CN112877282A - Method for culturing primary neuromuscular junction in vitro - Google Patents

Method for culturing primary neuromuscular junction in vitro Download PDF

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CN112877282A
CN112877282A CN202110180213.7A CN202110180213A CN112877282A CN 112877282 A CN112877282 A CN 112877282A CN 202110180213 A CN202110180213 A CN 202110180213A CN 112877282 A CN112877282 A CN 112877282A
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于舒
蔡二改
贺倩茹
孙俊杰
丁斐
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Abstract

The invention provides a method for culturing primary neuromuscular junctions in vitro, which comprises the steps of firstly obtaining primary spinal motoneurons and primary myoblasts, and inoculating the two cells into different chambers of a microfluid device. Different culture conditions are adopted for respective culture, the axon of the motor neuron is promoted to grow to a skeletal muscle cell cavity through a microchannel by utilizing fluid difference, and the axon is contacted with the skeletal muscle cell to form mature NMJ, so that the space-time control of the cell is realized, and the research on the local activities of the cell body, the axon and the NMJ of the motor neuron is facilitated. The present invention uses primary spinal cord motor neurons and primary myoblasts that better mimic the physiological tissues of the intact spinal cord and skeletal muscle. The mature and functional neuromuscular junction model can be established under the co-culture condition, is closer to the in vivo condition, provides an effective and measurable system for the in vitro biological research of NMJ, and has good application in the aspects of drug discovery, individualized medicine and disease pathology exploration.

Description

Method for culturing primary neuromuscular junction in vitro
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for culturing a primary neuromuscular junction in vitro and the like. The invention is useful for studying cellular and molecular mechanisms involved in motor neuron and muscle communications biology, including neuronal stimulation of axonal growth and guidance, synapse formation and muscle contraction activity, among others.
Background
Motor Neurons (MNs) extend axons long distances in different extracellular microenvironments, forming synapses with muscles-neuromuscular junctions (NMJ). NMJ formation and maintenance is dependent on internal and external signals that need to be coupled spatio-temporally with specificity and fidelity. This MN muscle communication is crucial for the formation and maintenance of NMJ, as well as for the survival and normal function of MN. This change in cell-cell communication can lead to synaptic disruption and axonal formation, a critical step in neurodegeneration. This two-way communication process is accomplished by both adhesion and secretion factors and is mediated through a ligand-receptor mechanism. However, the nature of these signals and the specific molecular mechanisms that regulate NMJ structure/function remain to be further understood. In particular, the mechanism of retrograde and antegrade signaling between neurons and muscles is poorly understood. Many of the difficulties in deciphering these mechanisms are due to the technical challenges of studying these complex intracellular and intercellular communications at the sub-cellular level.
The biggest challenge in the in vitro modeling of NMJ is the complexity of culturing two different types of cells, spinal motor neurons and skeletal muscle cells, obtained from different environments. The complexity of the two cell types, which usually require different culture conditions (media, growth and differentiation factors) when cultured separately, makes it difficult to establish NMJ co-culture models in vitro. For example, the different growth characteristics of muscle and neurons require suitable media that should allow for neuronal growth and muscle differentiation. Therefore, researchers can only obtain NMJ in vitro by the following method: inducing stem cell or transgenic cell in vitro, differentiating into motor neuron and skeletal muscle cell, and co-culturing to form NMJ; ② adopting motor neuron-like hybrid cell line (NSC-34) or myoblast cell line (C2C12, L6) to carry out in vitro induction and differentiation into motor neurons and skeletal muscle cells, and then co-culturing to form NMJ. However, the stem cell induction process is complex, the time is long, and most of various stem cell induction factors are expensive, so that the first method is difficult to popularize. Whereas cell lines are a population of monoclonal cells. Cell lines can only be regarded as living experimental materials with or retaining certain characteristics, and compared with primary culture cells, the cell lines have poor biological characteristics and cannot completely simulate the inherent structure of tissues. In addition, most studies are performed on static media that do not reproduce the dynamic flow conditions encountered in biological systems, lacking precise control over the cellular microenvironment (including diffusion and fluid flow). Therefore, there is a need to develop improved models of NMJ to overcome these problems.
