WO2023273197A1

WO2023273197A1 - Method and application for co-culture of dorsal root ganglion neurons and chondrocytes

Info

Publication number: WO2023273197A1
Application number: PCT/CN2021/138075
Authority: WO
Inventors: 杨帆; 高大双; 黄石书
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2021-06-29
Filing date: 2021-12-14
Publication date: 2023-01-05
Also published as: CN115537381A

Abstract

Provided are a method and an application for co-culture of dorsal root ganglion neurons and chondrocytes. The co-culture method comprises: constructing both dorsal root ganglion neuron spheroids not requiring scaffolding material and chondrocyte spheroids not requiring scaffolding material, and performing 3D co-culturing of the obtained neuron spheroids and chondrocyte spheroids; a culture medium used for constructing the dorsal root ganglion neuron spheroids and for 3D co-culturing is a complete culture medium for nerve cells, and the culture medium used for constructing the chondrocyte spheroids is a complete culture medium for chondrocytes. Also provided is a 3D co-culturing system for dorsal root ganglion neuron spheroids and chondrocyte spheroids, and a method for detecting nerve infiltration therefor. The co-culture method has simple operation and good cell physiology, can be used as a model for studies on pathogenic mechanisms of related diseases, and has broad application prospects.

Description

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

technical field

The invention belongs to the technical field of 3D co-culture of cells, and in particular relates to a method and application of co-culture of dorsal root ganglion nerve cells and chondrocytes.

Background technique

The dorsal root ganglion is a distended tubercle located in the dorsal root of the spinal cord near the medial surface of the intervertebral foramen, responsible for receiving all nerve impulses from the body receptors, including general somatosensory and visceral sensation, and sending them to the spinal cord and even the brain through afferent nerves. center.

Articular cartilage is composed of chondrocytes and cartilage matrix. The cartilage matrix is a network structure composed of collagen fibers and proteoglycans, and chondrocytes are filled in the network structure. Articular cartilage has neither nerves nor blood vessels, and its nutrition is mainly supplied by synovial fluid and arterial branches around the synovial layer of the joint capsule. Chondrocytes are scattered among these network structures to maintain the normal metabolism of articular cartilage. When arthritis occurs, articular cartilage lesion, loss of cartilage polysaccharides in the cartilage matrix, thinning of the cartilage matrix, proliferation of synoviocytes and infiltration of lymphocytes, accompanied by nerve infiltration of articular cartilage, resulting in irreparable articular cartilage, eventually leading to osteoarthritis inflammation.

The central nervous system and the peripheral nervous system play a significant role in the regulation of bone development and bone homeostasis, such as Semaphorins, Netrins, Slits and Ephrins families. The role of neurotrophic factors and axon growth-inducing factors in human osteoblasts and osteoclasts has been confirmed. These axon-guiding factors can induce and/or inhibit the growth of nerve fibers, thereby affecting the process of nerve endings infiltrating articular cartilage .

In recent years, the regulation of bone and cartilage by the nervous system has become a frontier hot spot 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 into cartilage and arthritis pain, and innovatively proposed the relationship between osteoclast-induced nerve infiltration in subchondral bone and osteoarthritis.

At present, there is no similar protocol for the co-culture of dorsal root ganglion nerve cells and chondrocytes in vitro. Therefore, how to provide a method for co-culture of dorsal root ganglion nerve cells and chondrocytes will provide a basis for the study of related mechanisms such as nerve infiltration in articular cartilage in vitro The job provisioning model has become an urgent problem to be solved.

technical problem

Aiming at the deficiencies and actual needs of the prior art, the present invention provides a method and application of co-cultivation of dorsal root ganglion nerve cells and chondrocytes. By co-culturing dorsal root ganglion nerve cells and chondrocytes in vitro, a The research model of nerve endings infiltrating articular cartilage creates conditions for the research on the pathogenic mechanism of arthritis and the screening of targeted drugs.

technical solution

In a first aspect, the present invention provides a method for co-cultivating dorsal root ganglion nerve cells and chondrocytes, the co-cultivating method comprising:

Dorsal root ganglion nerve cell spheres without scaffold materials and chondrocyte spheres without scaffold materials were respectively constructed, and then the obtained nerve cell spheres and chondrocyte spheres were co-cultured in 3D;

Wherein, the medium used for constructing the dorsal root ganglion nerve cell spheroid and 3D co-cultivation is a complete nerve cell medium, and the medium used for constructing the chondrocyte spheroid is a complete chondrocyte medium.

