CN106635990B

CN106635990B - Method for primary culture of dorsal root ganglion satellite glial cells

Info

Publication number: CN106635990B
Application number: CN201611170545.2A
Authority: CN
Inventors: 李力燕; 郭建辉; 马微; 杨金伟; 王先斌; 李兴统; 王通通; 代云飞; 张泰�; 李永进
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2016-12-16
Filing date: 2016-12-16
Publication date: 2020-09-08
Anticipated expiration: 2036-12-16
Also published as: CN106635990A

Abstract

The invention discloses a method for primary culture of dorsal root ganglion satellite glial cells, which comprises the following steps: disinfecting a newborn mouse or a rat, cutting off the head, killing, taking out a spine after dissection, removing muscles on the spine, cutting off the spine, cleaning blood vessels and the spinal cord under a stereoscopic microscope, taking out a DRG, and removing nerve fibers and capsules on the DRG; culturing the treated DRG by adopting a DRG-SGCs culture solution, inducing a large amount of satellite glial cells to migrate out of the DRG, adding trypsin for digestion after the culture is finished, then adding FBS for terminating the digestion, slightly blowing by adopting a Pasteur suction pipe, and transferring to a centrifuge tube for centrifugation; and (4) centrifuging, removing the supernatant, adding a culture solution, and continuously culturing to obtain the satellite glial cells. The cultured and purified satellite glial cells can survive for a long time in vitro, have high purity and stably survive, and can be passaged for multiple generations to form a cell network.

Description

Method for primary culture of dorsal root ganglion satellite glial cells

Technical Field

The invention belongs to the technical field of dorsal root ganglion satellite glial cell isolation culture, and particularly relates to a method for primarily culturing dorsal root ganglion satellite glial cells in vitro.

Background

Nerve cells of the Dorsal Root Ganglion (DRG) are primary afferent neurons of the somatosensory, in which the neurites enter the spinal cord to give rise to ascending fibers, which again form synaptic connections with neurons in other sites, such as the thalamus fascicles with neurons in the thalamus. These neurons and ascending fibers play an important role in the transmission of sensation from the periphery to the center. Satellite Glial Cells (SGCs), an important glial cell in peripheral nerves, are widely distributed in Dorsal Root Ganglia (DRGs) and Trigeminal Ganglia (TGs). the satellite glial cells surround 1-2 neurons to form a glial sheath, and a gap junction exists between two adjacent satellite glial cells, and communication between glial cells can be achieved through the gap junction. The culture of DRG-SGC provides a good model for researching the growing and developing mechanism and influencing factors of processes in the development process of a nervous system, how neurons form special synaptic connections, how an extremely complex neural network is formed to execute various physiological functions and the like. Therefore, the establishment of a stable, efficient and high-purity DRG-SGCs in-vitro primary culture model is of great significance.

At present, domestic research mostly focuses on in vitro separation and culture of neurons from DRG, and the common method is to take rat spinal cord dorsal root ganglia, prepare single cell suspension after enzyme digestion, and establish an in vitro dorsal root ganglia single cell culture system. For example, in "purification and culture of dorsal root ganglion neurons in rat spinal cord" of Yuxiadong et al, spinal cord dorsal root ganglia of newborn SD rats were collected and prepared into single cell suspensions by trypsin digestion, cells were purified by addition of cytarabine, and the cells were cultured in DF12 medium containing 10% fetal bovine serum and recombinant human glial cell-derived neurotrophic factor to observe the growth of neurons. Plum full wave et al obtain purified dorsal root ganglion neurons in vitro by methods of digesting DRG with trypsin and EDTA, alternately using DF-12 medium and DF-12 medium added with cytarabine for antimitotic culture and the like in 'model establishment of rat dorsal root ganglion neuron purification culture' and 'primary culture of Tatan Hongyu et al obtain high purity rat dorsal root ganglion neurons in vitro'. Zhang Jun et al in "culture and purification of dorsal root ganglion neurons of embryonic rat", and Suikan et al in "a novel method for primary culture of DRG neurons of rat", digested DRG in 0.25% trypsin solution for 20min (37 ℃), added Fetal Bovine Serum (FBS) to terminate digestion, then made into single cell suspension in DMEM, and removed fibroblasts by differential wall-sticking for 50min to obtain dorsal root ganglion single cell culture of rat. The above method can only isolate and culture neurons in vitro from DRG, but fails to isolate and culture satellite glial cells.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for primary culture of dorsal root ganglion satellite glial cells, which adopts mouse DRG tissues, directly cultures the satellite glial cells without a digestion process, has simple operation, can obtain a large amount of highly purified and stable dorsal root ganglion satellite glial cells, and can be used as an important model for research of developmental neurobiology, clinical neurology and the like.