Disclosure of Invention
The invention mainly aims to provide a microfluid primary spinal motor neuron-skeletal muscle cell co-culture model to solve the problem that a primary neuromuscular junction in the prior art cannot be established.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of culturing primary neuromuscular junctions in vitro comprising the steps of: obtaining primary myoblasts, inoculating the primary myoblasts into a porous culture plate coated by polylysine, performing myoblast differentiation culture, obtaining primary spinal cord motor neurons after the myoblasts are differentiated into mature myocytes, inoculating the primary spinal cord motor neurons into the porous culture plate, and continuously culturing to form mature primary neuromuscular junctions.
Further, the primary myoblasts and primary spinal motoneurons were taken from SD rats; the steps for obtaining primary myoblasts are as follows: newborn 1-day SD rats are sterilized by 75% ethanol and killed by decapitation; collecting limbs under sterile condition, removing skin, collecting gastrocnemius muscle, removing connective tissue such as fat and tendon, washing the tissue with 4 deg.C pre-cooled L15 culture medium for 3 times, and cutting the muscle tissue into 1mm pieces3Transferring the left and right fragments into a centrifuge tube, centrifuging at 1000rpm for 2min, removing supernatant, and repeating the step for 3 times to remove supernatant and floating tissues; followed by the addition of 0.1% type I collagenDigesting with enzyme for 25min, shaking for several times, discarding collagenase, digesting with 0.25% trypsin for 55min, shaking for several times, adding DMEM complete culture medium to terminate digestion, repeatedly beating, sucking supernatant, repeating the steps for three times, collecting all supernatants, centrifuging at 1000rpm for 5min, resuspending the DMEM complete culture medium, and filtering with 200-mesh and 400-mesh sieves respectively; collecting filtrate, centrifuging at 1000rpm for 10min, discarding supernatant, and completely culturing with DMEM to resuspend cells; the cell suspension was added to a petri dish without polylysine treatment and placed at 37 ℃ with 5% CO2Standing in an incubator for 60-80 min, gently shaking myoblasts which are not adhered to the wall, collecting the myoblasts into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding supernatant, and adjusting the cell density to 1.0 multiplied by 10 by a DMEM complete culture medium5Perml, 500. mu.l of cell suspension was inoculated into a polylysine-coated 24-well plate and placed at 37 ℃ in 5% CO2After the cell density reaches 70% -80% after about 3-4 days, changing into a myocyte differentiation culture medium to continue culturing for 5-7 days;
the method for obtaining the primary spinal cord motor neurons comprises the following steps: embryo 13.5 day SD rat, killed by cervical dislocation under anesthesia; after 75% ethanol disinfection, quickly taking out the embryo, and transferring the embryo to an L15 culture medium; under a dissecting microscope, separating vertebrae, obtaining spinal cords, digesting for 0.5h at 37 ℃ by using 0.125% trypsin, adding a complete culture medium (DMEM + 10% FBS + 1% PS) after digestion is finished, stopping digestion, blowing and beating the tissues for 10 times, collecting supernatant, blowing and beating for three times in the same way until all tissue blocks disappear, collecting all supernatant, centrifuging for 5min at 1100rpm, slightly suspending cell precipitates in an L15 culture medium, and filtering by using a 200-mesh screen to obtain a single cell suspension; an equal volume of density gradient centrifugate (4.165 ml) was added
Figure BDA0002941238600000031
-E Medium +0.75ml OptiPrepTM+0.085ml of B27); after centrifugation at 2000rpm for 20min, the intermediate phase was extracted and mixed with an equal volume of complete medium; after centrifugation at 1100rpm for 6min, the cells were resuspended in complete medium.
