A method and application of co-culturing dorsal root ganglion nerve cells and chondrocytes

Abstract

The present invention provides a method and application of co-culturing dorsal root ganglion nerve cells and chondrocytes, the co-culture method comprising: constructing dorsal root ganglion nerve cell balls without scaffold materials and chondrocyte balls without scaffold materials, respectively, and then 3D co-culturing the obtained nerve cell balls and chondrocyte balls; wherein the culture medium used for constructing the dorsal root ganglion nerve cell balls and 3D co-culture is a complete nerve cell culture medium, and the culture medium used for constructing the chondrocyte balls is a complete chondrocyte culture medium. The present invention also provides a 3D co-culture system of dorsal root ganglion nerve cell balls and chondrocyte balls and a method for detecting neural infiltration thereof. The co-culture method is simple to operate, and the cell physiological state is good. It can be used as a model in the study of the pathogenic mechanism of related diseases, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of 3D cell co-culture, and in particular relates to a method and application of co-culturing dorsal root ganglion nerve cells and chondrocytes. Background Art

[0002] The dorsal root ganglion is an expanded nodule of the dorsal root of the spinal cord located near the medial side of the intervertebral foramen. It is responsible for receiving all nerve impulses from the body's receptors, including general somatic sensations and visceral sensations, and transmitting them to the spinal cord and even the sensory center of the brain through afferent nerves.

[0003] Articular cartilage is composed of chondrocytes and cartilage matrix. The cartilage matrix is ​​a mesh structure composed of collagen fibers and proteoglycans, and chondrocytes fill the mesh structure. Articular cartilage has neither nerves nor blood vessels, and nutrition is mainly supplied by synovial fluid and arterial branches around the synovial layer of the joint capsule. Chondrocytes are scattered among these mesh structures to maintain the normal metabolism of articular cartilage. When arthritis occurs, articular cartilage is diseased, cartilage polysaccharides in the cartilage matrix are lost, the cartilage matrix becomes thinner, synovial cell hyperplasia and lymphocyte infiltration occur, accompanied by nerve infiltration of articular cartilage, making it impossible to repair articular cartilage, and eventually causing osteoarthritis.

[0004] The central and peripheral nervous systems play a significant regulatory role in bone development and bone homeostasis, such as the Semaphorins, Netrins, Slits and Ephrins families. The effects of neurotrophic factors and axon growth inducing factors on human osteoblasts and osteoclasts have been confirmed. These axon guidance factors can induce and / or inhibit the growth of nerve fibers, thereby affecting the process of nerve endings infiltrating articular cartilage.

[0005] In recent years, the regulation of bone and cartilage by the nervous system has become a hot topic in biomedicine. Research results and a series of clinical findings suggest that the nervous system plays an extremely important role in the occurrence, development and treatment of osteoarthritis. The latest research work reported that osteoclasts in subchondral bone induced nerve infiltration of cartilage and arthritis pain, and innovatively proposed the relationship between osteoclasts inducing nerve infiltration of subchondral bone and osteoarthritis.

[0006] Currently, there is no similar scheme for co-culturing dorsal root ganglion neurons and chondrocytes in vitro. Therefore, how to provide a method for co-culturing dorsal root ganglion neurons and chondrocytes to provide a model for the study of related mechanisms such as in vitro neural infiltration of articular cartilage has become an urgent problem to be solved. Summary of the invention

[0007] In view of the deficiencies of the prior art and actual needs, the present invention provides a method and application of co-culturing dorsal root ganglion neurons and chondrocytes. By co-culturing dorsal root ganglion neurons and chondrocytes in vitro, a research model of nerve endings infiltrating articular cartilage is established, creating conditions for the study of the pathogenic mechanism of arthritis and targeted drug screening.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for co-culturing dorsal root ganglion neurons and chondrocytes, the co-culturing method comprising:

[0010] Constructing dorsal root ganglion neurospheres without scaffold materials and chondrocyte spheres without scaffold materials respectively, and then 3D co-culturing the obtained neurospheres and chondrocyte spheres;

[0011] The culture medium used for constructing the dorsal root ganglion neurosphere and 3D co-culture is a complete neuronal culture medium, and the culture medium used for constructing the chondrocyte sphere is a complete chondrocyte culture medium.

[0012] In the present invention, by respectively constructing dorsal root ganglion neuron spheres that do not require scaffold materials and chondrocyte spheres that do not require scaffold materials, the neurons and chondrocytes can be grown without being disturbed by the external environment, the physiological state is closer to the natural state, and the influence of the material on the cytotoxicity is reduced, and the research results are more accurate; by optimizing the culture medium and the culture process used for culture, the growth state of the cell spheres is better, the probability of successful cell co-culture is higher, and the effect of neuron infiltration into articular cartilage is better.

