most significant factors hindering axonal regeneration and spinal cord repair. Scar tissue formation involves the following processes: 1. New capillary formation at the injury site; 2. Fibrosis; 3. Tissue remodeling. Under the influence of aseptic inflammation mediated by myelin fragments at the injury site, the activation, proliferation, differentiation, chemotactic migration, and synthesis and secretion of extracellular matrix by spinal cord fibroblasts are the main factors promoting fibrous scar formation. Furthermore, fibroblasts are involved in maintaining spinal cord tissue structure, repairing and remodeling after spinal cord injury, thereby affecting spinal cord function.

[0003] Scar formation is a chronic, progressive, and irreversible disease characterized by the activation and proliferation of fibroblasts, excessive deposition of extracellular matrix, and chronic interstitial inflammation. It is also the biggest obstacle to axonal regeneration in chronic spinal cord injury. Abnormal and persistent activation of spinal cord fibroblasts is considered a crucial process in the mechanism of spinal cord fibrosis scar formation. Simulating the mechanism of spinal cord fibrosis scar formation using a mouse spinal cord fibroblast in vitro model can provide new insights for treating or alleviating spinal cord fibrosis scarring.

[0004] However, there is currently no effective method for extracting primary mouse brain / spinal cord fibroblasts. This is due to several reasons: First, spinal cord fibroblasts do not exist in the normal spinal cord but are derived from other cell types. Second, spinal cord fibroblasts obtained using traditional extraction methods contain many impurity cells, have low purity, and grow slowly, making them unsuitable for in vitro experiments.

[0005] Existing culture methods, such as flow cytometry sorting or immunomagnetic bead sorting, require expensive equipment, magnetic beads, and antibodies. They also have high requirements for experimental conditions and operational techniques, resulting in poor economic efficiency and convenience, and are difficult to cultivate in general laboratories. Traditional culture methods require waiting for several passages of cells to obtain purified primary fibroblasts, greatly reducing the number of passages that can be used for subsequent experiments with fibroblasts.