The technical scheme of the invention is as follows: a method of primary culturing dorsal root ganglion satellite glial cells, the method operating as follows:

disinfecting a newborn mouse or a rat, cutting off the head, killing, taking out a spine after dissection, removing muscles on the spine, cutting off the spine, cleaning blood vessels and the spinal cord under a stereoscopic microscope, taking out a DRG, and removing nerve fibers and capsules on the DRG;

culturing the treated DRG by adopting a DRG-SGCs culture solution, inducing a large amount of satellite glial cells to migrate out of the DRG, carrying out cell passage after the culture is finished, adding trypsin for digestion, then adding FBS for terminating digestion, slightly blowing by adopting a Pasteur blowing-sucking tube, and transferring to a centrifuge tube for centrifugation;

centrifuging, removing supernatant, adding a DRG-SGCs culture solution, and continuously culturing to obtain satellite glial cells;

the DRG-SGCs culture solution comprises DMEM/F12, B27, penicillin-streptomycin double antibody, L-glutamine, BSA (bovine serum albumin), NRG 1-beta 1 (neuregulin), dexamethasone, Insulin, T3 (sodium triiodothyronine) and T4 (tetraiodothyronine).

The obtained high-purity satellite glial cell culture system is beneficial to the research on the biological characteristics and cell functions of specific cells. The dorsal root ganglion contains abundant neurons, satellite glial cells, Schwann cells and fibroblasts, and how to obtain the abundant satellite glial cells is still one of the difficulties in the separation and culture of the dorsal root ganglion.

In order to obtain purer and more uniform satellite glial cells, the dorsal root ganglion is first dissected and then the surface capsule and surrounding nerve fibers are completely stripped, which not only effectively removes fibroblasts and Schwann cells, but also facilitates the growth of the dorsal root ganglion. In the second culturing, L-glutamine is added to the culture medium, and not only can be used as an energy source of cultured cells, but also can be involved in protein synthesis and nucleic acid metabolism, and can inhibit the growth of other cells such as neurons. L-glutamine causes death of mature neurons due to excitotoxicity, and acts to promote growth of glial cells. Serum-free medium in combination with B27 additive inhibits the proliferation of fibroblasts and schwann cells and selectively promotes the survival of satellite glial cells, streptomycin prevents bacterial and fungal contamination during culture, and human recombinant neuregulin is an intercellular signal transduction protein produced by glial cells and neurons in brain tissue, which promotes the survival, migration and proliferation of glial cells. Dexamethasone, triiodothyronine and L-thyroxine are used as hormones to jointly regulate the growth and proliferation of the satellite glial cells, and the purification rate of the satellite glial cells can reach more than 95% by adopting the dissection method and the culture solution.

Preferably, each 50mL of DRG-SGCs culture solution contains 46-48 mL of DMEM/F12, 0.8-1.2 mL of B27, 0.4-0.6 mg of penicillin-streptomycin double antibody, 0.4-0.6 mg of L-glutamine, 15-20 mg of BSA, 1-1.2 ug of NRG 1-beta 1, 1.8-2 ug of dexamethasone, 280-300 ug of Insulin, 0.5-0.6 ug of T3 and 20-24 ug of T4.

Preferably, the newborn mouse or newborn rat is a suckling mouse born for 0-24 hours.

Animals currently used for culture of dorsal root ganglia include chickens, cats and adult mice, while human dorsal root ganglia are rarely used due to medical ethics and limited resources. For the culture of dorsal root ganglia of spinal cord of rat, embryo rat of 14-20 days of pregnancy is adopted, the dorsal root ganglia of embryo period belongs to the stage of more juvenile, and the rat is easy to survive compared with adult rat, but the defect that the number of obtained cells is small, the surface fascia of ganglia is not easy to strip, and the impurity cells such as fibroblasts are more. In the invention, the suckling mouse is preferably newborn within 24 hours, the dorsal root ganglion is mature compared with a fetal mouse, the shown capsule is easy to peel off, the purity of the required cells is higher, and the dorsal root ganglion is still in a juvenile stage and is also easy to obtain the cells. During the dorsal root ganglion separation, care is taken to perform gentle and rapid movements throughout the ice procedure when cleaning the spinal cord and blood vessels within the spine. Separation is difficult due to their depth and small size, while sampling time of dorsal root ganglia is a key contributor to successful culture of glial cells, and viability of cultured cells can only be guaranteed by reducing sampling time.