[0013] Preferably, the method for constructing dorsal root ganglion neurospheres without scaffold material comprises:

[0014] The dorsal root ganglion is separated, the surface connective tissue and the fibers at both ends are peeled off, the nerve cell bodies are retained, and the obtained ganglion tissue is placed in a culture plate for culture to obtain the dorsal root ganglion nerve cell sphere.

[0015] Preferably, after the dorsal root ganglia are isolated, they are placed in a neural cell basal culture medium.

[0016] Preferably, the temperature of the neural cell basal culture medium is 2-5°C, and the temperature may be, for example, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C or 5°C, and other specific point values ​​within this numerical range may be selected, which will not be described in detail here.

[0017] Preferably, the stripping of the connective tissue on the surface and the fibers at both ends is performed on ice.

[0018] Preferably, the time for stripping the connective tissue on the surface and the fibers at both ends does not exceed 1 hour.

[0019] Preferably, the culture plate comprises a low adhesion culture plate.

[0020] Preferably, the culturing step comprises:

[0021] Add complete neural cell culture medium to the culture plate, change the medium after culturing for 100 to 140 minutes, change half of the medium every 20 to 28 hours, and culture for 6 to 8 days. The culture time can be, for example, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes or 140 minutes, etc., the interval between medium changes can be, for example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours or 28 hours, etc., the culture time can be, for example, 6 days, 6.5 days, 7 days, 7.5 days or 8 days, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0022] Preferably, the complete neural cell culture medium comprises: neural cell basal medium, B-27, glutamine and antibiotics.

[0023] Preferably, the mass fraction of B-27 in the complete neural cell culture medium is 1% to 3%, for example, 1%, 1.5%, 2%, 2.5% or 3%, etc. Other specific points within this numerical range can be selected and will not be described here one by one. Preferably, it is 2%.

[0024] Preferably, the mass fraction of glutamine in the complete neural cell culture medium is 0.5% to 2%, for example, 0.5%, 1%, 1.5% or 2%, etc. Other specific values ​​within this numerical range can be selected and will not be described here one by one. Preferably, it is 1%.

[0025] Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete culture medium for neural cells is 0.5% to 2.5%, for example, 0.5%, 1%, 1.5%, 2% or 2.5%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here, preferably 1%.

[0026] Preferably, the complete neural cell culture medium comprises, by mass fraction, 1% to 3% B-27, 0.5% to 2% glutamine and 0.5% to 2.5% antibiotics, with the remainder being neural cell basal culture medium.

[0027] In the present invention, by optimizing the formula of the complete culture medium for nerve cells, the growth rate of nerve cells is faster, the growth state is better, and the infiltration ability to articular cartilage is stronger.

[0028] Preferably, the method for constructing chondrocyte spheres without scaffold materials comprises:

[0029] The articular cartilage is separated, digested by enzymatic hydrolysis, sieved, placed in a culture bottle for culture, and then the cell suspension is placed in a culture plate for further culture to obtain the chondrocyte sphere.

[0030] Preferably, the enzymatic digestion further includes a step of cutting the articular cartilage into pieces.

[0031] Preferably, the enzymatic digestion step comprises:

[0032] 0.2% to 0.3% trypsin digestion is 55 to 65 minutes, 0.15% to 0.25% type II collagenase digestion is 8 to 12 hours, the concentration of trypsin can be, for example, 0.2%, 0.25% or 0.3%, the digestion time of trypsin can be, for example, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes or 65 minutes, the concentration of type II collagenase can be, for example, 0.15%, 0.2% or 0.25%, the digestion time of type II collagenase can be, for example, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours, and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0033] Preferably, the pore size of the cell sieve used for screening is 80-120 μm, for example, it can be 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm or 120 μm, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0034] Preferably, the culturing step comprises:

[0035] Adjust the cell density to (1-2)×10 4 / mL, cultured in complete chondrocyte culture medium, after 45-50h, the medium was changed every 20-25h, and the cell density can be, for example, 1×10 4 / mL, 1.1×10 4 / mL, 1.2×10 4 / mL, 1.3×10 4 / mL, 1.4×10 4 / mL, 1.5×10 4 / mL, 1.6×10 4 / mL, 1.7×10 4 / mL, 1.8×10 4 / mL, 1.9×10 4 / mL or 2×10 4 / mL, the culture time can be, for example, 45h, 45.5h, 46h, 46.5h, 47h, 47.5h, 48h, 48.5h, 49h, 49.5h or 50h, and the liquid replacement interval can be, for example, 20h, 20.5h, 21h, 21.5h, 22h, 22.5h, 23h, 23.5h, 24h, 24.5h or 25h, and other specific point values ​​within this numerical range can be selected, which will not be described one by one here.