[0006] Currently, brain / spinal cord fibroblasts, which are the basis for research, are difficult to obtain. The culture cycle of primary brain / spinal cord fibroblasts is long, and cell extraction and purification are difficult and expensive. There is no recognized, public, or feasible method for the extraction of primary brain / spinal cord fibroblasts. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method and application for the extraction, purification and identification of fibroblasts, which addresses the shortcomings of the prior art. The method extracts fibroblasts with high purity, short culture period and simple operation.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for the extraction, purification and identification of fibroblasts, the method being as follows: S1. Extraction and purification of fibroblasts: S101. Separate the meninges or spinal meninges and cut them into small pieces. Mix the cut tissue with the tissue digestion solution and digest at 37°C for 30 minutes. Mix the tissue by blowing and blowing every 10 minutes. Filter the mixture through a sieve to obtain a suspension. S102. Add digestion termination solution to the suspension obtained in S101, centrifuge to obtain precipitate; add fibroblast culture medium to resuspend the precipitate to obtain resuspended solution. S103. Take 1 mL to 2 mL of polylysine solution and place it evenly in a culture flask. Let it stand at 37°C for 1 to 2 hours. Remove the excess polylysine solution and dry it at 37°C for 4 hours to obtain a culture flask pre-coated with polylysine. S104. Add 10 mL of phosphate buffer to the culture flask pre-coated with poly-L-lysine obtained in S103 for rinsing, discard the phosphate buffer, and spread the resuspension obtained in S102 evenly in the culture flask pre-coated with poly-L-lysine after rinsing. When the cell confluence reaches 90%, shake, discard the supernatant, and obtain purified fibroblasts. S2. Identification of fibroblast purity: S201. Place the purified fibroblasts obtained in S104 into a cell culture plate containing fibroblast culture medium and culture until the cell confluence reaches 90%. Discard the fibroblast culture medium to obtain cultured fibroblasts. Wash the cultured fibroblasts three times with phosphate buffer. Add 500 μL of paraformaldehyde fixative to each well of the cell culture plate and fix for 30 min. Discard the paraformaldehyde fixative to obtain fixed fibroblasts. S202. Wash the fixed fibroblasts obtained in S201 with phosphate buffer and shake. Repeat the washing and shaking operation with phosphate buffer for a total of 3 times. Discard the phosphate buffer to obtain fixed and washed fibroblasts. S203. Add 500 μL of immunostaining permeabilization solution to each well of the cell culture plate, permeate for 30 min, then discard the immunostaining permeabilization solution to obtain permeabilized fibroblasts. S204. Wash the permeabilized fibroblasts obtained in S203 with phosphate buffer and shake. Repeat the phosphate buffer washing and shaking operation three times. Discard the phosphate buffer to obtain permeabilized and washed fibroblasts. S205. Add 250 μL of goat serum blocking solution to each well of the cell culture plate and block for 1 h. Then add 250 μL of primary antibody solution to each well and culture at 4 °C for 12 h. Recover the primary antibody solution to obtain fibroblasts that specifically bind to the primary antibody. S206. Wash the fibroblasts that specifically bind to the primary antibody obtained in S205 with phosphate buffer and shake. Repeat the phosphate buffer washing and shaking operation for a total of 3 times. S207. Under light-protected conditions, add 250 μL of fluorescent secondary antibody solution to each well of the cell culture plate, culture at 37°C for 1 h, discard the fluorescent secondary antibody solution, and obtain fibroblasts that specifically bind to the fluorescent secondary antibody. S208. Under light-protected conditions, the fibroblasts that specifically bind to the fluorescent secondary antibody obtained in S207 are washed with phosphate buffer and shaken. The fibroblasts that specifically bind to the secondary antibody are then washed with phosphate buffer and shaken for a total of 3 times. S209. Under light-protected conditions, add 200 μL of DAPI to each well of the cell culture plate for nuclear staining to obtain nuclear-stained fibroblasts. Wash the nuclear-stained fibroblasts with phosphate buffer, shake, and repeat the phosphate buffer washing and shaking operation three times. Observe under a fluorescence microscope to complete the extraction, purification and identification of fibroblasts.

[0009] Preferably, the volume ratio of the tissue and the tissue digestion solution in S101 is 1:5; the tissue digestion solution is composed of the following raw materials in the following final contents: 0.25wt% EDTA-free pancreatic enzyme and 1mg / mL type IV collagenase; the pore size of the sieve is 40μm.

[0010] Preferably, the digestion termination solution in S102 is a DMEM / F-12 medium containing 10% fetal bovine serum; the volume ratio of the digestion termination solution to the fibroblast culture medium is 2:1; the centrifugation conditions are: centrifugation at 1500 rpm for 5 min; the fibroblast culture medium in S102 and S201 is prepared by adding the following raw materials to the DMEM / F-12 medium in the following final amounts: 10 wt% fetal bovine serum, 10 ng / mL TGF-β, and 1 wt% penicillin-streptomycin antibiotic solution.

[0011] Preferably, the mass concentration of the polylysine solution in S103 is 0.1 mg / mL; the culture conditions in S104 are: humidity of 95%, temperature of 37°C, and CO2 of 5%; the shaking conditions are: shaking at 37°C and 220 rpm for 24 h.

[0012] Preferably, the density of fibroblasts in the cell culture plate described in S201 is 2.5 × 10⁻⁶. 4 Cells / well; culture conditions: temperature 37℃, 5% CO2; the mass fraction of the paraformaldehyde fixative is 4%.

[0013] Preferably, the oscillation conditions in S202, S204, S206, S208 and S209 are: oscillation at 200 rpm for 5 min; the immunostaining permeation solution in S203 is Triton X-100 with a mass fraction of 0.2%.

