A high-efficiency method for separating primary cells of cochlear tissue based on physical filtration

Abstract

This invention discloses a highly efficient method for separating primary cells from cochlear tissue based on physical filtration. The method includes: S1: tissue pretreatment and viability preservation; S2: controlled mechanical dissociation; S3: automatic cell-impurity separation; b. differential centrifugation using a double-layer filtration centrifuge device; S4: impurity and cell removal and cell purification; S5: primary cell seeding and optimized culture. This separation method utilizes the natural differences in volume and mass between cells and bone fragments through a double-layer filtration centrifuge device and differential centrifugation filtration, simultaneously performing centrifugation sedimentation and filtration separation. Compared to traditional manual filtration or density gradient centrifugation, this device is simple to operate, has a fast separation speed, fundamentally avoids filter membrane clogging, and significantly improves separation efficiency, enabling the gentle and efficient separation of highly active primary cells from cochlear tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for efficient separation of primary cells from cochlear tissue based on physical filtration. This method is based on tissue engineering and cell culture technology and is used to efficiently separate highly active primary cells from the tiny and hard bony organ of the mammalian cochlea. Background Technology

[0002] Primary cochlear cells are a direct and precise research model for exploring the physiological mechanisms of hearing, assessing drug ototoxicity, elucidating the pathogenesis of hereditary deafness, and developing hair cell regeneration therapies. However, the cochlear tissue is naturally embedded deep within the temporal bone, and its unique anatomical characteristics—including its delicate and tiny structure, complex and diverse cell lineages, and being surrounded by dense and hard layers of bone—make the isolation and acquisition of highly active and pure primary functional cells (such as cochlear fibroblasts, cochlear mesenchymal stem cells, and auditory neuron precursor cells) a recognized technical challenge in the field.

[0003] Currently, the mainstream cell separation technologies in this field are mainly divided into two categories, but both have significant limitations: One is the direct enzymatic digestion method: This method relies on biological enzymes such as collagenase to digest cochlear tissue for a long time in order to release cells from the matrix. However, because the activity of the enzymatic digestion process is difficult to control precisely, excessively long digestion time or excessively strong enzyme activity can easily damage the integrity of the cell membrane and intracellular functional pathways, resulting in a significant reduction in the viability of the separated cells; at the same time, enzymatic digestion cannot effectively remove hard impurities such as bone fragments mixed in, which poses a hidden danger for subsequent cell purification and culture.

[0004] The second method is the traditional mechanical grinding method: using tools such as grinding rods and homogenizers to forcefully break down tissues and release cells physically. However, the shear force of this method depends entirely on the operator's experience, making it difficult to achieve standardized control. It is very easy to cause irreversible physical damage to cells and generate a large number of cell fragments. Moreover, during the breaking process, bone fragments are thoroughly mixed with target cells, making it impossible to effectively remove impurities in subsequent separation and purification steps, which seriously interferes with the accuracy and reproducibility of downstream experiments.

[0005] In summary, developing a novel separation technology that is both gentle and efficient, capable of precisely separating cochlear functional cells from bone impurities while preserving the activity and function of the cells to the maximum extent, has become a key technological need that urgently needs to be addressed in the fields of auditory biology and translational medicine. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for efficient separation of primary cells from cochlear tissue based on physical filtration, which solves the problems of the above-mentioned traditional technologies and can gently and efficiently separate highly active primary cells from cochlear tissue.

[0007] This invention is achieved using the following technical solution: A highly efficient method for separating primary cochlear tissue cells based on physical filtration includes the following steps: S1: Tissue pretreatment and viability maintenance; S2: Controllable mechanical dissociation; The pretreated cochlear tissue was transferred to a 3cm culture dish containing 1X PBS buffer, and the smooth curved surface of the centrifuge tube cap was used as a grinding tool for planar controlled grinding. S3: Automatic separation of cells and impurities; a. Transfer all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of a double-layer filtration centrifuge device; wherein, the bottom of the upper perforated centrifuge tube is provided with at least one micropore, which is used to allow single cells and red blood cells to pass through smoothly during centrifugation, while effectively retaining bone fragments and large-volume impurities of insufficiently ground tissue blocks; b. Perform differential centrifugal filtration on a double-layer filtration centrifuge device; S4: Impurity cell removal and cell purification; S5: Primary cell seeding and optimized culture.