[0036] Preferably, the step of continuing to culture comprises:

[0037] When the cell density is not less than 90%, digest with 0.2% to 0.3% trypsin for 2 to 5 min, collect the cells by centrifugation, and adjust the cell density to (1 to 3) × 10 4 / mL, the cell suspension is placed in a low-adhesion culture plate, the medium is changed every 20 to 28 hours, and the culture is carried out for 10 to 12 days. The concentration of trypsin can be, for example, 0.2%, 0.25% or 0.3%, and the digestion time of trypsin can be, for example, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min or 5 min. The cell density can be, for example, 1×10 4 / mL, 1.2×10 4 / mL, 1.4×10 4 / mL, 1.6×10 4 / mL, 1.8×10 4 / mL, 2×10 4 / mL, 2.2×10 4 / mL, 2.4×10 4 / mL, 2.6×10 4 / mL, 2.8×10 4 / mL or 3×10 4 / mL, the liquid replacement interval can be, for example, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h or 28h, and the culture time can be, for example, 10d, 10.5d, 11d, 11.5d or 12d, and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0038] In the present invention, by optimizing the culture method of chondrocyte spheres, the cells grow faster, have a better physiological state, are more easily infiltrated by nerve endings, and have a higher probability of successful co-culture.

[0039] Preferably, the complete chondrocyte culture medium comprises: DMEM / F12 culture medium, fetal bovine serum and antibiotics.

[0040] Preferably, the mass fraction of the fetal bovine serum in the complete chondrocyte culture medium is 8% to 12%, for example, it can be 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5% or 12%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here. It is preferably 10%.

[0041] Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete chondrocyte culture medium is 0.5% to 2.5%, for example, it can be 0.5%, 1%, 1.5%, 2% or 2.5%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here, preferably 1%.

[0042] Preferably, the complete chondrocyte culture medium comprises, by mass fraction, 8% to 12% fetal bovine serum and 0.5% to 2.5% antibiotics, with the remainder being DMEM / F12 culture medium.

[0043] Preferably, the 3D co-cultivation step comprises:

[0044] The neuron spheres and chondrocyte spheres are placed in the same culture plate and cultured with complete neuron culture medium. The medium is replaced by half every 20 to 28 hours, and a structurally stable 3D co-culture system is formed after 5 to 7 days. The interval between medium replacement can be, for example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours or 28 hours, and the culture time can be, for example, 5 days, 5.5 days, 6 days, 6.5 days or 7 days. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0045] Preferably, the culture time of the neurosphere is 6 to 8 days, for example, 6 days, 6.5 days, 7 days, 7.5 days or 8 days, and the culture time of the chondrocyte sphere is 10 to 12 days, for example, 10 days, 10.5 days, 11 days, 11.5 days or 12 days, and other specific point values ​​within this numerical range can be selected, which will not be described one by one here.

[0046] As a preferred technical solution, the method for co-culturing dorsal root ganglion neurons and chondrocytes of the present invention comprises the following steps:

[0047] (1) Construction of dorsal root ganglion neurospheres without scaffold materials:

[0048] The dorsal root ganglion is separated and placed in a neural cell basal culture medium at a temperature of 2 to 5°C. The surface connective tissue and fibers at both ends are peeled off on ice within 1 hour, and the neural cell bodies are retained. The obtained ganglion tissue is placed in a low-adhesion culture plate, and a neural cell complete culture medium is added. After culturing for 100 to 140 minutes, the medium is replaced by half every 20 to 28 hours, and the culture is carried out for 6 to 8 days to obtain the dorsal root ganglion neurosphere;

[0049] (2) Construction of chondrocyte spheres without scaffold materials:

[0050] Articular cartilage was separated and minced, digested with 0.2% to 0.3% trypsin for 55 to 65 min, and then digested with 0.15% to 0.25% type II collagenase for 8 to 12 h. The cells were sieved with a cell mesh with a pore size of 80 to 120 μm and placed in a culture bottle. The cell density was adjusted to (1 to 2) × 10 4 / mL, cultured in complete chondrocyte medium, changed medium after 45-50h, and changed medium every 20-25h. When the cell density was not less than 90%, digested with 0.2%-0.3% trypsin for 2-5min, collected cells by centrifugation, and adjusted the cell density to (1-3)×10 4 / mL, placing the cell suspension in a low-adhesion culture plate, changing the medium every 20 to 28 hours, and culturing for 10 to 12 days to obtain the chondrocyte spheres;

[0051] (3) 3D co-culture:

[0052] The neuron spheres cultured for 6-8 days and the chondrocyte spheres cultured for 10-12 days were placed in the same culture plate and cultured with complete neuron culture medium. Half of the medium was replaced every 20-28 hours, and a structurally stable 3D co-culture system was formed in 5-7 days.

[0053] In a second aspect, the present invention provides a 3D co-culture system of dorsal root ganglion neuron spheres and chondrocyte spheres obtained by co-culturing the dorsal root ganglion neuron and chondrocyte spheres according to the method for co-culturing dorsal root ganglion neuron and chondrocytes described in the first aspect.