The invention discloses a specific method for rapidly taking out DRG, which comprises the following steps:

first, the skin (from the caudal vertebra to the cervical vertebra) was cut open along the midline of the back of the newborn/embryonic rat with an ophthalmic scissors, and the skin was carefully separated to both sides with a hairspring forceps, exposing the vertebrae, at which time the contour of the spinal canal was clearly visible. The neonatal/embryonic mouse is then moved under a stereomicroscope and the vertebrae are cut off with an ophthalmic scissors along the spinal canal going from the caudal vertebra to the cervical vertebra, leaving the spinal cord fully exposed. The spinal cord was then gently removed with hairspring forceps, and the remaining meningeal tissue was cleared, at which time the circular or oval ganglia were seen to be fully exposed. Under the stereomicroscope, the ganglia are taken out one by one from the intervertebral foramen by using hairspring forceps.

Further, the processed DRG is inoculated in a six-hole culture plate in the step (2), DRG-SGCs culture solution is contained in the culture plate, 8-11 dorsal root nodes are inoculated in each hole, and then 5% CO is added at 36-38 DEG C₂Culturing in an incubator.

Further, step (2) stops the culture when the treated DRG occupies more than 85% of the bottom of the culture plate along with SGCs migrated from the DRG during the culture process.

Further, in the step (2), after the treated DRG is continuously cultured once by adopting a DRG-SGCs culture solution, the treated DRG is transferred to a new DRG-SGCs culture solution, and the satellite glial cells are induced to migrate out of the DRG again.

Further, after the culture in the step (2) is finished, 2.5% of trypsin is added for digestion for 2-4 min, and then 10% of FBS is added to stop digestion.

Further, the centrifugal rotating speed in the step (2) is 1000-1200 rmp, and the centrifugal time is 5-7 minutes.

The invention obtains the satellite glial cells from the DRG by directly culturing the DRG without a digestion process, the satellite glial cells continuously migrate out from the dorsal root ganglion along with the extension of the culture time, and the cells can be passed when the cells migrate out to a certain number. According to the method, a plurality of DRGs are not required to be digested and cultured, and only one complete DRG is required to be directly placed in the satellite glial cell culture solution provided by the invention for culture, so that a large number of satellite glial cells can be induced to migrate out of the DRG. And placing the DRG in another culture plate containing the satellite glial cell culture solution again, and inducing the satellite glial cells to migrate out of the DRG again. The satellite glial cells cultured by the method have the advantages of high number of migrated satellite glial cells, high purity, stable survival and capability of being passed for multiple generations.

The invention also provides a DRG-SGCs culture solution adopted in the method, wherein the culture solution is suitable for inducing the migration of the satellite glial cells from the DRG without a digestion step.

Compared with the prior art, the invention has the following beneficial effects: the invention simulates the physiological environment in the body of the satellite glial cell, and adopts a DRG tissue primary separation culture method to establish a separation culture system of the DRG-satellite glial cells of the newborn rat, so as to obtain a high-purity DRG-satellite glial cell purification culture system. The cultured and purified DRG-satellite glial cells can survive for a long time in vitro, have high purity, stably survive, can be passaged for multiple generations and form a cell network. The structure of the DRG-satellite glial cell under the in vitro culture condition is similar to that of the DRG-satellite glial cell in vivo, and the DRG-satellite glial cell can be used as an important model for research on developmental neurobiology, clinical neurology and the like.

Drawings

FIG. 1 is a dorsal root ganglion map;

FIG. 2 is a morphological diagram of satellite glial cell emigration from the dorsal root ganglion;

FIG. 3 is a morphological diagram of satellite glial cells after passaging;

FIG. 4 immunofluorescence cytochemistry assay satellite glial cell maps.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to the drawings and specific examples, but the present invention is not limited to the following technical solutions.

Example 1

1 materials and methods

1.1 Experimental animals

SD newborn rats born for 0-24 hours are purchased from the department of laboratory animals of Kunming medical university and approved by the medical ethics committee of Kunming medical university.