[0008] Further, in step S1, the specific steps include: After the cochlear tissue is removed, it is immediately placed in a nutrient-rich culture medium. Non-target tissues such as connective tissue are carefully trimmed and removed. After processing, the processed cochlear tissue is immersed in a sealed container filled with culture medium to maintain the physiological environment of the cells throughout the process and preserve cell activity to the greatest extent.

[0009] Furthermore, the nutrient-rich culture medium contains DMEM high-glucose medium, FBS, and antibiotic solution in a volume ratio of 9:1:0.1.

[0010] Furthermore, in step S2, the centrifuge tube cap is a 15ml centrifuge tube cap.

[0011] Furthermore, in step S3, the size of the micropores is 0.45mm-0.7mm.

[0012] Furthermore, in step S3, the double-layer filtration centrifuge device also includes a lower uncovered collection tube and a sealing connection component for sealing the upper perforated centrifuge tube and the lower uncovered collection tube. The sealing connection component is a sealing film or medical tape.

[0013] Furthermore, in step S3, the high-speed centrifugal force used in differential centrifugal filtration is 4347g.

[0014] Furthermore, in step S4, the specific steps include: a. Collect the cell suspension from the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer for specific lysis, and then add 2-3 times the volume of complete culture medium or 1X PBS buffer to neutralize the lysis buffer; c. Collect the target cells using a low-speed centrifugal force of 300g, and remove the lysate and cell debris from the supernatant.

[0015] Furthermore, the erythrocyte lysis buffer is G2015-500ML.

[0016] Furthermore, in step S5, the specific steps include: a. Resuspend the purified cells in complete culture medium and seed them into 12-well plate culture dishes; b. The first medium change should be performed 4 hours after inoculation, during the critical window period of cell adhesion. Before changing the medium, the culture plate should be gently washed with PBS buffer to remove unattached dead cells and residual impurities, so as to create a clean growth environment for primary cells and promote cell adhesion and proliferation.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The separation method of the present invention utilizes a double-layer filtration centrifugation device and differential centrifugation filtration principle. It takes advantage of the natural differences in volume and mass between cells and bone fragments to simultaneously carry out centrifugation sedimentation and filtration separation processes. Compared with traditional manual filtration or density gradient centrifugation methods, this device is easy to operate and has a fast separation speed. It fundamentally avoids the problem of filter membrane clogging and greatly improves separation efficiency. It can gently and efficiently separate highly active primary cells from cochlear tissue.

[0018] 2. The separation method of the present invention achieves a revolutionary improvement in cell viability and yield, and its features include the following: (1) In situ moisturizing strategy: The entire process from tissue isolation is carried out in the culture medium to provide continuous nutrition and environmental support for fragile primary cells and reduce cell stress damage; (2) Controllable planar grinding technology: using grinding tools with specific geometric shapes to precisely control mechanical force, significantly reducing physical damage to cells while breaking up bone structures; (3) Integrated rapid process: The entire process from tissue dissociation to cell seeding is compactly designed, which greatly shortens the exposure time of cells in adverse in vitro environments and maintains cell physiological activity; (4) Combined purification strategy: Through the dual purification steps of "physical filtration to remove bone fragments + chemical lysis to remove red blood cells", the two major sources of impurities are effectively removed, and a high-purity target cell population is obtained; (5) Standardization and repeatability: The method steps are clear, key parameters such as centrifugal force and time have been quantified, the device is produced in a standardized manner, which greatly reduces the impact of individual differences of operators on experimental results, ensures experimental stability and repeatability, and facilitates its application in scientific research and industry. Attached Figure Description

[0019] Figure 1 This is a diagram showing the separation process and results of the tissue suspension from cells to impurities after automatic cell-impurity separation in Example 1. Figure 1 A involves drilling a hole in a 1.5ml EP tube using a 1ml needle. Figure 1 In step B, the perforated EP tube is placed on top, and the normal, non-perforated EP tube is placed below. Both are then wrapped with transparent tape or sealing film. Figure 1 C represents the result after centrifugation: the upper EP tube contains bone tissue, and the lower EP tube contains cells. Figure 1 D represents the bone remaining in the upper EP canal, which is the bone mass of the two cochleas after centrifugation; Figure 2 These are microscopic comparison images of cells after isolation (before purification) in Example 1 and Comparative Examples 1-2. Figure 2 A is a cell micrograph from Example 1. Figure 2 B is a microscopic image of the cell from Comparative Example 1. Figure 2 C is a microscopic image of the cell in Comparative Example 2; Figure 3 The images and flow cytometry plots of live and dead cells from Example 1 and Comparative Example 1 are shown. Figure 3 A shows a photograph and flow cytometry plot of live and dead cells from Example 1. Figure 3 B shows a photograph and flow cytometry plot of live and dead cells from Comparative Example 1. Figure 4 This is a fluorescence result image of live and dead cell staining in Example 1. Detailed Implementation