[0054] In the present invention, the 3D co-culture system has a high success rate in preparation, and the infiltration degree of the neuron spheres into the chondrocyte spheres is relatively high. The system can be used as an in vitro cell research model for arthritis and has broad application prospects.

[0055] In a third aspect, the present invention provides a method for detecting neural infiltration of the dorsal root ganglion neurosphere and chondrocyte sphere 3D co-culture system according to the second aspect, the detection method comprising:

[0056] Samples of the system after co-culture were taken, fixed, and then frozen and sectioned. Immunofluorescence staining was performed and photos were taken under a microscope. The extent of nerve endings infiltration into the chondrocyte spheres was measured based on the length, area, and volume percentage of the nerve endings invading the chondrocyte spheres.

[0057] In the present invention, the detection method is simple to operate, has a high success rate, is easy to master, and promotes the promotion and use of related methods.

[0058] In a fourth aspect, the present invention provides the use of any one or a combination of at least two of the method for co-culturing dorsal root ganglion neurons and chondrocytes described in the first aspect, the 3D co-culture system of dorsal root ganglion neuron spheres and chondrocyte spheres described in the second aspect, or the neural infiltration detection method described in the third aspect in preparing an in vitro neural infiltration articular cartilage research model.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention improves the method for co-culturing the dorsal root ganglion nerve cells and chondrocytes and the formula of the culture medium used, thereby increasing the cell division and proliferation rate, improving the physiological metabolic level of the cells, making the physiological state closer to the natural state, and making the nerve endings more capable of infiltrating the chondrocytes. The co-culture system is a 3D stereoscopic model, which can analyze the mechanism of nerve endings infiltrating articular cartilage from a three-dimensional level, restore the pathological mechanism of arthritis to the greatest extent, and is applied to the relevant research on the pathogenic mechanism of arthritis and drug screening, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1A This is a picture of dorsal root ganglion neurospheres and chondrocyte spheres after 3D co-culture for 1 day in Example 1 of the present invention (scale bar = 200 μm);

[0062] Figure 1B This is a picture of dorsal root ganglion neurospheres and chondrocyte spheres after 3D co-culture for 3 days in Example 1 of the present invention (scale bar = 200 μm);

[0063] Figure 2 This is a picture of the dorsal root ganglion neurosphere constructed in Example 4 of the present invention (scale bar = 200 μm);

[0064] Figure 3 This is a picture of the chondrocyte sphere constructed in Example 5 of the present invention (scale bar = 200 μm);

[0065] Figure 4 This is a picture of the neural infiltration results of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres in Example 6 of the present invention (scale bar = 50 μm);

[0066] Figure 5A This is a picture of the neural infiltration results of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres in the control group in Example 7 of the present invention (scale bar = 50 μm);

[0067] Figure 5B This is a picture of the neural infiltration results of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres in the Sema3a gene knockdown group in Example 7 of the present invention (scale bar = 50 μm).

[0068] Figure 5C This is a picture showing the statistical results of the fluorescence intensity in the chondrocyte spheres in the control group and the Sema3a gene knockdown group in Example 7 of the present invention. DETAILED DESCRIPTION

[0069] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0070] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0071] Material:

[0072] Neurobasal culture medium was purchased from Gibco, catalog number 21103-049;

[0073] DMEM / F12 medium was purchased from Gibco, catalog number A4192001;

[0074] B-27 was purchased from Gibco, catalog number 17504-044;

[0075] Glutamine was purchased from Gibco, catalog number A2916801;

[0076] Antibiotics were purchased from Gibco at a concentration of 10,000 U / mL;

[0077] 96-well low-adhesion culture plates were purchased from Thermo, catalog number 174925;

[0078] Fetal bovine serum was purchased from Gibco, catalog number 10100139C;

[0079] Trypsin was purchased from Gibco, catalog number 25200056;

[0080] Collagenase type II was purchased from Gibco, catalog number 17101015;

[0081] Tuj1 primary antibody was purchased from Invitrogen, catalog number 480011;

[0082] Fluorescent secondary antibodies were purchased from Invitrogen, catalog number A-11032;

[0083] SD rats were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.;

[0084] siRNA transfection reagent K2 was purchased from biotex;

[0085] siRNA transfection medium was purchased from Gibco, catalog number 31985062;

[0086] siRNA was purchased from Santa Cruz Biotechnology, catalog number sc-36470.