In the present invention, by separately constructing dorsal root ganglion nerve cell spheres without scaffold materials and chondrocyte spheres without scaffold materials, nerve cells and chondrocytes can grow without interference from the external environment, and the physiological state is closer to natural State, and reduce the influence of materials on cytotoxicity, the research results are more accurate; by optimizing the culture medium and culture process, the growth state of cell spheres is better, the probability of successful co-culture of cells is higher, and the infiltration of nerve cells Articular cartilage works better.

Preferably, the method for constructing dorsal root ganglion nerve cell spheres without scaffold material comprises:

The dorsal root ganglion is separated, the connective tissue on the surface and the fibers at both ends are peeled off, the nerve cell body is reserved, and the obtained ganglion tissue is cultured in a culture plate to obtain the dorsal root ganglion nerve cell ball.

Preferably, after the dorsal root ganglion is isolated, it is placed in a nerve cell basal medium.

Preferably, the temperature of the neural cell basal medium is 2-5°C, for example, the temperature can be 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C or 5°C, etc. Other values within this range The specific point values can be selected, and will not be repeated here.

Preferably, the peeling of the connective tissue on the surface and the fibers at both ends is carried out on ice.

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

Preferably, the culture plate comprises a low adhesion culture plate.

Preferably, the step of cultivating comprises:

Add the complete medium of nerve cells to the culture plate, change the medium after 100-140 min of culture, change the medium in half every 20-28 h, and cultivate for 6-8 days. The culture time can be 100 min, 105 min, for example. min, 110 min, 115 min, 120 min, 125 min, 130 min, 135 min or 140 min, etc., the interval between liquid changes can be, for example, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h h, 27 h or 28 h, etc., the culture time can be, for example, 6 d, 6.5 d, 7 d, 7.5 d or 8 d, etc. Other specific point values within this value range can be selected, and will not be discussed here. repeat.

Preferably, the complete medium for nerve cells includes: basal medium for nerve cells, B-27, glutamine and antibiotics.

Preferably, the mass fraction of the B-27 in the complete culture medium of nerve cells is 1%~3%, for example, it can be 1%, 1.5%, 2%, 2.5% or 3%, etc., within this value range Other specific point values of can be selected, and will not be described here one by one, preferably 2%.

Preferably, the mass fraction of glutamine in the complete culture medium of nerve cells is 0.5%~2%, for example, it can be 0.5%, 1%, 1.5% or 2%, etc. Other specific values within this range The point value can be selected, so it will not be described here one by one, and it is preferably 1%.

Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete culture medium of the nerve cells 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 value range can be selected, and will not be described here one by one, preferably 1%.

Preferably, in terms of mass fraction, the complete culture medium for nerve cells includes: B-27 1%~3%, glutamine 0.5%~2% and antibiotics 0.5%~2.5%, and the balance is nerve cell basal culture medium .

In the present invention, by optimizing the formula of the complete culture medium for nerve cells, the growth speed of the nerve cells is faster, the growth state is better, and the ability to infiltrate the articular cartilage is stronger.

Preferably, the method for constructing chondrocyte spheres without scaffold material comprises:

The articular cartilage is separated, enzymatically digested, sieved, placed in a culture bottle for culture, and the cell suspension is placed in a culture plate for continuous culture to obtain the chondrocyte spheres.

Preferably, the step of shredding the articular cartilage is also included before the enzymatic digestion.

Preferably, the step of enzymatic digestion comprises:

Digest with 0.2%~0.3% trypsin for 55~65 minutes, and digest with 0.15%~0.25% type II collagenase for 8~12 hours. The concentration of trypsin can be 0.2%, 0.25% or 0.3%. For example, it can be 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min or 65 min, etc. The concentration of type II collagenase can be, for example, 0.15%, 0.2 % or 0.25%, etc., the digestion time of type II collagenase can be, for example, 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h or 12 h, etc. Others within this value range The specific point values can be selected, and will not be repeated here.

Preferably, the pore size of the cell sieve used in the sieving is 80-120 μm, such as 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 value range can be selected, so I won’t repeat them here.

Preferably, the step of cultivating comprises:

Adjust the cell density to (1~2)×10 ⁴ cells/mL, use chondrocyte complete medium for culture, change the medium every 20~25 h after 45~50 h, the cell density can be 1× 104/mL, ^1.1 ×104/mL, 1.2×104/mL, 1.3×104/mL, ^1.4 ^× ¹⁰⁴ /mL, 1.5×104/mL, ^1.6 ^× ¹⁰⁴ cells/mL, 1.7×10 ⁴ cells/mL, 1.8×10 ⁴ cells/mL, 1.9×10 ⁴ cells/mL or 2×10 ⁴ cells/mL, etc., the culture time can be 45 h, 45.5 h, 46 h, for example . , 23 h, 23.5 h, 24 h, 24.5 h or 25 h, etc., other specific point values within this value range can be selected, and will not be repeated here.

