KR101846732B1 - In vitro cultivation method for growing neuron - Google Patents

Abstract

The present invention relates to an apparatus and a method for culturing neurons in vitro. The apparatus according to the present invention treats nerve regeneration-associated factors or drugs to culture the neurons in which effects related to neuronal cell growth and nerve regeneration are easily evaluated in the vertical phase rather than in a plane phase, and thus axons of neurons are allowed to grow regularly in the measurable direction, thereby enabling the evaluation of the nerve regeneration-associated factors or drugs to be accurately performed. In addition, since the length of a nerve conduit according to the present invention can be manufactured in various sizes, it is possible to prepare a PDMS device and an in vitro cultivation method for neurons with various lengths.

Description

In vitro cultivation method for growing neuron < RTI ID = 0.0 >

The present invention relates to a method for culturing neurons in vitro .

If the peripheral nerve is injured by trauma, a method of direct segmentation of the severed nerve sections is performed. However, it is almost impossible to directly associate most of the nerves with each other. In cases where direct anastomosis is impossible, autologous nerve grafting is performed to restore the function. However, in the case of autologous nerve grafting, there is a disadvantage that it is difficult to match the size and shape of the nerve tissue to the damaged nerve tissue and the shape of the nerve tissue to be implanted, and there is a limitation in the harvestable nerve, have. Therefore, nerve conduit is used as a way to restore its function when a nerve defect site develops.

The nerve conduit connects both ends of the deficient nerve and acts as a pathway for nerve regeneration. It fixes both ends of the severed nerve in the nerve conduit and induces the connection of the nerve into the conduit. The use of nerve conduit can prevent penetration of scar tissue that interferes with nerve regeneration, can induce the direction of nerve regeneration in the right direction, keeps nerve regeneration substances secreted from the nerve itself in the conduit, Which can be shut off from the outside.

On the other hand, the dorsal root ganglion (DRG) is a mass of nerve cells with a diameter of 500 μm on the 300th stage, which facilitates the evaluation of effects related to nerve growth or nerve regeneration by treating factors or drugs related to nerve regeneration. However, as shown in FIG. 1, when the cells are cultured on the same plane as the cell culture dish, the axons of the DRG neurons are irregularly grown in an unmeasurable direction.

Korean Patent Publication No. 10-2014-0050900

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for culturing neurons in vitro using a porous neural duct having a microporous structure together with a microchannel.

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(A) weight / volume% (w / w) of PLGA and TG, which means the weight (g) of polylactic acid-glycolic acid copolymer (PLGA) which is a hydrophobic polymer which dissolves in 1 L of tetraglycol (TG) v%) is 10 to 40%, and the central portion of the capillary glass tube is heated to form a bottleneck point, thereby forming an upper channel and a lower channel inclined at discontinuous angles, Channel and a plurality of water-soluble glass fibers having a diameter of 10 to 20 占 퐉 are inserted axially densely in the upper channel of the glass tube so that the lower channel of the glass tube is immersed in the PLGA-TG solution, The PLGA-TG solution is allowed to penetrate into the gaps between the glass fibers, and the glass fiber infiltrated with the PLGA-TG solution is separated from the glass tube and completely immersed in the ultra-pure water DW The glass fiber is melted and the PLGA is cured in the glass fiber-dissolved space to form a plurality of microchannels having a diameter of 10 to 20 탆, and the PLGA-TG solution is immersed in the DW The TG reacts with the DW to form micropores in the microchannels. The microchannels formed with the micropores are frozen with liquid nitrogen and then cut and molded to form a nerve conduit having a diameter of 1.6 to 1.7 mm. Lt; RTI ID = 0.0 > a < / RTI > (B) inserting the nerve conduit into a PDMS chamber made of polydimethylsiloxane (PDMS), allowing the agarose completely dissolved in water to cool, and then penetrating into the space between the PDMS chamber and the nerve conduit so that the agarose is cured Preparing a PDMS device, preparing a multi-channel peristaltic pump on the PDMS device, connecting the PDMS device to a peristaltic pump with a tube, and connecting a media supply container to a lower portion of the PDMS device; (C) injecting neuron into a culture medium in the medium supply container; (D) supplying a culture medium in the culture medium supply container to the PDMS apparatus having the nerve conduit inserted thereto at a flow rate of 30 to 60 μl / min using the peristaltic pump to transfer neurons having a diameter of 300 to 500 μm to the nerve conduit A neuronal cell seeding step of seeding at the top; And (E) mixing the factor or medicament related to nerve regeneration with the culture medium in the medium supply container using the peristaltic pump, and then mixing the mixed culture medium with the neuron-seeded PDMS device at a rate of 5 to 20 μl / min And culturing the neuron so that the axon of the neuron grows downward by flowing the culture medium from the upper end to the lower end of the nerve conduit by gravity, Nerve cell in vitro culture method.
At this time, in the step (B), the T-flask provided with the filter cap is used as the culture medium supply container, so that circulation of the air is maintained, but it is prevented from being contaminated from the outside.
In addition, the factor relating to the nerve regeneration is characterized by being selected from the group consisting of nerve growth factor (NGF), neurotrophic factor (NTF).
The in vitro culture method of neurons according to the present invention may be that the axons of neurons grow along the microchannels inside the nerve conduit by seeding and culturing the neurons in the apparatus in which the neuron is inserted.