[0020] To address the problem that existing technologies cannot gently and efficiently separate highly active, high-purity primary cells, this invention provides a method for highly efficient separation of primary cochlear tissue cells based on physical filtration, comprising the following steps: S1 tissue pretreatment and viability maintenance: After the cochlear tissue is removed, it is immediately placed in a nutrient-rich culture medium. Non-target tissues such as connective tissue are carefully trimmed and removed. After processing, the processed cochlear tissue is immersed in a sealed container filled with culture medium to maintain the physiological environment of the cells throughout the process and preserve cell activity to the greatest extent.

[0021] In this embodiment, the nutrient-rich culture medium contains DMEM high-glucose medium, FBS, and a dual-antibiotic solution in a volume ratio of 9:1:0.1. The DMEM high-glucose medium is a serum- and antibiotic-free base solution used to support high-density, rapid cell proliferation. The FBS is fetal bovine serum used to supplement growth factors, hormones, adhesion factors, and lipids to promote cell adhesion, proliferation, and survival. The dual-antibiotic solution is a penicillin-streptomycin mixture used to inhibit bacterial contamination and ensure sterility during the culture process.

[0022] S2 Controllable Mechanical Dissociation: The pretreated cochlear tissue was transferred to a 3cm culture dish containing 1X PBS buffer. The smooth curved surface of the cap of a 15ml centrifuge tube was used as a grinding tool for planar controlled grinding. This step, by controlling the grinding force and direction, releases cells by physically breaking down the bony structure while avoiding excessive shearing damage to the cells by utilizing the smoothness of the tool, thus achieving precise dissociation that "breaks bone without damaging cells".

[0023] S3 cells - automatic impurity separation: a. Transfer all the tissue suspension obtained from grinding to the perforated centrifuge tube in the upper layer of a double-layer filtration centrifuge device; In this step, the double-layer filtration centrifuge device includes an upper perforated centrifuge tube, a lower open-top collection tube, and a sealing connection component for sealing the upper perforated centrifuge tube and the lower open-top collection tube. The bottom of the upper perforated centrifuge tube has at least one micropore, which allows single cells and red blood cells to pass through smoothly during centrifugation, while effectively trapping large impurities such as bone fragments and insufficiently ground tissue blocks. This double-layer filtration centrifuge device achieves highly efficient separation of cells and impurities through its structural design. The micropore size is 0.45mm-0.7mm, such as 0.45mm, 0.5mm, and 0.7mm.

[0024] In this embodiment, the upper perforated centrifuge tube uses a 1.5ml microcentrifuge tube (EP tube) as the base carrier, with at least one micropore formed at the bottom by puncturing the tube with a 1ml medical syringe needle. The pore size of this micropore is precisely controlled, allowing single cells and red blood cells to pass through during centrifugation while effectively trapping bone fragments and insufficiently ground tissue blocks. The lower open collection tube serves as the collection unit for target cells, receiving the cell suspension that has passed through the upper filter pores to ensure complete cell recovery. The sealing connection uses sealing film or medical tape to secure the interface between the upper and lower tubes airtightly, preventing liquid leakage during centrifugation and ensuring the stability of the separation process.

[0025] b. Differential centrifugal filtration is performed on the dual-layer filtration centrifuge. In this embodiment, the high-speed centrifugal force used in the differential centrifugal filtration is 4347g (approximately 1970 rpm).

[0026] This step utilizes a high-speed centrifugal force of 4347g (approximately 1970 rpm) to allow small particles such as single cells and red blood cells to pass through the micropores of the upper perforated centrifuge tube into the lower open collection tube. Larger impurities such as bone fragments and insufficiently ground tissue blocks are retained in the upper tube, thus achieving efficient physical separation of target cells from major solid impurities in one step.

[0027] S4 Impurity Cell Removal and Cell Purification: a. Collect the cell suspension in the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer (G2015-500 ml) for specific lysis, then add 2-3 times the volume of complete culture medium or 1X PBS buffer to neutralize the lysis buffer; c. Collect the target cells by centrifugation at 300 g, remove the lysis buffer and cell debris from the supernatant to further improve cell purity.