Preferably, the step of continuing to cultivate comprises:

After the cell density is not lower than 90%, digest with 0.2%~0.3% trypsin for 2~5 min, collect the cells by centrifugation, adjust the cell density to (1~ ³ )×104 cells/mL, and place the cell suspension in a low adhesion In the culture plate, change the medium every 20-28 hours, culture for 10-12 days, the concentration of trypsin can be 0.2%, 0.25% or 0.3%, etc., and the digestion time of trypsin can be 2 minutes, 2.5 minutes, for example , 3 min, 3.5 min, 4 min, 4.5 min or 5 min, etc., the cell density can be, for example, 1×10 ⁴ cells/mL, 1.2×10 ⁴ cells/mL, 1.4×10 ⁴ cells/mL, 1.6×10 ⁴ cells/mL pcs/mL, 1.8×10 ⁴ pcs/mL, 2×10 ⁴ pcs/mL, 2.2×10 ⁴ pcs/mL, 2.4×10 ⁴ pcs/mL, 2.6×10 ⁴ pcs/mL, 2.8×10 ⁴ pcs/mL mL or 3×10 ⁴ cells/mL, etc., the interval between changing the liquid can be 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h or 28 h, etc., and the culture time can be, for example, It is 10 d, 10.5 d, 11 d, 11.5 d or 12 d, etc. Other specific point values within this value range can be selected, so I won’t repeat them here.

In the present invention, by optimizing the culture method of chondrocyte spheres, the cells grow faster, have better physiological conditions, are more likely to be infiltrated by nerve endings, and have a higher probability of successful co-culture.

Preferably, the complete chondrocyte culture medium includes: DMEM/F12 medium, fetal bovine serum and antibiotics.

Preferably, the mass fraction of the fetal bovine serum in the complete chondrocyte 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 value range can be selected, and will not be repeated here, but 10% is preferred.

Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete chondrocyte medium is 0.5% to 2.5%, such as 0.5%, 1%, 1.5%, 2% or 2.5%, etc., other specific point values within this value range can be selected, and will not be described here one by one, preferably 1%.

Preferably, in terms of mass fraction, the complete medium for chondrocytes includes: 8%-12% fetal bovine serum and 0.5%-2.5% antibiotics, and the balance is DMEM/F12 medium.

Preferably, the step of described 3D co-culture comprises:

Place nerve cell spheroids and chondrocyte spheroids in the same culture plate, use the complete medium of nerve cells for culture, change the medium every 20-28 hours, and form a 3D co-culture system with stable structure every 5-7 days. The interval time can be, for example, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h or 28 h, etc., the culture time can be, for example, 5 d, 5.5 d, 6 d, 6.5 d or 7 d, etc. Other specific point values can be selected, and will not be repeated here.

Preferably, the culture time of the nerve cell sphere is 6-8 days, for example, it can be 6 days, 6.5 days d, 7 d, 7.5 d or 8 d, etc., the culture time of the chondrocyte spheres is 10 to 12 d, such as 10 d, 10.5 d, 11 d, 11.5 d or 12 d, etc. Other specific point values can be selected, and will not be repeated here.

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

(1) Construction of dorsal root ganglion nerve cell balls without scaffold materials:

Separate the dorsal root ganglion, place it in the basal culture medium of nerve cells at a temperature of 2-5°C, peel off the surface connective tissue and fibers at both ends on ice within 1 h, keep the nerve cell body, and place the obtained ganglion tissue in In the low-adhesion culture plate, add the complete medium of nerve cells, change the medium after culturing for 100-140 min, and change the medium every 20-28 h in half, and culture for 6-8 days to obtain the dorsal root ganglion nerve cell sphere;

(2) Construction of chondrocyte spheres without scaffold materials:

The articular cartilage was separated and shredded, digested with 0.2%-0.3% trypsin for 55-65 min, then digested with 0.15%-0.25% type II collagenase for 8-12 h, and sieved with a cell mesh with a pore size of 80-120 μm. Place in a culture flask, adjust the cell density to (1~ ² )×104 cells/mL, and use the complete chondrocyte medium for culture. After changing the medium for 45-50 hours, change the medium every 20-25 hours. After the density is not lower than 90%, digest with 0.2%~0.3% trypsin for 2~5 min, collect the cells by centrifugation, adjust the cell density to (1~ ³ )×104 cells/mL, and place the cell suspension in low-adhesion culture In the plate, the medium was changed every 20-28 h, and cultured for 10-12 days to obtain the chondrocyte spheres;

(3) 3D co-culture:

Will train 6~8 Nerve cell spheres in day d and chondrocyte spheres in culture for 10-12 days were placed in the same culture plate, cultured with complete nerve cell culture medium, half volume of medium was changed every 20-28 h, and a stable structure was formed in 5-7 days 3D co-culture system.