The present invention relates to a method of treating neuronal regeneration-associated factors or drugs to facilitate the evaluation of neurogenesis or neurogenesis-related effects by culturing neurons in a vertical phase rather than a plane, , The evaluation of the effect of the nerve regeneration-related factor or the drug can be accurately performed.
In addition, since the lengths of the neural ducts according to the present invention can be manufactured in various sizes, PDMS devices and nerve cell in vitro culture devices of various lengths can be prepared.

FIG. 1 shows a dorsal root ganglion (DRG) cultured on a flat surface.
Fig. 2 is a diagram showing PDMS chamber specifications (A, B) and a PDMS device (C) covered with a cover glass.
Figure 3 shows a nerve conduit of various lengths and a PDMS chamber of various lengths made accordingly.
Fig. 4 is a photograph showing a schematic diagram (A) of a nerve cell in vitro culture apparatus and a medium supply container (B).
5 is a view showing a configuration of a medium supply container.
6 is a view showing the location and shape of a spinal cord cut surface and DRG of a rat.
FIG. 7 is a view showing the ganglion ganglion separated from the spinal cord of the embryo at day 15 of the rat.
FIG. 8 is a photograph showing a seeding of the DRG exodus. FIG.
FIG. 9 is a photograph and a schematic diagram showing a seeding process of a DRG eating utensil using a pump.
10 is an illustration showing an actual use example of a device for seeding a DRG exodeviation to a nerve conduit.
FIG. 11 is a photograph showing axon staining using SMI-312 antibody after 3 days of DRG exodeviation. Scale bar = (A) 100 m, (B, C) 20 m.

The present invention provides a device comprising: i) a device having a nerve conduit inserted therein, the device comprising a microchannel having a diameter of 10 to 20 m; Ii) pumps; And ⅲ) provides an in vitro neuronal cell culture system consisting of a medium supply vessel containing the culture medium.

The nerve cell in-vitro culture apparatus may be configured such that the pump is connected to the upper part of a device having a nerve conduit containing the micro channel inserted therein, and a tube for supplying a culture medium containing the culture medium is connected to the lower part, The culture medium is supplied to the nerve conduit including the microchannel, and the nerve conduit including the microchannel is vertically disposed, so that the culture medium flows from the upper end to the lower end of the nerve conduit by gravity.

The culture medium may further comprise a nerve regeneration-related factor or a drug.

Wherein the nerve conduit comprises the steps of: i) dissolving the hydrophobic polymer in a hydrophobic solvent to prepare a nerve conduction polymer material; And ii) immersing the polymeric material in a hydrophilic solution to separate the hydrophobic solvent from the hydrophobic polymeric material to produce a nerve conduit comprising the porous polymer, wherein the polymeric material has a weight per volume (w (w / v%) of the PLGA and the TG means the weight (g) of the PLGA which dissolves in the TG 1 L. The weight / volume% (w / v%) of the PLGA and the TG is 10 to 40%
In addition, the TG may be a porous neural conduit in which water is separated from PLGA and separated from water in the step of immersing in water to form a porous structure in the PLGA.