[0028] Among them, red blood cell lysis buffer (G2015-500ML) is a laboratory reagent for selectively lysing red blood cells. It is produced by the brand Servicebio, with the product number G2015-500ML and a specification of 500 ml. Its core component is ammonium chloride (NH4Cl), combined with potassium bicarbonate and EDTA-Na2. By creating an osmotic pressure difference between the inside and outside of the red blood cells, water enters the cells, causing them to swell and rupture, thereby achieving lysis. Since nucleated cells are more tolerant to changes in osmotic pressure, they are not significantly damaged. Complete culture medium refers to a mature culture system that can be directly used for cell culture after adding serum, antibiotics, growth factors and other components to the basic culture medium. It can support cell growth and proliferation. It contains the following key components: (1) Basic culture medium: such as DMEM, RPMI-1640, which provides basic nutrition (amino acids, vitamins, inorganic salts, glucose, etc.). (2) Serum: the most commonly used is fetal bovine serum (FBS), which generally accounts for 5%-10% and provides key substances such as growth factors, hormones and adhesion factors. (3) Antibiotics: such as penicillin-streptomycin bispecific antibiotics, used to prevent bacterial contamination. (4) Glutamine: provides energy and nitrogen to cells, and is often added fresh before use because it is unstable in solution. In one example, the complete culture medium contains DMEM high-glucose medium, FBS and bispecific antibiotic solution in a volume ratio of 9:1:0.1. PBS buffer is a phosphate buffer solution, which is an isotonic buffer widely used in biochemical and cell biology experiments to maintain the pH stability of the solution and provide an osmotic pressure similar to that of human body fluids. Its core components include: Na2HPO4 (disodium hydrogen phosphate), KH2PO4 (potassium dihydrogen phosphate), NaCl (sodium chloride), KCl (potassium chloride), etc. S5 primary cell seeding and optimized culture: a. Resuspend the purified cells in complete culture medium and seed them into 12-well plates or other culture vessels; b. Perform the first medium change 4 hours after seeding, during the critical cell adhesion window. Before changing the medium, gently wash the culture plate with PBS buffer to remove unattached dead cells and residual impurities, creating a clean growth environment for primary cells and promoting cell adhesion and proliferation. The complete culture medium contains DMEM high-glucose medium (9:1:0.1 v / v), FBS, and antibiotic / antibiotic solution.

[0029] The core innovation of this invention lies in the design of a "differential centrifugation filtration device," which utilizes the natural differences in volume and mass between cells and bone fragments to simultaneously perform centrifugation sedimentation and filtration separation. Compared to traditional manual filtration or density gradient centrifugation, this device is simple to operate, has a fast separation speed, fundamentally avoids the problem of filter membrane clogging, and significantly improves separation efficiency. It also revolutionarily enhances cell activity and yield, with the following features: (1) In situ moisturizing strategy: The entire process from tissue isolation is carried out in the culture medium to provide continuous nutrition and environmental support for fragile primary cells and reduce cell stress damage; (2) Controllable planar grinding technology: using grinding tools with specific geometric shapes to precisely control mechanical force, significantly reducing physical damage to cells while breaking up bone structures; (3) Integrated rapid process: The entire process from tissue dissociation to cell seeding is compactly designed, which greatly shortens the exposure time of cells in adverse in vitro environments and maintains cell physiological activity; (4) Combined purification strategy: Through the dual purification steps of "physical filtration to remove bone fragments + chemical lysis to remove red blood cells", the two major sources of impurities are effectively removed, and a high-purity target cell population is obtained; (5) Standardization and repeatability: The method steps are clear, key parameters such as centrifugal force and time have been quantified, the device is produced in a standardized manner, which greatly reduces the impact of individual differences of operators on experimental results, ensures experimental stability and repeatability, and facilitates its application in scientific research and industry.

[0030] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0031] Example 1, S1 tissue pretreatment and viability maintenance: The cochlea is removed, and the surrounding tissues, including the auditory bullae and muscles, are carefully trimmed. The cochlea was immersed in a 1.5 ml EP tube filled with culture medium (containing DMEM high glucose medium:FBS:double antibody = 9:1:0.1, 50 ml) for 30 min to obtain pretreated cochlear tissue.