In a second aspect, the present invention provides a 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres cultured by the method for co-culturing dorsal root ganglion nerve cells and chondrocytes described in the first aspect.

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

In a third aspect, the present invention provides a method for detecting nerve invasion in the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres described in the second aspect, the detection method comprising:

The system samples after co-cultivation were taken, fixed, frozen and sectioned, and immunofluorescent staining was performed, and photographed with a microscope, and the degree of infiltration of chondrocyte spheres by nerve endings was measured according to the length, area and volume ratio of nerve endings invading chondrocyte spheres.

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

In a fourth aspect, the present invention provides the method for co-cultivating dorsal root ganglion nerve cells and chondrocytes described in the first aspect, the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres described in the second aspect, or Application of any one or a combination of at least two of the nerve invasion detection methods described in the third aspect in the preparation of an in vitro nerve invasion articular cartilage research model.

Beneficial effect

In the present invention, by improving the method of co-culture of dorsal root ganglion nerve cells and chondrocytes and the formulation of the medium used, the division and proliferation speed of cells is improved, the physiological metabolism level of cells is improved, and the physiological state is closer to that of In the natural state, nerve endings have a stronger ability to infiltrate 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 be applied to the pathogenesis of arthritis. It is of great significance in the related research of pathogenesis and drug screening.

Description of drawings

Fig. 1A is a picture of 3D co-cultured dorsal root ganglion nerve cell spheres and chondrocyte spheres after 1 day in Example 1 of the present invention (scale bar = 200 µm);

Fig. 1B is a picture of 3D co-cultured dorsal root ganglion nerve cell spheres and chondrocyte spheres in Example 1 of the present invention after 3 days (scale bar = 200 µm);

Figure 2 is a picture of the dorsal root ganglion nerve cell sphere constructed in Example 4 of the present invention (scale bar=200 µm);

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

Fig. 4 is a picture of the results of nerve infiltration in the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres in Example 6 of the present invention (scale bar = 50 μm);

Fig. 5A is a picture of the results of nerve infiltration in the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres in the control group in Example 7 of the present invention (scale bar = 50 μm);

Fig. 5B is a picture of the results of nerve infiltration in the 3D co-culture system of DRG nerve cell spheres and chondrocyte spheres in the Sema3a gene knockdown group in Example 7 of the present invention (scale bar = 50 μm).

5C is a picture of statistical results of fluorescence intensities in chondrocyte spheres in the control group and the Sema3a gene knockdown group in Example 7 of the present invention.

Embodiments of the present invention

In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products commercially available through regular channels.

Material:

Nerve cell basal medium Neurobasal was purchased from Gibco, Cat. No. 21103-049;

DMEM/F12 medium was purchased from Gibco, Cat. No. A4192001;

B-27 was purchased from Gibco, Cat. No. 17504-044;

Glutamine was purchased from Gibco, item number A2916801;

Antibiotics were purchased from Gibco with a concentration of 10000U/mL;

96-well low-adhesion culture plate was purchased from Thermo, Cat. No. 174925;

Fetal bovine serum was purchased from Gibco, product number 10100139C;

Trypsin was purchased from Gibco, product number 25200056;

Type II collagenase was purchased from Gibco, product number 17101015;

Tuj1 primary antibody was purchased from Invitrogen, Cat. No. 480011;

The fluorescent secondary antibody was purchased from Invitrogen, Cat. No. A-11032;

SD rats were from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.;

siRNA transfection reagent K2 was purchased from biotex;

siRNA transfection medium was purchased from Gibco, Cat. No. 31985062;

siRNA was purchased from Santa Cruz Biotechnology, Cat. No. sc-36470.