[0014] Preferably, the mass fraction of the goat serum in S205 is 10%.

[0015] Preferably, the nuclear staining time in S209 is 10 minutes.

[0016] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to isolate and extract meningeal and spinal fibroblasts from rat pups. Newborn rat pups were selected as the extraction subjects. Compared with adult mice, rat pups have higher cell activity, stronger cell stemness, and a higher number of passages, which greatly increases the number of passages available for subsequent experiments.

[0017] 2. This invention makes meningeal and spinal meningeal tissues easier to digest by combining multiple digestive enzymes and physical cutting methods, thereby reducing the time required for digestion of meningeal and spinal meningeal tissues.

[0018] 3. This invention combines selective culture, differential adhesion, and shaker oscillation methods to screen meningeal and spinal fibroblasts. This method can avoid the difficulties in cell screening caused by non-meningeal and spinal fibroblasts mixed in during primary cell culture.

[0019] 4. This invention uses T25 culture flasks pre-coated with poly-L-lysine to promote fibroblast adhesion. Fibroblasts are cultured in DMEM / F-12 medium containing TGF-β, which allows more fibroblasts to adhere earlier and enter the logarithmic growth phase more quickly. The fibroblasts extracted by this invention can cover 90% of the bottom of the T25 flask in about one day for passage or experimentation, with a purity of over 99%.

[0020] 5. The method for extraction, purification and identification of fibroblasts of the present invention is simple to operate, highly efficient, economical and convenient.

[0021] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 This is a diagram of the spinal cord tissue of a young mouse in Embodiment 1 of the present invention.

[0023] Figure 2 This is an optical microscope image of spinal fibroblasts in baby rats in Example 1 of this invention.

[0024] Figure 3 This is an optical microscope image of spinal fibroblasts purified from pups in Example 1 of this invention.

[0025] Figure 4 This is an optical microscope image of spinal fibroblasts from pups after passage in Example 1 of this invention.

[0026] Figure 5 This is an immunofluorescence identification image of PDGFRβ, a surface marker of spinal fibroblasts in rat pups, in Example 1 of this invention.

[0027] Figure 6 This is an immunofluorescence identification image of Fibronectin, a surface marker of spinal fibroblasts in pups, in Example 1 of this invention.

[0028] Figure 7 This is an immunofluorescence negative identification image of GFAP, a surface marker of astrocytes in rat pups, in Example 1 of this invention.

[0029] Figure 8 This is an immunofluorescence negative identification image of OLIG2, a surface marker of oligodendrocytes in pups, in Example 1 of this invention.

[0030] Figure 9 This is a histogram of fibroblast purity calculated using ImageJ software in Example 1 of this invention.

[0031] Figure 10 This is a brain tissue diagram of a baby mouse in Embodiment 2 of the present invention. Detailed Implementation