Further, inoculating the primary spinal motor neurons into the porous culture plate, and continuing to cultureThe cultivation steps are as follows: adjusting primary spinal motor neuron density to 2.0 × 105Perml, 500. mu.l were inoculated into a 24-well myocyte culture plate and placed at 37 ℃ in 5% CO2After the cells are attached to the wall, the cells are replaced by a neuron growth medium (NB + 2% B27+ 1% Gluttamax + 1% PS +5ng/ml BDNF) for another 7 days to form mature primary neuromuscular junctions.
A method for culturing primary neuromuscular junctions in vitro based on a microfluidic device, comprising the steps of: obtaining primary spinal motor neurons and primary myoblasts, inoculating the two cells into two different chambers of a microfluid device, respectively culturing by adopting different culture conditions, and promoting the motor neurons and skeletal muscle cells to be connected through axons in microchannels by utilizing fluid difference, thereby establishing the microfluid primary neuromuscular junction.
Further, the primary spinal motor neurons and primary myoblasts were taken from SD rats; after primary myoblast inoculation, 5% CO at 37 ℃2After culturing for 1h in the incubator, adding 200 mul of myocyte complete culture medium into each hole on the side of the microfluidic myocyte, changing to myocyte differentiation culture medium on the 2 nd day, wherein the volume is 180 mul, changing the liquid once every day later, wherein the myocyte differentiation culture medium is the same, but the liquid volume is changed, 180 mul is used on the 2 nd to 3 rd day, and 200 mul is used on the 4 th to 7 th day;
primary spinal motoneuron inoculation was followed by 5% CO at 37 deg.C2After 1 hour of culture in the incubator, 200. mu.l of the complete neuron culture medium was added to each well, and after 4 hours, 200. mu.l of the neuron growth medium was changed, and after every day, the medium was changed, but the volume of the medium was changed, 200. mu.l on days 2 to 3, 180. mu.l on days 4 to 5, and 200. mu.l on days 6 to 7.
Further, the muscle cell complete medium is DMEM + 20% FBS + 1% PS; the muscle cell differentiation medium is DMEM + 2% HS + 1% PS +10nM insulin +20ng/ml BDNF. High concentration of FBS (20% FBS) in complete medium favors myoblast adherence, while low concentration of HS (2% HS) and insulin in differentiation medium favors myoblast differentiation to mature myocytes. In addition, high concentrations of BDNF in the differentiation medium (20ng/ml BDNF) favoured recruitment of motor neuron axons in the microchannel to the myocyte compartment growth extension in the presence of fluid differences.
Further, the complete neuron culture medium is DMEM + 10% FBS + 1% PS; the neuron growth medium is NB + 2% B27+ 1% Gluttamax + 1% PS +5ng/ml BDNF. 10% FBS in complete medium favors neuronal anchorage, while low concentrations of BDNF in growth medium (5ng/ml BDNF) favor motor neuron growth and neurite extension.
Has the advantages that: the invention obtains primary spinal motor neurons and primary myoblasts, and inoculates the two cells in two different chambers of a microfluidic device. Different culture conditions are adopted for respective culture, and the fluid difference is utilized to promote motor neurons and skeletal muscle cells to be connected through axons in microchannels, so that the space-time control of the cells is realized, and the study on the local activities of cell bodies, axons and NMJ is facilitated. The present invention uses primary spinal motor neurons and primary myoblasts that are easier to extract and better mimic the physiological tissues in the intact spinal cord and muscles. The mature and functional neuromuscular junction model can be established under the co-culture condition, is closer to the in vivo condition, provides an effective and measurable system for the in vitro biological research of NMJ, and has good application in the aspects of drug discovery, individualized medicine and disease pathology exploration.
Drawings
FIG. 1 is an image under a light microscope after primary myoblasts were cultured for 5 days with myocyte differentiation medium;
FIG. 2 is an image under a light microscope of primary myoblasts seeded and co-cultured with primary spinal motoneurons for 7 days after differentiation into mature skeletal muscle cells;
FIG. 3 is a NMJ structural diagram formed after 7 days of coculture of primary skeletal muscle cells and primary spinal cord motoneurons, and after the coculture of the neuromuscular markers, beta-III tubulin, a neuron marker, and alpha-bungarotoxin (alpha-BTX), a postsynaptic membrane AchR specific binder, using immunofluorescence staining techniques.