1.2 instruments required for DRG extraction

Stereomicroscope, ophthalmology micro straight forceps, ophthalmology micro bent forceps, small scissors, big scissors, flat sharp forceps and big and small hairspring forceps.

1.3 Primary reagents for DRG-SGC culture

DMEM/F12 (1: 1) medium, B-27 additive (50X), diabody (penicillin and streptomycin solution), L-glutamine, BSA (30mg/mL), dexamethasone (25ug/mL), bovine Insulin (5mg/mL) were purchased from biosharp; t3(10ug/mL), T4(400ug/mL), NRG-. beta.1 from abcam, fetal bovine serum, 0.25% pancreatic enzyme from Life Technology, USA; l-lysine and PBS were purchased from Genview, USA;

1.3 isolation of neonatal rat DRG and culture of DRG-SGC

One day old newborn SD rats (purchased from university of Kunming medical laboratory animal school) were killed by decapitation 3-5 minutes after 75% alcohol sterilization, with the rats back upHolding flat sharp forceps with left hand to fix rat, holding scissors with right hand to cut back skin and rib of rat, taking out spine, removing muscle on spine, cutting spine, cleaning blood vessel and spinal cord under stereomicroscope, clamping DRG (see figure 1) with ophthalmic microscopic forceps, removing nerve fiber on DRG and peeling capsule on DRG surface, placing processed DRG into culture dish containing DRG-SGC culture solution (see table 1), inoculating the obtained DRG into six-hole plate, each hole having ten dorsal root segments, placing into 37 deg.C and 5% CO₂Culturing in an incubator.

TABLE 1 DRG-SGC culture fluid formulation

1. A HEPES solution may be added to the medium: is a weak acid, the Chinese name is hydroxyethyl piperazinethiosulfonic acid, and the main function is to prevent the pH of the culture medium from rapidly changing. Under the open culture conditions, the medium was removed from the 5% CO2 environment during cell observation, CO2 gas rapidly escaped, pH rapidly increased, and pH was maintained at about 7.0 with HEPES.

2. The content of BSA added into the culture medium can be changed, and the content of BSA added into the culture medium can be in the range of 15-30 mg.

3. The culture medium can be added with no streptomycin double antibody, except that the first time of taking dorsal root segments from rats for culture, the culture medium can be selectively added with double antibody, and the double antibody can be not added after the culture is carried out for a period of time without pollution.

1.4 subculture and Observation of SGC

And (3) transferring the DRG to another six-hole plate culture dish coated with polylysine when the SGC migrated from the DRG occupies more than 85% of the bottom of the culture dish, adding a culture solution, and putting the culture solution into an incubator for culture. 0.5mL of 0.25% pancreatin was added to each well, digested at 37 ℃ for 3-5 minutes, and the outermost SGC was observed to float under a microscope. Digestion was stopped by adding an equal volume of 10% FBS, and cells were observed to be blown off the plate by gently pipetting 10-15 times with a Pasteur pipette. Transferring the liquid into a 15mL centrifuge tube, centrifuging with a centrifuge of 1200rmp for 5-7After a minute, the supernatant was discarded, and 3mLDRG-SGCs culture medium was added, and the cells were transferred to a 24-well plate (300. mu.l/well) containing a slide and coated. Adding 5% CO at 37 deg.C₂Culturing in an incubator, and changing the culture solution every other day. The morphology and growth of the cells were observed under an inverted microscope and identified as satellite glial cells.

2 results

2.1 growth status of Primary culture of dorsal root ganglion emigration of satellite glial cells

When dorsal root ganglia are cultured in DRG-SGCs culture medium, DRG adheres to the bottom of the petri dish and SGCs migrate out from around the DRG, increasing with the duration of culture. The SGCs are radially distributed around the DRG, with the SGCs having protuberances formed. (see FIG. 2).

In fig. 2, the migration rate of the satellite glial cells from the dorsal root ganglion is higher with the increase of the culture time, and the migration rate and the morphology of the satellite glial cells are observed at 3 days, 7 days, 14 days and 21 days after the culture of the dorsal root ganglion.

2.2 morphology of satellite glial cells after passage (see FIG. 3)

FIG. 3 shows the morphology of the satellite glial cells after passage, with the cells being full and elliptical, with projections on both poles of the cell, and with the projections interconnected.