[0032] Example 1 This embodiment describes a method for the extraction, purification, and identification of spinal meningeal fibroblasts. The method is as follows: S1. Extraction and purification of spinal meningeal fibroblasts: S101. After euthanizing 3-day-old pups by cervical dislocation, immerse them in -20°C frozen alcohol for disinfection. On a tray containing ice, cut open the skin on the back and the muscles on both sides of the spine, and sever the spine at the legs. Using a syringe, draw serum-free DMEM / F-12 culture medium and insert it into the medullary cavity to flush out the spinal cord completely using water pressure. Figure 1 As shown, spinal cord tissue of pups was obtained. The obtained spinal cord tissue of pups was immersed in serum-free DMEM / F-12 medium at 4°C and placed on a clean bench. Under a stereomicroscope, the spinal membranes (dura mater and pia mater) attached to the surface of the spinal cord tissue were carefully separated using micro forceps and micro scissors. The separated spinal membranes were transferred to a new 35 mm diameter culture dish and the separated spinal membranes were cut into small pieces with sterile micro scissors. S102. After mixing the shredded spinal membrane with the tissue digestion solution, digest it at 37°C for 30 minutes, mixing it by blowing every 10 minutes, and then filtering it through a sieve with a pore size of 40μm to obtain a suspension. The volume ratio of the shredded spinal membrane to the tissue digestion fluid is 1:5; The tissue digestion solution is composed of the following raw materials in the following final amounts: 0.25 wt% EDTA-free pancreatic enzyme and 1 mg / mL type IV collagenase; S103. Transfer the suspension obtained in S102 to a new 15mL centrifuge tube, add 10mL of digestion termination solution, and centrifuge at 1500rpm for 5min to obtain a precipitate; resuspend the precipitate in 5mL of fibroblast culture medium to obtain a resuspended solution; observe the spinal fibroblasts in the obtained suspension using an optical microscope, such as... Figure 2 As shown, spindle-shaped cells are spinal meningeal fibroblasts, while round cells are other cell types, mainly astrocytes. The fibroblast culture medium was prepared by adding the following ingredients to DMEM / F-12 medium in the following final amounts: 10 wt% fetal bovine serum, 10 ng / mL TGF-β, and 1 wt% penicillin-streptomycin solution. S104. Take 1 mL of polylysine solution with a mass concentration of 0.1 mg / mL and place it evenly in a culture flask. Let it stand at 37°C for 1 h, remove the excess polylysine solution, and then dry it at 37°C for 4 h to obtain a culture flask pre-coated with polylysine. S105. Add 10 mL of phosphate buffer to the poly-L-lysine pre-coated culture flask obtained in S104 for rinsing, then discard the phosphate buffer. Spread the resuspension obtained in S103 evenly in the rinsed poly-L-lysine pre-coated culture flask. Incubate in a 95% humidity, 37℃, 5% CO2 (gas environment: 95% air and 5% CO2) incubator until the cell confluence reaches 90%. Shake at 37℃ and 220 rpm for 24 hours, then discard the supernatant to remove poorly adhered impurity cells. Figure 3 As shown, purified spinal meningeal fibroblasts were obtained. The spinal meningeal fibroblasts were spindle-shaped and fish-like, which is consistent with the morphological characteristics of fibroblasts. S2. Purity identification of spinal meningeal fibroblasts: S201, The purified spinal fibroblasts obtained in S105 were subjected to a process of 2.5 × 10⁻⁶... 4 Cells were evenly seeded at a density of [number] cells / well in a 24-well cell culture plate containing fibroblast culture medium. The cells were cultured at 37°C and 5% CO2 until 90% confluence was achieved. The fibroblast culture medium was then discarded. Figure 4 As shown, cultured spinal meningeal fibroblasts were obtained. With passage, the number of contaminating cells decreased and the purity of fibroblasts gradually increased. After washing the cultured spinal meningeal fibroblasts three times with phosphate buffer, 500 μL of 4% paraformaldehyde fixative was added to each well of the cell culture plate. After fixation for 30 min, the paraformaldehyde fixative was discarded to obtain fixed spinal meningeal fibroblasts. S202. The fixed spinal fibroblasts obtained in S201 are washed with phosphate buffer and shaken at 200 rpm for 5 min. The fixed spinal fibroblasts are then washed with phosphate buffer and shaken for a total of 3 times. The phosphate buffer is then discarded to obtain the fixed and washed spinal fibroblasts. S203. Add 500 μL of 0.2% Triton X-100 to each well of the cell culture plate, permeate for 30 min, then discard the Triton X-100 to obtain permeabilized spinal fibroblasts. S204. Wash the permeabilized spinal membrane obtained in S203 with phosphate buffer and shake at 200 rpm for 5 min. Repeat the phosphate buffer washing and shaking operation on the permeabilized fibroblasts for a total of 3 times. Discard the phosphate buffer to obtain permeabilized and washed spinal membrane fibroblasts. S205. Add 250 μL of 10% goat serum blocking solution to each well of the cell culture plate. After blocking for 1 h, add 250 μL of a 1:200 mixture of fibronectin antibody and platelet-derived growth factor receptor β (PDGFRβ) antibody to the positive control wells. Add 250 μL of a 1:200 mixture of oligodendrocyte transcription factor 2 (OLIG2) antibody and glial fibrillary acidic protein (GFAP) antibody to the negative control wells to identify whether astrocytes and oligodendrocytes are mixed in the sample. Repeat each group 3 times. Incubate the cell culture plate at 4°C for 12 h. Recover the antibody solution from each well to obtain spinal fibroblasts that specifically bind to the primary antibody. S206. The spinal fibroblasts that specifically bind to the primary antibody obtained in S205 are washed with phosphate buffer and shaken at 200 rpm for 5 min. The spinal fibroblasts that specifically bind to the primary antibody are then washed with phosphate buffer and shaken for a total of 3 times. S207. Under light-protected conditions, add 250 μL of a mixture of goat anti-mouse Cy3 red fluorescent secondary antibody and goat anti-rabbit Alexa Fluor 488 green fluorescent secondary antibody at a volume ratio of 1:200 to each well of the cell culture plate, and culture at 37°C for 1 h. Discard the fluorescent secondary antibody solution to obtain spinal fibroblasts that specifically bind to the fluorescent secondary antibody. S208. Under light-protected conditions, the spinal fibroblasts that specifically bind to the fluorescent secondary antibody obtained in S207 were washed with phosphate buffer and shaken at 200 rpm for 5 min. The spinal fibroblasts that specifically bind to the secondary antibody were then washed with phosphate buffer and shaken for a total of 3 times. S209. Under light-protected conditions, add 200 μL of DAPI to each well of the cell culture plate for nuclear staining for 10 min to obtain nuclear-stained spinal meningeal fibroblasts. Wash the nuclear-stained spinal meningeal fibroblasts with phosphate buffer and shake at 200 rpm for 5 min. Repeat the phosphate buffer washing and shaking operation three times to complete the extraction, purification and identification of spinal meningeal fibroblasts.