FIG. 4 is a schematic view of the structure of a microfluidic device in example 2;
fig. 5 is a schematic diagram of the flow pattern of a microfluidic NMJ growth liquid phase;
FIG. 6 is an image under a light mirror of motor neuron axons passing through microchannels to myocyte compartments in contact with myocytes after 7 days of co-culture of primary spinal motor neurons and primary myoblasts in a microfluidic device;
FIG. 7 is a representation of NMJ structure formed after 7 days of co-culture of primary spinal motoneurons and primary myoblasts in a microfluidic device, following the labeling of the neuromuscular co-culture with the neuronal marker β -III tubulin, the postsynaptic sarcolemum AchR specific binder α -bungarotoxin (α -BTX) using immunofluorescence staining techniques (panel b is a partial enlargement of panel a).
Detailed Description
The invention is further illustrated by the following examples and figures.
The microfluidic device used in The present invention (The microfluidic chamber, XONA 2-component SND 150) was purchased from Xona Microfluidics LLC, USA. The device is a culture system made of optically transparent Polydimethylsiloxane (PDMS), and is provided with bilateral culture chambers which are communicated by a microchannel with the length of 150 microns, and the microchannel can only allow the neuron axon to pass through and is suitable for the separation culture of the neuron soma and the axon.
Example 1:
1) isolation culture of primary myoblasts
Newborn 1 day sd (sprague dawley) rats were sterilized with 75% ethanol and sacrificed by decapitation. Collecting limbs under sterile condition, removing skin, collecting gastrocnemius muscle, removing connective tissue such as fat and tendon, washing the tissue with 4 deg.C pre-cooled L15 culture medium for 3 times, and cutting the muscle tissue into 1mm pieces3The left and right pieces were transferred to a centrifuge tube, centrifuged at 1000rpm for 2min, the supernatant removed, and the procedure repeated 3 times to remove supernatant and floating tissue. Adding 0.1% type I collagenase, digesting for 25min while shaking for several times, discarding collagenase, digesting for 55min with 0.25% trypsin while shaking for several times, adding DMEM complete culture medium (DMEM + 20% FBS + 1% PS) to stop digestion, repeatedly beating, sucking supernatant, repeating the steps for three times, collecting all supernatants, and washing with 1000r collagenasepm centrifugation for 5min, DMEM complete medium heavy suspension, respectively using 200 and 400 mesh screen filtration. The filtrate was collected, centrifuged at 1000rpm for 10min, the supernatant was discarded and the cells were resuspended in complete medium. The cell suspension was added to a petri dish without polylysine treatment and placed at 37 ℃ with 5% CO2Standing in an incubator for 60-80 min, gently shaking myoblasts which are not adhered to the wall, collecting the myoblasts into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding supernatant, and adjusting the cell density to 1.0 multiplied by 10 by using complete culture medium5Perml, 500. mu.l of cell suspension was inoculated into a polylysine-coated 24-well plate and placed at 37 ℃ in 5% CO2After the cell density reaches 70% -80% after about 3-4 days, the cell is changed into a myocyte differentiation culture medium (DMEM + 2% HS + 1% PS +10nM insulin) to continue culturing for 5-7 days.