The term "polymer material" means a solution prepared by dissolving a hydrophobic polymer in a hydrophobic solvent. In the present invention, PLGA is used as a hydrophobic polymer and PLGA-TG solution prepared using TG as a hydrophobic solvent.

The hydrophobic polymer may be polylactic acid-co-glycolic acid (PLGA), and the hydrophobic solvent may be tetraglycol (TG). According to the present invention, when PLGA is mixed with TG, PLGA is melted once with TG, and then the solution is maintained at room temperature. Therefore, the polymer material can be used without melting again.

The weight / volume% (w / v%) of the PLGA and the TG means the weight (g) of the PLGA dissolved in the TG 1L, and the weight / volume% (w / v%) is 10 to 40% 15% to 25%, and most optimally 20%. If it is less than the above range, there is a problem that porosity increases greatly due to excessive use of TG, and in the opposite case, formation of sufficient pores may be difficult.

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Inserting a plurality of glass fibers into a container having upper and lower channels; Injecting a polymer material into a container into which the plurality of glass fibers are inserted; Applying a vacuum from the channel to permeate the polymeric material between the glass fibers; Separating the glass fibers from the vessel; And dissolving the glass fiber by immersing the separated glass fiber in water, but the present invention is not limited thereto.

The lower channel has a diameter smaller than that of the upper channel, so that the glass fiber injected into the container can be kept filled without flowing out in the container.

 The container may be inclined at a discontinuous angle, and more specifically, the container may be formed by forming upper and lower channels inclined at discontinuous angles, but is not limited thereto.

Since the interval between the inserted glass fibers is constant due to the container inclined at the discontinuous angle and the upper and lower channels thereof, the interval of the microchannels formed in the space where the glass fibers are melted is also constant. That is, the nerve conduit manufactured according to the present invention can form microchannels at regular intervals, thereby inducing nerve regeneration in the same direction.

The polymer material may be in a solution state at room temperature.

The method for manufacturing a nerve conduit using the glass fiber comprises the steps of: cooling the nerve conduit formed after the step of dissolving the glass fiber with liquid nitrogen; And cutting and shaping the cooled nerve conduit, but the present invention is not limited thereto.

The container may be made of a transparent material through which the penetration of the polymer solution can be visually confirmed, and is preferably made of glass, but is not limited thereto.

The application of the vacuum may be repeated a plurality of times. More specifically, the vacuum may be repeatedly applied to the inside of the glass tube using a syringe, but is not limited thereto.

The nerve conduit may be a microchannel formed in the axial direction of the nerve conduit as the glass fiber is inserted in the axial direction of the container. More specifically, after the glass fiber is axially inserted into the upper channel of the vessel (glass tube), the polymer material (PLGA-TG solution) is injected into the vessel, the vacuum is applied to penetrate the glass fiber, And immersed in water (DW) to melt all of the glass fibers, thereby forming microchannels of hydrophobic polymer (PLGA) in the space where the glass fibers were melted. That is, by inserting the glass fiber in the axial direction of the container, and melting the glass fiber, a nerve conduit in which microchannels are formed axially in the space where the glass fiber is melted is manufactured.

The term "microchannel" means an empty space having a size of 10 to 20 mu m formed in a space in which glass fibers are melted.

The nerve conduit may be one in which micropores are formed in the nerve conduit as TG is dissolved in water. More specifically, in the process of immersing the glass fiber into which the polymer material (PLGA-TG solution) is impregnated with water DW, TG is reacted (dissolved) with the water DW and is discharged from the nerve conduit, .

The term "micropores" means micropores formed in the microchannel as TG dissolves in the DW and exits from the nerve conduit. The nerve conduits manufactured according to the present invention are easily fluid-exchanged by microchannels when applied in vivo. TG exiting from the nerve conduit was more dense than DW (1.09 g / ml) and submerged under DW.

In an embodiment of the present invention, a multichannel peristaltic pump is connected to an upper portion of a PDMS device in which a nerve conduit is inserted using a silicone tube, and a T-flask is modified on the lower side A media reservoir was connected to make a nerve cell in vitro culture device.

The device into which the nerve conduit is inserted may be a nerve conduit inserted into a chamber made of polydimethylsiloxane (PDMS).