[0033] Markers on the surface of spinal meningeal fibroblasts were observed under a fluorescence microscope, and the results were as follows: Figure 5 As shown, the spinal meningeal fibroblast surface marker PDGFRβ is mainly present on the fibroblast membrane surface, and staining reveals it as a fibroblast outline; as Figure 6As shown, Fibronectin is mainly found at cell-cell junctions, and therefore in the Merge diagram, it is primarily present at the cell-cell interface (yellow area in the Merge diagram); for example... Figure 7 and Figure 8 As shown, both the astrocyte surface marker GFAP and the oligodendrocyte surface marker OLIG2 were negative; the few positive results were due to nonspecific staining. Fibroblasts extracted using the method provided in this invention, such as... Figure 9 As shown, 99.27% ​​of the cells expressed the fibroblast-specific marker PDGFRβ, while only a small amount of the astrocyte marker GFAP and the oligodendrocyte marker OLIG2 were expressed non-specifically. This demonstrates that this method can obtain spinal meningeal fibroblasts with extremely high purity, which can be used for subsequent studies on the formation of spinal cord scars.

[0034] Example 2 This embodiment describes a method for the extraction, purification, and identification of meningeal fibroblasts. The method is as follows: S1. Extraction and purification of meningeal fibroblasts: S101. After euthanizing 2-day-old pups by cervical dislocation, immerse them in -20°C frozen alcohol for disinfection. On a tray containing ice, cut open the skin on the pup's head, cut along the midcranial suture, and carefully remove the intact brain tissue. Figure 10 As shown, the brain tissue of the pups was obtained. The obtained brain tissue of the pups was immersed in Hank's balanced salt solution at 4°C and placed on a clean bench. Under a stereomicroscope, the meninges (dura mater and arachnoid mater) attached to the surface of the brain tissue were carefully separated using micro forceps and micro scissors. The separated meninges were transferred to a new 35 mm diameter culture dish and the separated meninges were cut into pieces with sterile micro scissors. S102. After mixing the shredded meninges with the tissue digestion solution, digest at 37°C for 30 minutes, mixing by blowing every 10 minutes, and then filtering through a sieve with a pore size of 40μm to obtain a suspension. The volume ratio of the shredded meninges to the tissue digestive fluid is 1:5; The tissue digestion solution is composed of the following raw materials in the following final amounts: 0.25 wt% EDTA-free pancreatic enzyme and 1 mg / mL type IV collagenase; S103. Transfer the suspension obtained in S102 to a new 15mL centrifuge tube, add 10mL of digestion termination solution, centrifuge at 1500rpm for 5min to obtain the precipitate; add 5mL of fibroblast culture medium to resuspend the precipitate to obtain the resuspension. The fibroblast culture medium was prepared by adding the following ingredients to DMEM / F-12 medium in the following final amounts: 10 wt% fetal bovine serum, 10 ng / mL TGF-β, and 1 wt% penicillin-streptomycin solution. S104. Take 2 mL of polylysine solution with a mass concentration of 0.1 mg / mL and place it evenly in a culture flask. Let it stand at 37°C for 2 hours. Remove the excess polylysine solution and dry it at 37°C for 4 hours to obtain a culture flask pre-coated with polylysine (in this embodiment, 2.5 mL of polylysine solution can also be taken and let it stand at 37°C for 2.5 hours). S105. Add 10 mL of phosphate buffer to the culture flask pre-coated with poly-L-lysine obtained in S104 for rinsing, discard the phosphate buffer, and spread the resuspension obtained in S103 evenly in the rinsed culture flask pre-coated with poly-L-lysine. Incubate in a 95% humidity, 37℃ temperature, and 5% CO2 (gas environment is 95% air and 