[0032] S2 Controllable Mechanical Dissociation: The pretreated cochlear tissue was transferred to a 3cm culture dish containing 0.1ml of 1X PBS buffer and planar controlled grinding was performed using the smooth curved surface of a 15ml centrifuge tube cap as a grinding tool.

[0033] S3 cells - automatic impurity separation: a. Transfer all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of a double-layer filtration centrifuge apparatus; b. Perform differential centrifugation filtration on the double-layer filtration centrifuge apparatus, centrifuge for 1 min, maximum speed, 4347 g, 4℃.

[0034] Construction of a double-layer filtration centrifuge apparatus: Prepare a perforated EP tube. Using a 1ml needle, invert the needle with the tip facing upwards. Then, invert the EP tube and poke a small hole (i.e., a micropore, with a size of 0.45mm) at the bottom. Next, prepare an EP tube without a hole (i.e., an EP tube without a hole, with the cap cut off). Place the perforated EP tube on top and the EP tube without a hole on the bottom, then seal it tightly with sealing film or bandage.

[0035] After the above steps, the separation process and results are as follows: Figure 1 The diagram shown illustrates the separation process and results of the tissue suspension after automatic cell-impurity separation. Figure 1 A involves drilling a hole in a 1.5ml EP tube using a 1ml needle. Figure 1 In step B, place the EP tube with the hole on top, and the normal EP tube without the hole (with the EP tube cap cut off) below. Wrap both with transparent tape or sealing film. Figure 1 C represents the result after centrifugation: the upper EP tube contains bone tissue, and the lower EP tube contains cells. Figure 1 D represents the bone remaining in the upper EP canal, which is the bone mass after centrifugation of both cochleas.

[0036] S4 Impurity Cell Removal and Cell Purification: a. Aspirate the cell suspension from the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer (G2015-500 ml) for specific lysis, mix well for 3-5 min, then add 2 times the volume of complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) to neutralize the lysis buffer; c. Collect the target cells by low-speed centrifugation at 300 g for 5 min, remove the lysis buffer and cell debris from the supernatant to further improve cell purity. Neutrophils are 430 g, and primary liver cells are 50 g.

[0037] S5 primary cell seeding and optimized culture: a. Remove the supernatant and resuspend the purified cells in complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) and seed them into 12-well plates or other culture vessels; b. Perform the first medium change 4 hours after seeding, during the critical window of cell adhesion. Before changing the medium, gently wash the culture plate twice with PBS buffer to remove unattached dead cells and residual impurities, creating a clean growth environment for primary cells and promoting cell adhesion and proliferation, before placing them back into the culture medium.

[0038] Example 2, S1 tissue pretreatment and viability maintenance: The cochlea is removed, and the surrounding tissues, including the auditory bullae and muscles, are carefully trimmed. The cochlea was immersed in a 1.5 ml EP tube filled with culture medium (containing DMEM high glucose medium:FBS:double antibody = 9:1:0.1, 50 ml) for 30 min to obtain pretreated cochlear tissue.

[0039] S2 Controllable Mechanical Dissociation The pretreated cochlear tissue was transferred to a 3cm culture dish containing 0.1ml of 1X PBS buffer and planar controlled grinding was performed using the smooth curved surface of a 15ml centrifuge tube cap as a grinding tool.

[0040] S3 cells - automatic impurity separation a. Transfer all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of a double-layer filtration centrifuge apparatus; b. Perform differential centrifugation filtration on the double-layer filtration centrifuge apparatus, centrifuge for 1 min, maximum speed, 4347 g, 4℃.

[0041] To prepare a double-layer filtration centrifuge apparatus: Prepare a perforated EP tube. Using a 2ml needle, invert the needle with the tip facing upwards. Then, invert the EP tube and poke a small hole (i.e., a micropore, 0.5mm in size) at the bottom. Next, prepare an unperforated EP tube (i.e., an EP tube without a hole, with the cap cut off). Place the perforated EP tube on top and the unperforated EP tube on the bottom, then seal it tightly with sealing film or bandage.

[0042] S4 Impurity Cell Removal and Cell Purification: a. Aspirate the cell suspension from the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer (G2015-500 ml) for specific lysis, mix well for 3-5 min, then add 2 times the volume of complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) to neutralize the lysis buffer; c. Collect the target cells by low-speed centrifugation at 300 g for 5 min, remove the lysis buffer and cell debris from the supernatant to further improve cell purity. Neutrophils are 430 g, and primary liver cells are 50 g.