In an embodiment of the present invention, a chamber made of polydimethylsiloxane (PDMS) is prepared, a nerve conduit is inserted, the space between the PDMS chamber and the nerve conduit is filled with agarose, Thereby creating a device having a nerve conduit inserted therein. The length of the nerve conduit according to the present invention can be manufactured in various sizes, and PDMS devices having various lengths can be prepared.

The nerve cell may be any one or more selected from the group consisting of dorsal root ganglion (DRG), nociceptive-specific neuron (NS), wide dynamic range neuron (WDR) But is not limited thereto.

Preferably, the diameter of the nerve cell is larger than the diameter of the microchannel of 10 to 20 mu m, and the diameter of the nerve cell may be 300 to 500 mu m. When the diameter of the neuron is smaller than the diameter of the microchannel, the microchannel enters the microchannel along the culture medium into which the neuron is introduced.

The term "nerve regeneration-related factor" refers to factors related to growth of axons of nerve cells. The nerve regeneration-related factors include nerve growth factor (NGF), neurotrophic factor (NTF) , But the present invention is not limited thereto.

The term "drug" refers to an unknown substance that is thought to have an effect on growth, such as the axon of a neuron. The drug may include, but is not limited to, a chemically synthesized substance, a natural product extract, and a nucleotide.

The present invention is connected to a nerve cell in vitro to neurons by using a culture apparatus as a method of culturing in in vitro, ⅰ) pump to the top of the device with nerve conduit is inserted including micro-channel according to the invention into a tube Connecting the culture medium supply container containing the culture medium to the lower portion with a tube; Ii) injecting neural cells into the culture medium in the medium supply container; Iii) seeding the neuron by supplying the culture medium in the culture medium supply container to the device having the nerve conduit inserted therein using the pump; Ⅳ) a neuronal cell in vitro culture comprising the step of using the pump the nerve cells within the culture medium medium supply container using a seeded (seeding), the nerve conduit is supplied to the inserted device culturing nerve cells to provide.

The flow rate of the culture medium in step iii) may be 30 to 60 μl / min. If the flow rate is higher than the numerical range of the flow rate, it may enter the microchannel of the nerve conduit along with the culture medium into which the neuron flows at a high rate, In some cases, neurons may be difficult to reach the nerve conduit along the tube.

In step iv), the flow rate of the culture medium may be 5 to 20 μl / min. If the flow rate is higher than the numerical range of the flow rate, it may enter the microchannel of the nerve conduit along with the culture medium into which the neuron flows at a high rate, , It may be impossible to measure the degree of axonal growth by growing axons of nerve cells in an unspecific direction as in the case of culturing in the plane.

In an embodiment of the present invention, an explant of dorsal root ganglion (DRG) isolated from a 15-day-old embryo of a rat is mixed in a culture medium, and then 50 μl / min) to the upper part of the nerve conduit. Because the diameter of the nerve conduit is 1.6 to 1.7 mm and the diameter of the microchannel is 10 to 20 μm, the DRG exodeviation with a diameter of about 300 to 500 μm is located at the top of the nerve conduit without entering the nerve conduit. The DRG meal was seeded at the top of the nerve conduit, and a culture medium containing a factor or drug associated with nerve regeneration was added to the medium supply container. The culture medium flowed from the upper part of the nerve conduit to the lower part. The inflow rate was 10 μl The peristaltic pumps were actuated to keep the DRG outlets from entering the nerve conduit.

The culture medium flows from the upper end to the lower end of the nerve conduit by gravity, so that the axon of the DRG can grow downward, and the axon can grow in an unspecified direction during DRG culture without flowing in the culture medium.

The culture medium may further comprise a nerve regeneration-related factor.

The nerve regeneration-related factor may be any one selected from the group consisting of nerve growth factor (NGF), neurotrophic factor (NTF), but is not limited thereto.

The culture medium may further comprise a drug.

The culture medium according to the present invention can be continuously and easily delivered to nerve cells by including a mixture of factors involved in axon growth of nerve cells or drugs exhibiting similar effects.

The nerve cell in vitro culture apparatus or culture method according to the present invention may be one in which axons of nerve cells are grown along microchannels inside a nerve conduit by seeding and culturing nerve cells in a device in which the nerve conduit is inserted have.