2) Isolation and co-culture of primary spinal motor neurons with primary myocytes
Embryos day 13.5 SD rats were sacrificed by cervical dislocation under anesthesia. After 75% ethanol sterilization, embryos were quickly removed and transferred to L15 medium. Under a dissecting microscope, vertebrae were separated, spinal cords were obtained, digested with 0.125% trypsin at 37 ℃ for 0.5h, after completion of digestion, complete medium (DMEM + 10% FBS + 1% PS) was added to terminate digestion, tissues were blown 10 times, supernatants were collected, and blown three times in this manner until all tissue mass disappeared, all supernatants were collected, after centrifugation at 1100rpm for 5min, cell pellets were gently suspended in L15 medium and filtered with a 200 mesh screen to obtain a single cell suspension. An equal volume of density gradient centrifugate (4.165 ml) was added
Figure BDA0002941238600000061
-E Medium +0.75ml OptiPrepTM+0.085ml of B27). After centrifugation at 2000rpm for 20min, the intermediate phase was extracted and mixed with an equal volume of complete medium. After centrifugation at 1100rpm for 6min, the cells were resuspended in complete medium and the cell density adjusted to 2.0X 105Perml, 500. mu.l were inoculated into a 24-well myocyte culture plate and placed at 37 ℃ in 5% CO2After the cells are attached to the wall for about 4 hours, the cells are replaced by a neuron growth medium (NB + 2% B27+ 1% Gluttamax + 1% PS +5ng/ml BDNF), and the cells are cultured for 7 daysMature NMJ is formed.
Example 2:
1) isolation of primary skeletal muscle cells and plating of the muscle cell side of microfluidics
Newborn 1 day sd (sprague dawley) rats were sterilized with 75% ethanol and sacrificed by decapitation. Collecting limbs under sterile condition, removing skin, collecting gastrocnemius muscle, removing connective tissue such as fat and tendon, washing the tissue with 4 deg.C pre-cooled L15 culture medium for 3 times, and cutting the muscle tissue into 1mm pieces3The left and right pieces were transferred to a centrifuge tube, centrifuged at 1000rpm for 2min, the supernatant removed, and the procedure repeated 3 times to remove supernatant and floating tissue. Adding 0.1% type I collagenase for digestion for 25min, shaking for several times, discarding collagenase, digesting with 0.25% trypsin for 55min, shaking for several times, adding DMEM complete culture medium (DMEM + 20% FBS + 1% PS) to stop digestion, repeatedly blowing, sucking supernatant, repeating the steps for three times, collecting all supernatants, centrifuging at 1000rpm for 5min, resuspending the DMEM complete culture medium, and filtering by using 200-mesh and 400-mesh sieves respectively. The filtrate was collected, centrifuged at 1000rpm for 10min, the supernatant was discarded and the cells were resuspended in complete medium. The cell suspension was added to a petri dish without polylysine treatment and placed at 37 ℃ with 5% CO2Standing in an incubator for 60-80 min, gently shaking myoblasts which are not adhered to the wall, collecting the myoblasts into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding supernatant, and adjusting the cell density to 800 multiplied by 10 by complete culture medium4Perml, 10. mu.l of cell suspension was seeded on the right side (myocyte side) of polylysine-coated microfluidics at 37 ℃ with 5% CO2Cultured in an incubator.
2) Isolation of primary spinal motor neurons and inoculation of microfluidic motor neuron side
Embryos day 13.5 SD rats were sacrificed by cervical dislocation under anesthesia. After 75% ethanol sterilization, embryos were quickly removed and transferred to L15 medium. Dissecting under microscope, separating vertebrae, obtaining spinal cord, digesting with 0.125% trypsin at 37 deg.C for 0.5 hr, adding complete culture medium (DMEM + 10% FBS + 1% PS) to terminate digestion, blowing tissue for 10 times, collecting supernatant, blowing for three times until all tissue blocks disappear, collecting all supernatant, centrifuging at 1100rpm for 5min, and collecting supernatantThe cell pellet was gently suspended in L15 medium and filtered through a 200 mesh screen to obtain a single cell suspension. An equal volume of density gradient centrifugate (4.165 ml) was added
Figure BDA0002941238600000071
-E Medium +0.75ml OptiPrepTM+0.085ml of B27). After centrifugation at 2000rpm for 20min, the intermediate phase was extracted and mixed with an equal volume of complete medium. After centrifugation at 1100rpm for 6min, the cells were resuspended in complete medium and cell density adjusted to 3000X 104Mu.l were inoculated into the left side of polylysine-coated microfluidics (motor neuron side) at 37 ℃ with 5% CO2Cultured in an incubator.