5% CO2) incubator until the cell confluence is 90%. Shake at 37℃ and 220 rpm for 24 hours, then discard the supernatant to remove poorly adhered impurity cells and obtain purified meningeal fibroblasts. S2. Purity identification of meningeal fibroblasts: S201, The purified meningeal fibroblasts obtained in S105 were subjected to a process of 2.5 × 10⁻⁶... 4 Meningeal fibroblasts were seeded at a density of cells / well in a 24-well cell culture plate containing fibroblast culture medium and cultured at 37°C and 5% CO2 until the cell confluence reached 90%. The fibroblast culture medium was then discarded to obtain cultured meningeal fibroblasts. After washing the cultured meningeal fibroblasts three times with phosphate buffer, 500 μL of 4% paraformaldehyde fixative was added to each well of the culture plate and fixed for 30 min. The paraformaldehyde fixative was then discarded to obtain fixed meningeal fibroblasts. S202. The fixed meningeal fibroblasts obtained in S201 are washed with phosphate buffer and shaken at 200 rpm for 5 min. The fixed meningeal fibroblasts are then washed and shaken with phosphate buffer for a total of 3 times. The phosphate buffer is then discarded to obtain the fixed and washed meningeal fibroblasts. S203. Add 500 μL of 0.2% Triton X-100 to each well of the cell culture plate, permeate for 30 min, then discard the Triton X-100 to obtain permeated meningeal fibroblasts. S204. Wash the permeabilized meninges obtained in S203 with phosphate buffer and shake at 200 rpm for 5 min. Repeat the phosphate buffer washing and shaking operation on the permeabilized fibroblasts for a total of 3 times. Discard the phosphate buffer to obtain permeabilized and washed meningeal fibroblasts. S205. Add 250 μL of 10% goat serum blocking solution to each well of the cell culture plate. After blocking for 1 h, add 250 μL of a 1:200 mixture of Fibronectin antibody and PDGFRβ antibody to the positive control well; add 250 μL of a 1:200 mixture of OLIG2 antibody and GFAP antibody (each antibody is diluted 1:200) to the negative control well. This is used to identify whether astrocytes and oligodendrocytes are mixed in the sample. Each group is repeated 3 times. Incubate the cell culture plate at 4°C for 12 h, and recover the antibody solution from each well to obtain meningeal fibroblasts that specifically bind to the primary antibody. S206. Wash the meningeal fibroblasts that specifically bind to the primary antibody obtained in S205 with phosphate buffer and shake at 200 rpm for 5 min. Repeat the phosphate buffer washing and shaking operation for a total of 3 times. S207. Under light-protected conditions, add 250 μL of a mixture of goat anti-mouse Cy3 red fluorescent secondary antibody and goat anti-rabbit Alexa Fluor 488 green fluorescent secondary antibody at a volume ratio of 1:200 to each well of the cell culture plate, and culture at 37°C for 1 h. Discard the fluorescent secondary antibody solution to obtain meningeal fibroblasts that specifically bind to the fluorescent secondary antibody. S208. Under light-protected conditions, the meningeal fibroblasts that specifically bind to the fluorescent secondary antibody obtained in S207 were washed with phosphate buffer and shaken at 200 rpm for 5 min. The meningeal fibroblasts that specifically bind to the secondary antibody were then washed with phosphate buffer and shaken for a total of 3 times. S209. Under light-protected conditions, add 200 μL of DAPI to each well of the cell culture plate for nuclear staining for 10 min to obtain nuclear-stained meningeal fibroblasts. Wash the nuclear-stained meningeal fibroblasts with phosphate buffer and shake at 200 rpm for 5 min. Repeat the phosphate buffer washing and shaking operation three times in total. Observe under a fluorescence microscope to complete the extraction, purification and identification of meningeal fibroblasts.