2.3 identification of satellite glial cells by immunofluorescence cytochemistry

Immunofluorescence staining detects positive expression of the satellite glial markers GS and GFAP after passage. The cells after passage are suggested to be satellite glial cells, and the purity of the cells reaches more than 95%, which is shown in figure 4.

In the first row of FIG. 4, DAPI-labeled cells are nuclei, GS is a specific marker for satellite glial cells, and Merge is the sum of the first two images of the first row; in the second row, DAPI-labeled nuclei of cells, GFAP-labeled glial cells, and Merge-labeled sum of the first two in the second row.

In summary, the experimental results show that the new-born rat can obtain satellite glial cells with higher purity and convenient separation of dorsal root ganglia. The identification test shows that the DRG-SGCs maintain the glial cell morphology positive for GFAP and GS immunofluorescence in vitro. The culture method for culturing the satellite glial cells has the advantages of high purity, simple purified cells, short time and multiple passages of dorsal root nodes. The method for culturing the DRG-SGCs provides a basis for research related to the satellite glial cells and provides a more favorable experimental basis for research of related diseases.

Claims

1. A method for primary culture of dorsal root ganglion satellite glial cells, the method operating as follows:

sterilizing and dissecting dead newborn mice or rats, taking out spines, removing muscles on the spines, cutting the spines, cleaning blood vessels and spinal cords under a stereoscopic microscope, taking out DRGs, and removing nerve fibers and capsules on the DRGs;

culturing the treated DRG by adopting a DRG-SGCs culture solution, inducing the satellite glial cells to migrate out of the DRG, carrying out cell passage after the culture is finished, adding trypsin for digestion, then adding FBS for terminating the digestion, slightly blowing and beating by adopting a Pasteur blowing and sucking tube, and transferring to a centrifuge tube for centrifugation;

centrifuging, removing supernatant, adding a DRG-SGCs culture solution, inoculating the culture solution to a polylysine-coated six-hole culture plate, and continuously culturing to obtain satellite glial cells;

the DRG-SGCs culture solution comprises DMEM/F12, B27, penicillin-streptomycin double antibody, L-glutamine, bovine serum albumin BSA, neuregulin NRG 1-beta 1, dexamethasone, Insulin, triiodothyronine sodium T3 and tetraiodothyronine T4;

each 50mL of DRG-SGCs culture solution contains 46-48 mL of DMEM/F12, 0.8-1.2 mL of B27, 0.4-0.6 mg of penicillin-streptomycin double antibody, 0.4-0.6 mg of L-glutamine, 15-20 mg of BSA, 1-1.2 ug of NRG 1-beta 1, 1.8-2 ug of dexamethasone, 280-300 ug of Insulin, 0.5-0.6 ug of T3 and 20-24 ug of T4.

2. The method of primary culturing dorsal root ganglion satellite glial cells according to claim 1, wherein the neonatal mouse or rat is a suckling mouse born for 0-24 hours.

3. The method for primary culture of dorsal root ganglion glial cells according to claim 1, wherein the DRG treated in step (2) is inoculated into a six-well plate containing a DRG-SGCs culture medium, 8 to 11 dorsal root ganglion cells are inoculated into each well, and then the temperature is 36 to 38 ℃ and 5% CO is added₂Culturing in an incubator.

4. The method of primary culturing dorsal root ganglion glial cells according to claim 1, wherein step (2) comprises stopping culturing the treated DRG during the culturing period when the percentage of glial cells migrated from the DRG in the bottom of the six-well culture plate is more than 85%.

5. The method of primary culture of dorsal root ganglion glial cells according to claim 1, wherein in step (2), the treated DRG is continuously cultured once in the DRG-SGCs culture medium and then transferred to a new DRG-SGCs culture medium, which again induces the migration of glial cells from the DRG.

6. The method of primary culturing dorsal root ganglion satellite glial cells according to claim 1, wherein the digestion is terminated by adding 2.5% trypsin for 2-4 min after the culture in step (2) is completed, and then adding 10% FBS to terminate the digestion.

7. The method for primary culture of dorsal root ganglion glial cells according to claim 1, wherein the centrifugation in step (2) is performed at a rotation speed of 1000 to 1200rmp for 5 to 7 minutes.

8. A culture of DRG-SGCs for use in a method according to any one of claims 1 to 7.