[0087] Example 1

[0088] This embodiment provides a 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres, wherein the co-culture system is prepared by the following method:

[0089] (1) Construction of dorsal root ganglion neurospheres without scaffold materials:

[0090] The dorsal root ganglia of newborn SD rats were isolated and placed in a neural cell basal medium at a temperature of 4°C. The surface connective tissue and fibers at both ends were peeled off on ice within 1 hour, and the neural cell bodies were retained. The obtained ganglion tissue was placed in a 96-well low-adhesion culture plate, and 100 μL of neural cell complete medium was added. The medium was changed after culturing for 120 minutes, and half of the medium was changed every 24 hours for 8 days to obtain the dorsal root ganglion neurospheres.

[0091] (2) Construction of chondrocyte spheres without scaffold materials:

[0092] The articular cartilage was separated and cut into pieces, digested with 0.25% trypsin for 60 min, and then digested with 0.2% type II collagenase for 10 h. The digested cartilage tissue was blown away, sieved with a cell mesh with a pore size of 100 μm, and placed in a culture flask. The cell density was adjusted to 1×10 4 / mL, cultured in complete chondrocyte medium, changed medium after 48h, and changed medium every 24h. When the cell density was not less than 90%, digested with 0.25% trypsin for 3min, collected cells by centrifugation, and adjusted the cell density to 2×10 4 / mL, take 100 μL of cell suspension and place it in a 96-well low-adhesion culture plate, change the medium every 24 hours, and culture for 10 days to obtain the chondrocyte spheres;

[0093] (3) 3D co-culture:

[0094] The neuronal cell spheres cultured for 8 days and the chondrocyte spheres cultured for 10 days were placed in the same culture plate and cultured with complete neuronal cell culture medium. Half of the medium was replaced every 24 hours. The cell spheres began to fuse after 2 days, and a structurally stable 3D co-culture system of neuronal cell spheres and chondrocyte spheres was formed on the 7th day.

[0095] Wherein, the complete culture medium for neural cells comprises, by mass fraction, 2% B-27, 1% glutamine and 1% antibiotics, and the remainder is the basic culture medium for neural cells; the complete culture medium for chondrocytes comprises, by mass fraction, 10% fetal bovine serum and 1% antibiotics, and the remainder is DMEM / F12 culture medium.

[0096] The pictures of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres after 1d and 3d co-culture are shown in the figure below. Figure 1A and Figure 1B As shown in the figure, after 1 day of co-culture, the dorsal root ganglion neurosphere and chondrocyte sphere had clear outlines and no fusion occurred. However, after 3 days of co-culture, the two began to fuse and the nerve endings infiltrated into the chondrocyte sphere.

[0097] Example 2

[0098] This embodiment provides a 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres, wherein the co-culture system is prepared by the following method:

[0099] (1) Construction of dorsal root ganglion neurospheres without scaffold materials:

[0100] The dorsal root ganglia of newborn SD rats were isolated and placed in a neural cell basal culture medium at a temperature of 2°C. The surface connective tissue and fibers at both ends were peeled off on ice within 1 hour, and the neural cell bodies were retained. The obtained ganglion tissue was placed in a 96-well low-adhesion culture plate, and 100 μL of neural cell complete culture medium was added. The medium was changed after culturing for 100 minutes, and half of the medium was changed every 20 hours. After culturing for 7 days, the dorsal root ganglion neurospheres were obtained;

[0101] (2) Construction of chondrocyte spheres without scaffold materials:

[0102] The articular cartilage was separated and cut into pieces, digested with 0.2% trypsin for 65 min, and then digested with 0.25% type II collagenase for 8 h. The digested cartilage tissue was blown away, sieved with a cell mesh with a pore size of 80 μm, and placed in a culture flask. The cell density was adjusted to 1.5×104 / mL, cultured in complete chondrocyte medium, changed medium after 50h, and changed medium every 25h. When the cell density was not less than 90%, digested with 0.3% trypsin for 2min, collected cells by centrifugation, and adjusted the cell density to 1×10 4 / mL, take 100 μL of cell suspension and place it in a 96-well low-adhesion culture plate, change the medium every 28 hours, and culture for 12 days to obtain the chondrocyte spheres;

[0103] (3) 3D co-culture:

[0104] The neuronal cell spheres cultured for 6 days and the chondrocyte spheres cultured for 11 days were placed in the same culture plate and cultured with complete neuronal cell culture medium. Half of the medium was replaced every 28 hours. After 2 days, the cell spheres began to fuse, and a structurally stable 3D co-culture system of neuronal cell spheres and chondrocyte spheres was formed on the 5th day.

[0105] Wherein, the complete culture medium for neural cells comprises, by mass fraction, 1% B-27, 2% glutamine and 2.5% antibiotics, and the remainder is the basic culture medium for neural cells; the complete culture medium for chondrocytes comprises, by mass fraction, 8% fetal bovine serum and 2.5% antibiotics, and the remainder is DMEM / F12 culture medium.

[0106] The pictures of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres after co-culture are similar to those in Example 1 and will not be described again here.