3) Primary skeletal muscle cell and motor neuron microfluid coculture
After primary myoblast inoculation, 5% CO at 37 ℃2After 1h of culture in the incubator, 200. mu.l of myocyte complete medium (DMEM + 20% FBS + 1% PS) was added to each well of the microfluidic myocyte side, and the cell was replaced with myocyte differentiation medium (DMEM + 2% HS + 1% PS +10nM insulin +20ng/ml BDNF) at day 2 in a volume of 180. mu.l. The culture medium was changed every day, and the culture medium was muscle cell differentiation medium. To create a fluid differential in the microfluidic device to promote recruitment of motor neuron axons on the myocyte side, the fluid volume was 180 μ l on days 2-3 and 200 μ l on days 4-7.
After primary motoneuron inoculation, 5% CO was added at 37 deg.C2After 1 hour of culture in the incubator of (1), 200. mu.l of a neuron complete medium (DMEM + 10% FBS + 1% PS) was added to each well, and after 4 hours, the medium was changed to a neuron growth medium (NB + 2% B27+ 1% Gluttamax + 1% PS +5ng/ml BDNF), and then the medium was changed once a day, and the medium was a neuron growth medium, wherein the volume of the medium was 200. mu.l for days 2 to 3, 180. mu.l for days 4 to 5, and 200. mu.l for days 6 to 7.
As a result:
1. after the primary myoblasts are cultured for 5 days by using a myocyte differentiation medium, a considerable part of the cells are proliferated, fused with each other and gradually arranged in parallel along one direction in a regular way under a light microscope, and are fused into multinuclear myotubes, wherein the myotubes are smooth and long-strip-shaped (figure 1, the scale is 50 mu m).
2. After differentiation of primary myoblasts into mature skeletal muscle cells, primary spinal motoneurons were seeded and co-cultured for 7 days, with the result that star, cone or sphere motor neurons (black arrows) were visualized under the light microscope, and long processes were emitted from the neuronal cell bodies, with a portion of the processes spanning the skeletal muscle myotubes (white arrows) (fig. 2, scale 20 μm).
3. After primary skeletal muscle cells and primary spinal cord motor neurons are co-cultured for 7 days, an immunofluorescence staining technology is adopted, and a synapse structure formed after the neuromuscular co-culture is identified by using a neuron marker beta-III tubulin and a postsynaptic muscle membrane AchR specific conjugate alpha-bungarotoxin (alpha-BTX). As a result, it was found that motor neuron processes extended and contacted myotubes to form mature neuromuscular junctions (fig. 3, scale 25 μm).
4. After 7 days of microfluidic co-culture, the soma and dendrites of the motor neurons were visualized under a light microscope on the motor neuron side of the microfluidics, primary skeletal muscle cells on the muscle cell side of the microfluidics, a microfluidic 150 μm microchannel was visualized in the middle of the visual field, and part of the motor neuron axons passed through the microchannel to the muscle cell side and contacted with the muscle cells (fig. 6, scale 50 μm).
5. After 7 days of microfluidic co-culture, the synaptic structure formed after neuromuscular co-culture is identified by using an immunofluorescence staining technique and a neuron marker beta-III tubulin and a postsynaptic muscle membrane AchR specific binding substance alpha-bungarotoxin (alpha-BTX). The results showed that there were a large number of motor neuron axons in the field near the microchannel, and these axons that passed through the microchannel to the muscle cell side contacted the skeletal muscle cells to form a mature neuromuscular junction (fig. 7, panel b is a partial enlargement of panel a, panel a scale 250 μm, panel b scale 75 μm).
6. After the micro-fluid co-culture is carried out for 7 days, the living cell real-time observation technology is adopted, the arrangement rule and the arrangement with the consistent inclination direction of myotubes in the visual field close to the micro-channel are found, and the stable and rhythmic contraction phenomenon appears on part of myotubes under the condition of no stimulation, so that the muscle innervated by nerves has good contraction function.