Example 1

This embodiment provides a 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres, which is prepared by the following method:

The dorsal root ganglion of newborn SD rats was isolated, placed in the nerve cell basal medium at a temperature of 4 °C, and the connective tissue on the surface and the fibers at both ends were peeled off on ice within 1 h, and the nerve cell body was preserved, and the obtained ganglion The tissue was placed in a 96-well low-adhesion culture plate, 100 μL of complete nerve cell culture medium was added, the medium was changed after 120 min of culture, and half of the medium was changed every 24 h, and cultured for 8 days to obtain the dorsal root ganglion nerve cell sphere ;

(2) Construction of chondrocyte spheres without scaffold materials:

The articular cartilage was separated and shredded, digested with 0.25% trypsin for 60 min, 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 bottle In the medium, the cell density was adjusted to 1×10 ⁴ cells/mL, cultured with chondrocyte complete medium, and the medium was changed every 24 h after 48 h. After the cell density was not lower than 90%, 0.25% trypsin After digestion for 3 min, the cells were collected by centrifugation, and the cell density was adjusted to ² ×104 cells/mL. 100 μL of the cell suspension was placed in a 96-well low-adhesion culture plate, and the medium was changed every 24 h. After culturing for 10 days, the obtained Chondrocyte spheres;

(3) 3D co-culture:

The nerve cell spheroids cultured for 8 days and the chondrocyte spheroids cultured for 10 days were placed in the same culture plate, cultured in the complete medium of nerve cells, and half of the medium was changed every 24 h. After 2 days, the cell spheroids began to fuse. d Formed a structurally stable 3D co-culture system of nerve cell spheres and chondrocyte spheres.

Wherein, in terms of mass fraction, the complete culture medium for nerve cells includes: B-27 2%, glutamine 1% and antibiotics 1%, and the balance is nerve cell basal medium; in terms of mass fraction, the chondrocytes The complete medium includes: 10% fetal bovine serum and 1% antibiotics, and the balance is DMEM/F12 medium.

The pictures of the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres for 1 day and 3 days of co-culture are shown in Figure 1A and Figure 1B, respectively. It can be seen from the figure that after 1 day of co-culture, the dorsal root ganglion nerve cell spheres and chondrocyte spheres had clear outlines and no fusion occurred, while after 3 days of co-culture d Afterwards, the two began to fuse, and the nerve endings infiltrated into the chondrocyte balls.

Example 2

The dorsal root ganglion of newborn SD rats was isolated, placed in the nerve cell basal medium at a temperature of 2°C, and the connective tissue on the surface and the fibers at both ends were peeled off on ice within 1 h, and the nerve cell body was retained, and the obtained ganglion The tissue was placed in a 96-well low-adhesion culture plate, 100 μL of complete nerve cell culture medium was added, the medium was changed after 100 min of culture, and half of the medium was changed every 20 h, and cultured for 7 days to obtain the dorsal root ganglion nerve cell sphere ;

(2) Construction of chondrocyte spheres without scaffold materials:

The articular cartilage was separated and shredded, digested with 0.2% trypsin for 65 min, 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 bottle In the medium, the cell density was adjusted to 1.5×10 ⁴ cells/mL, cultured with chondrocyte complete medium, and the medium was changed every 25 h after 50 h, the cell density was not lower than 90%, 0.3% trypsin After digestion for 2 min, the cells were collected by centrifugation, and the cell density was adjusted to ¹ ×104 cells/mL. 100 μL of the cell suspension was placed in a 96-well low-adhesion culture plate, and the medium was changed every 28 h. After culturing for 12 days, the obtained Chondrocyte spheres;

(3) 3D co-culture:

The neurocyte spheroids cultured for 6 d and the chondrocyte spheroids cultured for 11 d were placed in the same culture plate, cultured with the complete medium of neurons, and half of the medium was changed every 28 h. After 2 d, the cell spheroids began to fuse. d Formed a structurally stable 3D co-culture system of nerve cell spheres and chondrocyte spheres.

Wherein, in terms of mass fraction, the complete culture medium of nerve cells includes: B-27 1%, glutamine 2% and antibiotics 2.5%, and the balance is nerve cell basal medium; in terms of mass fraction, the chondrocyte The complete medium includes: 8% fetal bovine serum and 2.5% antibiotics, and the balance is DMEM/F12 medium.

The pictures of the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres after co-culture are similar to those in Example 1, and will not be repeated here.