[0107] Example 3

[0108] This embodiment provides a 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres, wherein the co-culture system is prepared by the following method:

[0109] (1) Construction of dorsal root ganglion neurospheres without scaffold materials:

[0110] The dorsal root ganglia of newborn SD rats were isolated and placed in a neural cell basal culture medium at a temperature of 5°C. The surface connective tissue and fibers at both ends were peeled off on ice within 1 hour, and the neural cell bodies were retained. The obtained ganglion tissue was placed in a 96-well low-adhesion culture plate, and 100 μL of neural cell complete culture medium was added. The medium was changed after culturing for 140 minutes, and half of the medium was changed every 28 hours. After culturing for 6 days, the dorsal root ganglion neurospheres were obtained;

[0111] (2) Construction of chondrocyte spheres without scaffold materials:

[0112] The articular cartilage was separated and cut into pieces, digested with 0.3% trypsin for 55 min, and then digested with 0.15% type II collagenase for 12 h. The digested cartilage tissue was blown away, sieved with a cell mesh with a pore size of 120 μm, and placed in a culture flask. The cell density was adjusted to 2×10 4 / mL, cultured with complete chondrocyte medium, changed medium after 45h, and changed medium every 20h. When the cell density was not less than 90%, 0.2% trypsin was used for digestion for 5min, and cells were collected by centrifugation. The cell density was adjusted to 3×10 4 / mL, 100 μL of cell suspension was placed in a 96-well low-adhesion culture plate, the medium was changed every 20 hours, and the culture was carried out for 11 days to obtain the chondrocyte spheres;

[0113] (3) 3D co-culture:

[0114] The neuronal cell spheres cultured for 7 days and the chondrocyte spheres cultured for 12 days were placed in the same culture plate and cultured with complete neuronal cell culture medium. Half of the medium was replaced every 20 hours. After 3 days, the cell spheres began to fuse, and a structurally stable 3D co-culture system of neuronal cell spheres and chondrocyte spheres was formed after 6 days.

[0115] Wherein, the complete culture medium for neural cells comprises, by mass fraction, 3% B-27, 0.5% glutamine and 0.5% antibiotics, and the remainder is the basic culture medium for neural cells; the complete culture medium for chondrocytes comprises, by mass fraction, 12% fetal bovine serum and 0.5% antibiotics, and the remainder is DMEM / F12 culture medium.

[0116] The pictures of the 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres after co-culture are similar to those in Example 1 and will not be described again here.

[0117] Example 4

[0118] The only difference from Example 1 is that the formula of the complete culture medium for neural cells used in this example is as follows in terms of mass fraction: 10% fetal bovine serum, 2% B-27, 1% glutamine and 1% antibiotics, and the remainder is basal culture medium.

[0119] The remaining materials and preparation methods are the same as those in Example 1.

[0120] The pictures of the constructed dorsal root ganglion neurospheres are shown in Figure 2. Figure 2 As shown in the figure, it can be seen that after culturing with the culture medium in this example, stable suspended neuron spheres cannot be formed, and the cells will differentiate and adhere to the wall prematurely under the action of serum, which is not conducive to the subsequent 3D co-culture with chondrocyte spheres.

[0121] Example 5

[0122] The only difference from Example 1 is that in step (2) of this example, when constructing chondrocyte spheres without scaffold materials, 100 μL of cell suspension is placed in a common cell culture plate and cultured on a shaker with a rotation speed of 20 revolutions per minute. The medium is changed every 20 hours and cultured for 11 days to obtain the chondrocyte spheres. The remaining materials and preparation methods are the same as those in Example 1.

[0123] The pictures of the constructed chondrocyte spheres are shown in Figure 3 As shown in the figure, it can be seen that after using the ordinary culture plate with the shaking table shaking culture method, it is impossible to form suspended chondrocyte spheres, and they grow prematurely attached to the wall, which is not conducive to the subsequent 3D co-culture with dorsal root ganglion neuron spheres.

[0124] Example 6

[0125] In this example, the neural infiltration of the dorsal root ganglion neurosphere and chondrocyte sphere 3D co-culture system prepared in Example 1 was detected, and the steps were as follows:

[0126] (1) Take samples of the neurosphere and chondrocyte sphere 3D co-culture system after 7 days, remove the culture medium, and rinse with 1× PBS three times;

[0127] (2) Add 100 μL of 4% paraformaldehyde solution for 20 min and rinse three times with 1× PBS;

[0128] (3) Add 200 μL of 30% sucrose solution and incubate at 4°C for 24 h;

[0129] (4) Take out the sample, place it in a sample tank, add OCT embedding medium, and freeze embed at -20°C;

[0130] (5) Take out the embedded sample and use a cryostat to make frozen sections with a thickness of 20 μm, which are attached to the surface of an adhesive slide;

[0131] (6) After the samples were washed three times with PBS, they were blocked with 1% goat serum for 1 h;

[0132] (7) Remove the blocking solution, add Tuj1 primary antibody diluted 1:500, incubate at room temperature for 90 min, and then wash three times with PBST, each time for 10 min;

[0133] (8) Add fluorescent secondary antibody diluted 1:1000, incubate at room temperature for 60 min, and then wash three times with PBST, each time for 10 min;

[0134] (9) After drying and sealing, the slides were photographed using a confocal microscope. Tuj1 staining was used to mark the length, area, and volume percentage of the nerve endings invading the chondrocyte spheres, and the degree of nerve ending infiltration into the chondrocyte spheres was measured.