7. After the micro-fluid co-culture is carried out for 7 days, the primary myocytes at the myocyte side of the micro-fluid are firstly incubated for 30min at 37 ℃ by adopting an intracellular calcium ion fluorescent probe Fluo-4(2.0 mu M), then 200mM glutamic acid is added at the neuron side of the micro-fluid to induce the excitability of the neurons, and the change of the calcium ion concentration in skeletal muscle cells at the myocyte side of the micro-fluid is observed by adopting a fluorescent living cell real-time observation technology. The results indicate that glutamate-induced neuronal excitability can be transmitted to skeletal muscle cells through the neuromuscular junction, suggesting maturation of neuromuscular junction function in this model.
All of the above culture conditions were invented for SD rat spinal cord motor neurons, myoblast differentiation culture and XONA 2-component SND 150 microfluidic devices, and if the cell type or microfluidic device is changed, the culture conditions will change accordingly.
The invention designs culture conditions suitable for differentiation culture of spinal motor neurons and myoblasts of SD rats, and controls the direction of fluid difference by utilizing the liquid volume difference in bilateral culture chambers of a microfluidic device at different periods of axon extension, thereby recruiting motor neuron axons and maintaining the formation and maturation of NMJ. Without the combined effects of these conditions, two cells cannot be connected by axons in microchannels.

Claims (8)

1. A method for culturing primary neuromuscular junctions in vitro is characterized by comprising the following steps: obtaining primary myoblasts, inoculating the primary myoblasts into a porous culture plate coated by polylysine, performing myoblast differentiation culture, obtaining primary spinal cord motor neurons after the myoblasts are differentiated into mature myocytes, inoculating the primary spinal cord motor neurons into the porous culture plate, and continuously culturing to form mature primary neuromuscular junctions.
2. The method of claim 1, wherein the primary myoblasts and primary spinal motoneurons are obtained from SD rats; the steps for obtaining primary myoblasts are as follows: newborn 1-day SD rat, 75% BSterilizing with alcohol, and killing broken ends; collecting limbs under sterile condition, removing skin, collecting gastrocnemius muscle, removing connective tissue such as fat and tendon, washing the tissue with 4 deg.C pre-cooled L15 culture medium for 3 times, and cutting the muscle tissue into 1mm pieces3Transferring the left and right fragments into a centrifuge tube, centrifuging at 1000rpm for 2min, removing supernatant, and repeating the step for 3 times to remove supernatant and floating tissues; adding 0.1% type I collagenase for digestion for 25min, shaking for several times, discarding collagenase, then digesting for 55min with 0.25% trypsin, shaking for several times, adding DMEM complete culture medium to stop digestion, repeatedly blowing, sucking supernatant, collecting all supernatants for three times, centrifuging for 5min at 1000rpm, resuspending the DMEM complete culture medium, and filtering by using 200-mesh and 400-mesh screens respectively; collecting filtrate, centrifuging at 1000rpm for 10min, discarding supernatant, and completely culturing with DMEM to resuspend cells; the cell suspension was added to a petri dish without polylysine treatment and placed at 37 ℃ with 5% CO2Standing in an incubator for 60-80 min, gently shaking myoblasts which are not adhered to the wall, collecting the myoblasts into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding supernatant, and adjusting the cell density to 1.0 multiplied by 10 by a DMEM complete culture medium5Perml, 500. mu.l of cell suspension was inoculated into a polylysine-coated 24-well plate and placed at 37 ℃ in 5% CO2Culturing in an incubator for about 3-4 days until the cell density reaches 70-80%, and replacing a myocyte differentiation culture medium for continuous culture for 5-7 days;
the method for obtaining the primary spinal cord motor neurons comprises the following steps: embryo 13.5 day SD rat, killed by cervical dislocation under anesthesia; after 75% ethanol disinfection, quickly taking out the embryo, and transferring the embryo to an L15 culture medium; under a dissecting microscope, separating vertebrae, obtaining spinal cords, digesting for 0.5h at 37 ℃ by using 0.125% trypsin, adding a complete culture medium to terminate digestion after digestion is completed, blowing and beating the tissues for 10 times, collecting supernatant, blowing and beating for three times in this way until all tissue blocks disappear, collecting all supernatant, centrifuging for 5min at 1100rpm, gently suspending cell precipitates in an L15 culture medium, and filtering by using a 200-mesh screen to obtain a single-cell suspension; adding an equal volume of density gradient centrifugate; after centrifugation at 2000rpm for 20min, the intermediate phase was extracted and mixed with an equal volume of complete medium; after centrifugation at 1100rpm for 6min, the cells were resuspended in complete medium.