[0035] The method for extraction, purification, and identification of meningeal fibroblasts in this embodiment is simple to operate and highly efficient. It can extract mouse primary meningeal fibroblasts without expensive instruments and equipment, with a purity of over 99% and a short culture period, providing a foundation for subsequent experimental research. The purity of fibroblasts can be identified by using several commonly used laboratory antibodies for cell immunofluorescence.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for the extraction, purification, and identification of fibroblasts, characterized in that, The method is as follows: S1. Extraction and purification of fibroblasts: S101. Separate the meninges or spinal meninges and cut them into small pieces. Mix the cut tissue with the tissue digestion solution and digest at 37°C for 30 minutes. Mix the tissue by blowing and blowing every 10 minutes. Filter the mixture through a sieve to obtain a suspension. S102. Add digestion termination solution to the suspension obtained in S101, centrifuge to obtain precipitate, add fibroblast culture medium to resuspend the precipitate to obtain resuspended solution. S103. Take 1 mL to 2 mL of polylysine solution and place it evenly in a culture flask. After standing at 37°C for 1 to 2 hours, remove the excess polylysine solution and dry it at 37°C for 4 hours to obtain a culture flask pre-coated with polylysine. S104. Add 10 mL of phosphate buffer to the culture flask pre-coated with poly-L-lysine obtained in S103 for rinsing, discard the phosphate buffer, and spread the resuspension obtained in S102 evenly in the culture flask pre-coated with poly-L-lysine after rinsing. When the cell confluence reaches 90%, shake, discard the supernatant, and obtain purified fibroblasts. S2. Identification of fibroblast purity: S201. Place the purified fibroblasts obtained in S104 into a cell culture plate containing fibroblast culture medium and culture until the cell confluence reaches 90%. Discard the fibroblast culture medium to obtain cultured fibroblasts. Wash the cultured fibroblasts three times with phosphate buffer. Add 500 μL of paraformaldehyde fixative to each well of the cell culture plate and fix for 30 min. Discard the paraformaldehyde fixative to obtain fixed fibroblasts. S202. Wash the fixed fibroblasts obtained in S201 with phosphate buffer and shake. Repeat the washing and shaking operation with phosphate buffer for a total of 3 times. Discard the phosphate buffer to obtain fixed and washed fibroblasts. S203. Add 500 μL of immunostaining permeabilization solution to each well of the cell culture plate, permeate for 30 min, then discard the immunostaining permeabilization solution to obtain permeabilized fibroblasts. S204. Wash the permeabilized fibroblasts obtained in S203 with phosphate buffer and shake. Repeat the phosphate buffer washing and shaking operation three times. Discard the phosphate buffer to obtain permeabilized and washed fibroblasts. S205. Add 250 μL of goat serum blocking solution to each well of the cell culture plate and block for 1 h. Then add 250 μL of primary antibody solution to each well and culture at 4 °C for 12 h. Recover the primary antibody solution to obtain fibroblasts that specifically bind to the primary antibody. S206. Wash the fibroblasts that specifically bind to the primary antibody obtained in S205 with phosphate buffer and shake. Repeat the phosphate buffer washing and shaking operation for a total of 3 times. S207. Under light-protected conditions, add 250 μL of fluorescent secondary antibody solution to each well of the cell culture plate, culture at 37°C for 1 h, discard the fluorescent secondary antibody solution, and obtain fibroblasts that specifically bind to the fluorescent secondary antibody. S208. Under light-protected conditions, the fibroblasts that specifically bind to the fluorescent secondary antibody obtained in S207 are washed with phosphate buffer and shaken. The fibroblasts that specifically bind to the secondary antibody are then washed with phosphate buffer and shaken for a total of 3 times. S209. Under light-protected conditions, add 200 μL of DAPI to each well of the cell culture plate for nuclear staining to obtain nuclear-stained fibroblasts. Wash the nuclear-stained fibroblasts with phosphate buffer, shake, and repeat the phosphate buffer washing and shaking operation three times. Observe under a fluorescence microscope to complete the extraction, purification and identification of fibroblasts.

2. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The volume ratio of the tissue and the tissue digestion solution in S101 is 1:5; the tissue digestion solution is composed of the following raw materials in the following final contents: 0.25wt% EDTA-free pancreatic enzyme and 1mg / mL type IV collagenase; the pore size of the sieve is 40μm.

3. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The digestion termination solution described in S102 is a DMEM / F-12 medium containing 10% fetal bovine serum; the volume ratio of the digestion termination solution to the fibroblast culture medium is 2:1; the centrifugation conditions are: centrifugation at 1500 rpm for 5 min; the fibroblast culture medium described in S102 and S201 is prepared by adding the following raw materials to the DMEM / F-12 medium in the following final amounts: 10 wt% fetal bovine serum, 10 ng / mL TGF-β, and 1 wt% penicillin-streptomycin bispecific antibody solution.

4. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The mass concentration of the poly-L-lysine solution in S103 is 0.1 mg / mL; the culture conditions in S104 are: humidity 95%, temperature 37℃, 5% CO2; the shaking conditions are: shaking at 37℃ and 220 rpm for 24 h.

5. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The density of fibroblasts in the cell culture plate described in S201 is 2.5 × 10⁻⁶. 4 Cells / well; culture conditions: temperature 37℃, 5% CO2; the mass fraction of the paraformaldehyde fixative is 4%.

6. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The shaking conditions in S202, S204, S206, S208 and S209 are: shaking at 200 rpm for 5 min; the immunostaining permeabilization solution in S203 is Triton X-100 with a mass fraction of 0.2%.

7. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The goat serum described in S205 has a mass fraction of 10%.

8. The method for extraction, purification, and identification of fibroblasts according to claim 1, characterized in that, The nuclear staining time described in S209 is 10 minutes.

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