Example 3

The dorsal root ganglion of newborn SD rats was isolated, placed in the nerve cell basal medium at a temperature of 5°C, and the connective tissue on the surface and fibers at both ends were peeled off on ice within 1 h, and the nerve cell body was retained, and the obtained ganglion The tissue was placed in a 96-well low-adhesion culture plate, 100 μL of complete nerve cell culture medium was added, the medium was changed after 140 min of culture, and half of the medium was changed every 28 h, and cultured for 6 days to obtain the dorsal root ganglion nerve cell sphere ;

(2) Construction of chondrocyte spheres without scaffold materials:

The articular cartilage was separated and shredded, digested with 0.3% trypsin for 55 min, 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 bottle In the medium, the cell density was adjusted to 2×10 ⁴ cells/mL, cultured with chondrocyte complete medium, and the medium was changed every 20 h after 45 h. After the cell density was not lower than 90%, 0.2% trypsin After digestion for 5 min, the cells were collected by centrifugation, and the cell density was adjusted to 3×10 ⁴ cells/mL. 100 μL of the cell suspension was removed and placed in a 96-well low-adhesion culture plate. The medium was changed every 20 h, and cultured for 11 days to obtain the obtained Chondrocyte spheres;

(3) 3D co-culture:

The chondrocyte spheres cultured for 7 days and the chondrocyte spheres cultured for 12 days were placed in the same culture plate, cultured with the complete medium of nerve cells, and half of the medium was changed every 20 h. After 3 days, the cell spheres began to fuse. d Formed a structurally stable 3D co-culture system of nerve cell spheres and chondrocyte spheres.

Wherein, in terms of mass fraction, the complete medium of nerve cells includes: B-27 3%, glutamine 0.5% and antibiotics 0.5%, and the balance is nerve cell basal medium; in terms of mass fraction, the chondrocytes The complete medium includes: 12% fetal bovine serum and 0.5% antibiotics, and the balance is DMEM/F12 medium.

Example 4

The only difference from Example 1 is that the formula of the complete culture medium for nerve cells used in this example is as follows in terms of mass fraction: 10% fetal bovine serum, B-272%, 1% glutamine and 1% antibiotics, and the remaining The amount is the basal medium.

All the other materials and preparation method are the same as in Example 1.

The picture of the constructed dorsal root ganglion nerve cell sphere is shown in Figure 2. It can be seen from the figure that after culturing with the medium in this example, a stable suspended nerve cell sphere cannot be formed, and it will differentiate in advance under the action of serum Adhering to the wall, it is not conducive to the subsequent 3D co-culture with chondrocyte spheres.

Example 5

The only difference from Example 1 is that when constructing chondrocyte spheres without scaffold materials in step (2) of this example, 100 μL of the cell suspension was placed in an ordinary cell culture plate and cultured on a shaker. The rotational speed was 20 revolutions per minute, the medium was changed every 20 h, and the chondrocyte spheres were obtained by culturing for 11 days. The rest of the materials and preparation methods were the same as in Example 1.

The picture of the constructed chondrocyte spheres is shown in Figure 3. It can be seen from the figure that the suspension chondrocyte spheres cannot be formed after using the ordinary culture plate and the culture method of shaker vibration, and the growth of adherent cells is early, which is not conducive to the follow-up and dorsal root ganglion. 3D co-culture of neurospheres.

Example 6

This example detects the nerve infiltration in the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres prepared in Example 1, and the steps are as follows:

(1) Take the system samples of the 3D co-culture system of nerve cell spheres and chondrocyte spheres after 7 days, remove the medium, and rinse with 1×PBS for 3 times;

(2) Add 100 μL of 4% paraformaldehyde solution to fix for 20 min, and rinse with 1×PBS for 3 times;

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

(4) Take out the sample, put it in the sample tank, add OCT embedding agent, and freeze it at -20°C for embedding;

(5) Take out the embedded sample, and use a cryostat to make a frozen section with a thickness of 20 μm, and attach it to the surface of the adhesive slide;

(6) After the samples were washed 3 times with PBS, they were blocked with 1% goat serum for 1 h;

(7) Remove the blocking solution, add Tuj1 primary antibody diluted at 1:500, incubate at room temperature for 90 min, wash with PBST 3 times, 10 min each time;

(8) Add fluorescent secondary antibody diluted at 1:1000, incubate at room temperature for 60 min, wash with PBST 3 times, 10 min each time;

(9) After drying and mounting the slides, take pictures with a confocal microscope. The length, area, and volume ratio of nerve endings invading chondrocyte spheres were marked by Tuj1 staining to measure the degree of nerve endings infiltrating chondrocyte spheres.

The result is shown in Figure 4. It can be seen from the figure that the cell spheres on the upper side of the picture are chondrocyte spheres, and the lower side are nerve cell spheres. In the chondrocyte spheres, the nerve endings labeled with Tuj1 can be observed, which shows that the nerve endings marked by Tuj1 have successfully infiltrated the cartilage. cell ball.