[0135] The results are as follows Figure 4 As shown in the figure, the cell ball on the upper side of the picture is a chondrocyte ball, and the lower side is a nerve cell ball. Nerve endings marked with fluorescent staining Tuj1 can be observed in the chondrocyte ball, which indicates that the nerve endings marked by Tuj1 have successfully infiltrated the chondrocyte ball.

[0136] Example 7

[0137] In this example, the dorsal root ganglion neurosphere and chondrocyte sphere 3D co-culture system prepared in Example 1 was used as a model to study the effect of cartilage secretion factors on neuronal cell infiltration into chondrocytes, as follows:

[0138] The preparation method of the co-culture system of the control group was the same as that in Example 1. In the process of preparing chondrocyte spheres in the co-culture system of the experimental group, after the chondrocytes were inoculated into the culture flask and the medium was changed, when the cell density was not less than 90%, siRNA was transfected to knock down the Sema3a gene, and the steps were as follows:

[0139] (1) Add 1 μg siRNA to 100 μL siRNA transfection medium to prepare solution A; add 8 μL siRNA transfection reagent K2 to 100 μL siRNA transfection medium to prepare solution B;

[0140] (2) Add solution A to solution B, gently mix the solution by pipetting up and down, and incubate at room temperature for 30 min;

[0141] (3) Wash the cells once with 2 mL of siRNA transfection medium, aspirate the medium, add 0.8 mL of siRNA transfection medium to the mixture of solution A and solution B, mix gently, add to the washed cells, and incubate at 37°C in a CO2 incubator for 5 to 7 h;

[0142] (4) Add 2 mL of complete chondrocyte culture medium and culture for 48 h;

[0143] (5) Digest with 0.25% trypsin for 3 min, collect cells by centrifugation, and adjust the cell concentration to 2×10 4 / mL, take 100 μL of cell suspension and place it in a 96-well low-adhesion culture plate, change the medium every 24 hours, and culture for 10 days to obtain structurally stable chondrocyte spheres with knocked-down Sema3a gene.

[0144] The subsequent 3D co-culture was the same as in Example 1.

[0145] The method in Example 6 was used to detect the neural infiltration of the neurosphere and chondrocyte sphere 3D co-culture system. The results are as follows: Figure 5A and Figure 5B As shown. Figure 5A and Figure 5B It can be seen that after knocking down the Sema3a gene in chondrocytes, the degree of infiltration of nerve endings in the chondrocyte spheres increased significantly. The fluorescence intensity in the image was statistically analyzed using Graphpad Prism8. The results are as follows Figure 5C As shown. Figure 5C It can be seen that after Sema3a gene knockdown, the fluorescence intensity in the chondrocyte spheres increased significantly, which was statistically significant. The above results show that conventional molecular cell biology research methods are also applicable to the co-culture system in the present invention, which can be used in the study of related mechanisms and is a good research model.

[0146] In summary, the present invention improves the cell division and proliferation ability by optimizing the culture medium formula and the culture method, makes it easier to form cell spheres, and the physiological metabolism is very close to that in the natural state, so it can be used in related research; the co-culture system is a 3D stereoscopic model, and the infiltration mechanism of nerve endings to articular cartilage can be analyzed from multiple levels. Conventional molecular cell biology research methods are also applicable to co-cultured cell spheres, and can be used as a cell model in related research on the pathogenesis of arthritis and drug screening, and has broad application prospects.