3. The method of claim 2, wherein said myocyte completion medium is DMEM + 20% FBS + 1% PS; the muscle cell differentiation medium is DMEM + 2% HS + 1% PS +10nM insulin; the complete neuron culture medium is DMEM + 10% FBS + 1% PS; the density gradient centrifugate is 4.165ml
Figure FDA0002941238590000021
-E Medium +0.75ml OptiPrepTM+0.085ml B27。
4. The method of claim 2, wherein primary spinal motor neurons are seeded into the multi-well culture plate and the culturing is continued by the steps of: adjusting primary spinal cord motor neuron density to 2.0 × 10 using complete medium5Perml, 500. mu.l were inoculated into a 24-well myocyte culture plate and placed at 37 ℃ in 5% CO2After the cells are attached to the wall, the cells are replaced by a neuron growth medium which is NB + 2% B27+ 1% Glutamax + 1% PS +5ng/ml BDNF for about 4 hours, and the cells are continuously cultured for 7 days to form a mature primary neuromuscular junction.
5. A method for culturing primary neuromuscular junctions in vitro based on a microfluidic device is characterized by comprising the following steps: obtaining primary spinal cord motor neurons and primary myoblasts, inoculating the two cells into two different chambers of a microfluid device, respectively culturing by adopting different culture conditions, and promoting the motor neurons and skeletal muscle cells to be connected through axons in microchannels by utilizing fluid difference, thereby establishing a microfluid primary neuromuscular junction model.
6. The method of claim 5, wherein the primary spinal cord is transported by the primary neuromuscular junction in vitro using a microfluidic deviceThe motor neurons and primary myoblasts were obtained from SD rats; after primary myoblast inoculation, 5% CO at 37 ℃2After culturing for 1h in the incubator, adding 200 mul of myocyte complete culture medium into each hole on the side of the microfluidic myocyte, changing to myocyte differentiation culture medium on the 2 nd day, wherein the volume is 180 mul, changing the liquid once every day later, wherein the myocyte differentiation culture medium is the same, but the liquid volume is changed, 180 mul is used on the 2 nd to 3 rd day, and 200 mul is used on the 4 th to 7 th day;
primary spinal motoneuron inoculation was followed by 5% CO at 37 deg.C2After 1h of culture in the incubator, 200. mu.l of neuron complete culture medium is added to each well on the side of the microfluidic neuron, 200. mu.l of neuron growth medium is replaced after 4h, and then the liquid is replaced once a day, wherein the neuron growth medium is obtained, but the liquid volume is changed, 200. mu.l is obtained from 2 days to 3 days, 180. mu.l is obtained from 4 days to 5 days, and 200. mu.l is obtained from 6 days to 7 days.
7. The method of claim 6, wherein the myocyte completion medium is DMEM + 20% FBS + 1% PS; the muscle cell differentiation medium is DMEM + 2% HS + 1% PS +10nM insulin +20ng/ml BDNF.
8. The method of claim 6, wherein the neuronal complete medium is DMEM + 10% FBS + 1% PS; the neuron growth medium is NB + 2% B27+ 1% Gluttamax + 1% PS +5ng/ml BDNF.
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