Example 7

In this example, the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres prepared in Example 1 was used as a model to study the influence of cartilage-secreted factors on the infiltration of chondrocytes by nerve cells, as follows:

The preparation method of the co-culture system of the control group was the same as that in Example 1. During the preparation of the chondrocyte spheres of the co-culture system of the experimental group, after the chondrocytes were inoculated into the culture flask and the liquid was changed, when the cell density was not lower than 90%, Transfect siRNA knockdown Sema3a gene, the steps are as follows:

(1) Add 1 µg siRNA to 100 µL siRNA transfection medium to make solution A; add 8 µL siRNA transfection reagent K2 to 100 µL siRNA transfection medium to make solution B;

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

(3) Wash the cells once with 2 mL siRNA transfection medium, aspirate the medium, add 0.8 mL siRNA transfection medium to the mixture containing solution A and solution B, mix gently, and add the washed cells in a carbon dioxide incubator at 37°C for 5–7 h;

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

(5) Digest with 0.25% trypsin for 3 min, collect cells by centrifugation, adjust the cell concentration to ² ×104/mL, take 100 μL of cell suspension and place it in a 96-well low-adhesion culture plate, and change the medium every 24 h , cultivated for 10 days, and obtained chondrocyte spheres with stable structure and Sema3a gene knockdown.

Subsequent 3D co-cultivation was the same as in Example 1.

The method in Example 6 was used to detect the nerve infiltration in the 3D co-culture system of nerve cell spheroids and chondrocyte spheroids, and the results are shown in FIG. 5A and FIG. 5B . It can be seen from Figure 5A and Figure 5B that after knocking down the Sema3a gene in chondrocytes, the degree of nerve terminal infiltration of nerve cell balls into chondrocyte balls was significantly increased. Use Graphpad Prism8 counts the fluorescence intensity in the picture, and the results are shown in Figure 5C. It can be seen from Figure 5C that after Sema3a gene knockdown, the fluorescence intensity in chondrocyte spheres increased significantly, which was statistically significant. The above results show that conventional molecular and cell biology research methods are also applicable to the co-culture system in the present invention, and can be used in the research of related mechanisms, which is a good research model.

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

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention can only be implemented depending on the above-mentioned detailed methods. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

A method for co-cultivating dorsal root ganglion nerve cells and chondrocytes, characterized in that the co-culturing method comprises:

Dorsal root ganglion nerve cell spheres without scaffold materials and chondrocyte spheres without scaffold materials were respectively constructed, and then the obtained nerve cell spheres and chondrocyte spheres were co-cultured in 3D;

Wherein, the medium used for constructing the dorsal root ganglion nerve cell spheroid and 3D co-cultivation is a complete nerve cell medium, and the medium used for constructing the chondrocyte spheroid is a complete chondrocyte medium.
The method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to claim 1, wherein the method for constructing dorsal root ganglion nerve cell balls without scaffold material comprises:

Isolating the dorsal root ganglion, peeling off the surface connective tissue and fibers at both ends, retaining the nerve cell body, placing the obtained ganglion tissue in a culture plate for culture, and obtaining the dorsal root ganglion nerve cell ball;

Preferably, after the dorsal root ganglion is isolated, it is placed in a nerve cell basal medium;

Preferably, the temperature of the neural cell basal medium is 2-5°C;

Preferably, the connective tissue on the peeling surface and the fibers at both ends are carried out on ice;

Preferably, the time for stripping the connective tissue on the surface and the fibers at both ends is no more than 1 h;

Preferably, the culture plate comprises a low adhesion culture plate;

Preferably, the step of cultivating comprises:

Add the complete medium of nerve cells to the culture plate, and change the medium after culturing for 100-140 min, and change the medium every 20-28 h in half, and culture for 6-8 days.
The method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to claim 1 or 2, wherein the complete culture medium for nerve cells comprises: nerve cell basal medium, B-27, glutamine and antibiotic;

Preferably, the mass fraction of the B-27 in the complete culture medium of the nerve cells is 1% to 3%, preferably 2%;

Preferably, the mass fraction of glutamine in the complete culture medium for nerve cells is 0.5% to 2%, preferably 1%;

Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete culture medium of the nerve cells is 0.5% to 2.5%, preferably 1%;

Preferably, in terms of mass fraction, the complete culture medium for nerve cells includes: B-27 1%~3%, glutamine 0.5%~2% and antibiotics 0.5%~2.5%, and the balance is nerve cell basal culture medium .
The method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 3, wherein the method for constructing chondrocyte spheres without scaffold material comprises:

Separating the articular cartilage, enzymatically digesting it, sieving it, placing it in a culture bottle for cultivation, and then placing the cell suspension in a culture plate to continue culturing to obtain the chondrocyte spheres;