[0147] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for co-culturing dorsal root ganglion neurons and chondrocytes, characterized in that: The co-cultivation method comprises: (1) Construction of dorsal root ganglion neurospheres without scaffold materials: The dorsal root ganglion was isolated and placed in a neural cell basal culture medium at a temperature of 2-5°C. The surface connective tissue and fibers at both ends were peeled off on ice within 1 hour, and the neuronal cell bodies were retained. The obtained ganglion tissue was placed in a low-adhesion culture plate, and a neural cell complete culture medium was added. The medium was changed after culturing for 100-140 minutes, and half of the medium was changed every 20-28 hours. The culture was carried out for 6-8 days to obtain the dorsal root ganglion neurospheres. (2) Construction of chondrocyte spheres without scaffold materials: Articular cartilage was separated and minced, digested with 0.2%-0.3% trypsin for 55-65 min, and then digested with 0.15%-0.25% type II collagenase for 8-12 h. The cells were sieved with a cell mesh with a pore size of 80-120 μm and placed in a culture flask. The cell density was adjusted to (1-2)×10 4 Cells were cultured in complete chondrocyte medium for 45-50 h and then every 20-25 h. When the cell density was not less than 90%, the cells were digested with 0.2%-0.3% trypsin for 2-5 min and collected by centrifugation. The cell density was adjusted to (1-3)×10 4 / mL, placing the cell suspension in a low-adhesion culture plate, changing the medium every 20-28 hours, and culturing for 10-12 days to obtain the chondrocyte spheres; (3) 3D co-culture: The neurospheres cultured for 6-8 days and the chondrocyte spheres cultured for 10-12 days were placed in the same culture plate and cultured with complete neuronal cell culture medium. Half of the medium was replaced every 20-28 hours, and a stable 3D co-culture system was formed in 5-7 days. The complete neural cell culture medium comprises, by mass fraction, 1% to 3% B-27, 0.5% to 2% glutamine, and 0.5% to 2.5% antibiotics, and the remainder is a neural cell basal culture medium; The complete chondrocyte culture medium comprises, by mass fraction, 8% to 12% fetal bovine serum and 0.5% to 2.5% antibiotics, with the remainder being DMEM / F12 culture medium.

2. The method for co-culturing dorsal root ganglion neurons and chondrocytes according to claim 1, characterized in that: The mass fraction of the B-27 in the complete neural cell culture medium is 2%.

3. The method for co-culturing dorsal root ganglion neurons and chondrocytes according to claim 1, characterized in that: The mass fraction of the glutamine in the complete neural cell culture medium is 1%.

4. The method for co-culturing dorsal root ganglion neurons and chondrocytes according to claim 1, characterized in that: The antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete culture medium for neural cells is 1%.

5. The method for co-culturing dorsal root ganglion neurons and chondrocytes according to claim 1, characterized in that: The mass fraction of the fetal bovine serum in the complete chondrocyte culture medium is 10%.

6. A method for detecting neural infiltration in a 3D co-culture system of dorsal root ganglion neurospheres and chondrocyte spheres, characterized in that: The detection method comprises: Take samples of the co-cultured system, fix them, and then perform frozen sections, immunofluorescence staining, and take pictures under a microscope. The extent of nerve endings infiltrating the chondrocyte spheres is measured based on the length, area, and volume ratio of the nerve endings invading the chondrocyte spheres. The co-cultivation method comprises: (1) Construction of dorsal root ganglion neurospheres without scaffold materials: The dorsal root ganglion was isolated and placed in a neural cell basal culture medium at a temperature of 2-5°C. The surface connective tissue and fibers at both ends were peeled off on ice within 1 hour, and the neuronal cell bodies were retained. The obtained ganglion tissue was placed in a low-adhesion culture plate, and a neural cell complete culture medium was added. The medium was changed after culturing for 100-140 minutes, and half of the medium was changed every 20-28 hours. The culture was carried out for 6-8 days to obtain the dorsal root ganglion neurospheres. (2) Construction of chondrocyte spheres without scaffold materials: Articular cartilage was separated and minced, digested with 0.2%-0.3% trypsin for 55-65 min, and then digested with 0.15%-0.25% type II collagenase for 8-12 h. The cells were sieved with a cell mesh with a pore size of 80-120 μm and placed in a culture flask. The cell density was adjusted to (1-2)×10 4 Cells were cultured in complete chondrocyte medium for 45-50 h and then every 20-25 h. When the cell density was not less than 90%, the cells were digested with 0.2%-0.3% trypsin for 2-5 min and collected by centrifugation. The cell density was adjusted to (1-3)×10 4 / mL, placing the cell suspension in a low-adhesion culture plate, changing the medium every 20-28 hours, and culturing for 10-12 days to obtain the chondrocyte spheres; (3) 3D co-culture: The neurospheres cultured for 6-8 days and the chondrocyte spheres cultured for 10-12 days were placed in the same culture plate and cultured with complete neuronal cell culture medium. Half of the medium was replaced every 20-28 hours, and a stable 3D co-culture system was formed in 5-7 days. The complete neural cell culture medium comprises, by mass fraction, 1% to 3% B-27, 0.5% to 2% glutamine, and 0.5% to 2.5% antibiotics, and the remainder is a neural cell basal culture medium; The complete chondrocyte culture medium comprises, by mass fraction, 8% to 12% fetal bovine serum and 0.5% to 2.5% antibiotics, with the remainder being DMEM / F12 culture medium.

7. Use of the method for co-culturing dorsal root ganglion neurons and chondrocytes according to any one of claims 1 to 5 in preparing an in vitro neural infiltration articular cartilage research model.

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