[0043] S5 primary cell seeding and optimized culture: a. Remove the supernatant and resuspend the purified cells in complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) and seed them into 12-well plates or other culture vessels; b. Perform the first medium change 4 hours after seeding, during the critical window of cell adhesion. Before changing the medium, gently wash the culture plate twice with PBS buffer to remove unattached dead cells and residual impurities, creating a clean growth environment for primary cells and promoting cell adhesion and proliferation, before placing them back into the culture medium.

[0044] Example 3, S1 tissue pretreatment and viability maintenance: The cochlea is removed, and the surrounding tissues, including the auditory bullae and muscles, are carefully trimmed. The cochlea was immersed in a 1.5 ml EP tube filled with culture medium (containing DMEM high glucose medium:FBS:double antibody = 9:1:0.1, 50 ml) for 30 min to obtain pretreated cochlear tissue.

[0045] S2 Controllable Mechanical Dissociation: The pretreated cochlear tissue was transferred to a 3cm culture dish containing 0.1ml of 1X PBS buffer and planar controlled grinding was performed using the smooth curved surface of a 15ml centrifuge tube cap as a grinding tool.

[0046] S3 cells - automatic impurity separation: a. Transfer all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of a double-layer filtration centrifuge apparatus; b. Perform differential centrifugation filtration on the double-layer filtration centrifuge apparatus, centrifuge for 1 min, maximum speed, 4347 g, 4℃.

[0047] To prepare a double-layer filtration centrifuge apparatus: Prepare a perforated EP tube. Using a 10ml needle, invert the needle with the tip facing upwards. Then, invert the EP tube and poke a small hole (i.e., a micropore, 0.7mm in size) at the bottom. Next, prepare an EP tube without a hole (i.e., an EP tube without a hole, with the cap cut off). Place the perforated EP tube on top and the EP tube without a hole on the bottom, then seal it tightly with sealing film or bandage.

[0048] S4 Impurity Cell Removal and Cell Purification: a. Aspirate the cell suspension from the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer (G2015-500 ml) for specific lysis, mix well for 3-5 min, then add 2 times the volume of complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) to neutralize the lysis buffer; c. Collect the target cells by low-speed centrifugation at 300 g for 5 min, remove the lysis buffer and cell debris from the supernatant to further improve cell purity. Neutrophils are 430 g, and primary liver cells are 50 g.

[0049] S5 primary cell seeding and optimized culture: a. Remove the supernatant and resuspend the purified cells in complete culture medium (containing DMEM high glucose medium:FBS:antibody = 9:1:0.1, 50 ml) and seed them into 12-well plates or other culture vessels; b. Perform the first medium change 4 hours after seeding, during the critical window of cell adhesion. Before changing the medium, gently wash the culture plate twice with PBS buffer to remove unattached dead cells and residual impurities, creating a clean growth environment for primary cells and promoting cell adhesion and proliferation, before placing them back into the culture medium.

[0050] Comparative Example 1 differs from Example 1 in that it does not go through step S2, but uses a traditional collagenase digestion group. The other steps are the same as in Example 1, and will not be repeated here.

[0051] In Comparative Example 2, unlike Example 1, the step S3 of "transferring all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of the double-layer filtration centrifuge device" is omitted. Instead, all the tissue suspension obtained from grinding is transferred to the centrifuge tube (excluding the upper perforated centrifuge tube). The other steps are the same as in Example 1 and will not be repeated here.

[0052] Performance testing: 1. Perform microscopic examinations on the isolated (but unpurified) cells: like Figure 2 The images shown are microscopic comparisons of cells after isolation (before purification) in Example 1 and Comparative Examples 1-2. Figure 2 A is a microscopic image of the cells from Example 1. The image shows a clean field of view, mainly consisting of transparent, living cells without bone fragments. The cells are spindle-shaped and adherent to the cell surface. Figure 2 B is a microscopic image of the cells from Comparative Example 1, obtained using conventional collagenase digestion. The image shows that, due to the presence of bone, although the background is exceptionally clean, the cell count is very low, with few adherent cells and a shrunken morphology. Figure 2 C is a microscopic image of the cells in Comparative Example 2. The image shows a large number of highly refractive bone fragments in the field of view. The cells are covered or compressed by the fragments, the background is also very dirty, there are many cell fragments, occasional cell clumps, almost no normal adherent cells, and a large number of dead cells (round bright bodies) floating in the field of view.