Preferably, the step of shredding the articular cartilage is also included before the enzymatic digestion;

Preferably, the step of enzymatic digestion comprises:

0.2%~0.3% trypsin digestion for 55~65 min, 0.15%~0.25% type II collagenase digestion for 8~12 h;

Preferably, the pore size of the cell sieve used in the sieving is 80-120 μm;

Preferably, the step of cultivating comprises:

Adjust the cell density to (1~ 2 )×104 cells/mL, use the complete chondrocyte medium for culture, change the medium every 20~25 hours after changing the medium for 45~50 h;

Preferably, the step of continuing to cultivate comprises:

After the cell density is not lower than 90%, digest with 0.2%~0.3% trypsin for 2~5 min, collect the cells by centrifugation, adjust the cell density to (1~ 3 )×104 cells/mL, and place the cell suspension in a low adhesion In the culture plate, the medium was changed every 20-28 h, and cultured for 10-12 days.
The method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 4, wherein the chondrocyte complete medium comprises: DMEM/F12 medium, fetal calf serum and antibiotics ;

Preferably, the mass fraction of the fetal bovine serum in the complete chondrocyte medium is 8% to 12%, preferably 10%;

Preferably, the antibiotics include penicillin and streptomycin, and the mass fraction of the antibiotics in the complete chondrocyte medium is 0.5% to 2.5%, preferably 1%;

Preferably, in terms of mass fraction, the complete medium for chondrocytes includes: 8%-12% fetal bovine serum and 0.5%-2.5% antibiotics, and the balance is DMEM/F12 medium.
The method for co-cultivating dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 5, wherein the 3D co-culturing step comprises:

Put the nerve cell spheres and chondrocyte spheres in the same culture plate, use the complete medium of nerve cells for culture, every 20~28 Change the medium once at half the volume, and form a 3D co-culture system with stable structure in 5-7 days;

Preferably, the culture time of the nerve cell spheres is 6-8 days, and the culture time of the chondrocyte spheres is 10-12 days.
The method for co-culture of dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 6, wherein the co-culture method comprises:

(1) Construction of dorsal root ganglion nerve cell balls without scaffold materials:

Separate the dorsal root ganglion, place it in the basal culture medium of nerve cells at a temperature of 2-5°C, peel off the surface connective tissue and fibers at both ends on ice within 1 h, keep the nerve cell body, and place the obtained ganglion tissue in In the low-adhesion culture plate, add the complete medium of nerve cells, change the medium after culturing for 100-140 min, and change the medium every 20-28 h in half, and culture for 6-8 days to obtain the dorsal root ganglion nerve cell sphere;

(2) Construction of chondrocyte spheres without scaffold materials:

The articular cartilage was separated and shredded, digested with 0.2%-0.3% trypsin for 55-65 min, then digested with 0.15%-0.25% type II collagenase for 8-12 h, and sieved with a cell mesh with a pore size of 80-120 μm. Place in a culture flask, adjust the cell density to (1~ 2 )×104 cells/mL, use chondrocyte complete medium for culture, change the medium every 20~25 h after 45~50 h, the cells After the density is not lower than 90%, digest with 0.2%~0.3% trypsin for 2~5 min, collect the cells by centrifugation, adjust the cell density to (1~ 3 )×104 cells/mL, and place the cell suspension in low-adhesion culture In the plate, the medium was changed every 20-28 h, and cultured for 10-12 days to obtain the chondrocyte spheres;

(3) 3D co-culture:

The nerve cell 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 the complete medium of nerve cells, every 20-28 days. Half the amount of medium was changed once in h, and a 3D co-culture system with stable structure was formed in 5-7 days.
A 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres obtained by the method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 7.
A method for detecting nerve infiltration of the dorsal root ganglion nerve cell sphere and chondrocyte sphere 3D co-culture system according to claim 8, wherein the detection method comprises:

The system samples after co-cultivation were taken, fixed, frozen and sectioned, and immunofluorescent staining was performed, and photographed with a microscope, and the degree of infiltration of chondrocyte spheres by nerve endings was measured according to the length, area and volume ratio of nerve endings invading chondrocyte spheres.
The method for co-culturing dorsal root ganglion nerve cells and chondrocytes according to any one of claims 1 to 7, the 3D co-culture system of dorsal root ganglion nerve cell spheres and chondrocyte spheres according to claim 8, or claim 9 Application of any one or a combination of at least two of the nerve invasion detection methods in the preparation of an in vitro nerve invasion articular cartilage research model.