[0053] 2. Cell viability staining and flow cytometry results: like Figure 3 The images and flow cytometry plots of live and dead cells from Example 1 and Comparative Example 1 are shown, in which... Figure 3 Image A shows a photograph and flow cytometry plot of live and dead cells from Example 1. The figure shows that 94% of the primary isolated cells are live. Figure 3 B shows the staining images and flow cytometry plots of live and dead cells in Comparative Example 1. The figures show that the traditional collagenase digestion method resulted in a small cell count, with live cells accounting for approximately 75%. Additionally, Comparative Example 2 did not undergo separation in step S3 to avoid damaging the flow cytometer and causing blockages; therefore, flow cytometry analysis was not performed in this case.

[0054] 3. Fluorescence assay for staining live and dead cells: like Figure 4 The fluorescence results of staining live and dead cells in Example 1 are shown in the figure, which shows that the proportion of live cells is nearly 100%.

[0055] The performance test results of Examples 2-3 are the same as those of Example 1, and will not be repeated here.

[0056] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for efficient separation of primary cochlear tissue cells based on physical filtration, characterized in that, Includes the following steps: S1: Tissue pretreatment and viability maintenance; S2: Controllable mechanical dissociation; The pretreated cochlear tissue was transferred to a 3cm culture dish containing 1X PBS buffer, and the smooth curved surface of the centrifuge tube cap was used as a grinding tool for planar controlled grinding. S3: Automatic separation of cells and impurities; a. Transfer all the tissue suspension obtained from grinding to the upper perforated centrifuge tube of a double-layer filtration centrifuge device; wherein, the bottom of the upper perforated centrifuge tube is provided with at least one micropore, which is used to allow single cells and red blood cells to pass through smoothly during centrifugation, while effectively retaining bone fragments and large-volume impurities of insufficiently ground tissue blocks; b. Perform differential centrifugal filtration on a double-layer filtration centrifuge device; S4: Impurity cell removal and cell purification; S5: Primary cell seeding and optimized culture.

2. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 1, characterized in that, In step S1, the specific steps include: After the cochlear tissue is removed, it is immediately placed in a nutrient-rich culture medium. Non-target tissues such as connective tissue are carefully trimmed and removed. After processing, the processed cochlear tissue is immersed in a sealed container filled with culture medium to maintain the physiological environment of the cells throughout the process and preserve cell activity to the greatest extent.

3. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 2, characterized in that, The nutrient-rich culture medium contains DMEM high-glucose medium, FBS, and antibiotic solution in a volume ratio of 9:1:0.

1.

4. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 1, characterized in that, In step S2, the centrifuge tube cap is a 15ml centrifuge tube cap.

5. The method for efficient separation of primary cochlear tissue cells based on physical filtration according to claim 1, characterized in that, In step S3, the size of the micropores is 0.45mm-0.7mm.

6. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 1, characterized in that, In step S3, the double-layer filtration centrifuge device further includes a lower uncovered collection tube and a sealing connection component for sealing the upper perforated centrifuge tube and the lower uncovered collection tube. The sealing connection component is a sealing film or medical tape.

7. The method for efficient separation of primary cochlear tissue cells based on physical filtration according to claim 1, characterized in that, In step S3, the high-speed centrifugal force used in differential centrifugal filtration is 4347g.

8. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 1, characterized in that, In step S4, the specific steps include: a. Collect the cell suspension from the lower collection tube; b. If the red blood cell content in the suspension is high, add 1 ml of red blood cell lysis buffer for specific lysis, and then add 2-3 times the volume of complete culture medium or 1X PBS buffer to neutralize the lysis buffer; c. Collect the target cells using a low-speed centrifugal force of 300g and remove the lysate and cell debris from the supernatant.

9. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 8, characterized in that, The erythrocyte lysis buffer was G2015-500ML.

10. The method for efficient separation of primary cochlear cells based on physical filtration according to claim 1, characterized in that, In step S5, the specific steps include: a. Resuspend the purified cells in complete culture medium and seed them into 12-well plates; b. The first medium change should be performed 4 hours after inoculation during the critical window of cell adhesion. Before changing the medium, the culture plate should be gently washed with PBS buffer to remove dead cells and residual impurities that have not adhered to the plate, so as to create a clean growth environment for primary cells and promote cell adhesion and proliferation.

Images