In one embodiment of the present invention, the DRG cultured for 3 days using the nerve cell in vitro culture apparatus according to the present invention, the DRG outgrowth remains at the top of the nerve conduit and the axons grown from the DRG are axially And it was confirmed that they were oriented along the fine channels.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

≪ Example 1 > Production of nerve conduit using glass fiber

(Density: 1.09 g / ml, Sigma-Aldrich, USA) containing polylactic acid-glycolic acid copolymer (PLGA) (lactic acid to glycolic acid mol%, 85:15) which is a hydrophobic polymer and hydrophobic solvent tetraglycol ) Was mixed to a weight-to-volume (w / v) ratio of 20% (w / v) and then dissolved at 60 ° C for 18 hours to prepare a 20% (w / v) PLGA-TG solution (polymeric material).

The central portion of the capillary glass tube having an inner diameter of 1.6 mm and a length of 13 cm was heated to form a bottleneck point to form upper and lower channels inclined at discontinuous angles. At this time, the lower channel was formed to have a smaller diameter than the upper channel. Thereafter, 7000 to 8500 strands of water-soluble glass fiber (50P ₂ O ₅ -20CaO-30Na ₂ O mol% (1100 ° C, 800rpm)) having a diameter of 10 to 20 μm were cut in units of 5 to 6 cm, Direction.

A pressure device manufactured by connecting a Luer lock syringe with a 2-way valve to a silicone tube having an inner diameter of 0.8 mm and a length of 15 cm was prepared by inserting a pressure device manufactured in the upper channel of the glass tube into which the glass fiber was inserted.

After the bottom channel of the glass tube was immersed in a 20% (w / v) PLGA-TG solution at room temperature, a vacuum was repeatedly applied to the inside of the glass tube using a syringe, and a 20% (w / v) PLGA- To penetrate completely into the gap between.

In this embodiment, as described above, the width of the lower channel is narrowed at a discontinuous angle with respect to the upper channel. When the angle is continuous, the interval between the glass fibers gradually becomes narrow, which makes it difficult to maintain a constant interval between the glass fibers. In the case where the intervals of the glass fibers are not constant, the nerve regeneration direction formed by the glass fibers changes depending on the channel, and therefore, there arises a problem that nerve regeneration in the same direction becomes difficult.

After the glass fiber infiltrated with the PLGA-TG solution was separated from the glass tube using a wire having a diameter of 1.5 mm and a length of 15 cm, it was completely immersed in distilled water (DW) at 10 to 20 ° C for more than 24 hours, In the space where the fibers are melted and the glass fiber is melted, about 7,000 ~ 8,500 microchannels (microchannel diameter: 16.54 ± 3.6 μm) (microchannel number: 7,777 ± 716.2) Respectively. Glass fibers were dissolved in a DW of 10 to 20 占 폚, and PLGA was cured to form microchannels. Also, in the process of immersing the glass fiber infiltrated with the PLGA-TG solution in the DW, TG was reacted with DW (dissolved in DW), and the micropores were formed in the microchannels as they escaped from the microchannels. TG exiting from the nerve conduit was more dense than DW, so it fell into a hazy shape under DW.

The porous microchannel made of PLGA, in which glass fiber and TG were removed by DW treatment, was soaked in liquid nitrogen for about 30 seconds and then cut and shaped into a size suitable for the purpose of use.

< Example  2> Neuron in vitro  Production of culture equipment

A chamber made of polydimethylsiloxane (PDMS) according to the standards of FIGS. 2A and 2B was prepared.

The nerve conduit of Example 1, which had been formed to a diameter of 1.6 to 1.7 mm and a length of 16 mm, was inserted into the prepared PDMS chamber, and 4% agarose completely dissolved in water was cooled to 40 to 50 ° C. And penetrated into the space between the conduits. The agarose was allowed to stand at room temperature for 5 minutes so that the agarose was completely cured, and then the agarose and the surface of the PDMS chamber surface were uniformly polished, covered with a cover glass, and sealed to produce a PDMS device (FIG. 2C). At this time, care was taken to prevent the agarose from penetrating the upper and lower ends of the nerve conduit and blocking the microchannel. The length of the nerve conduit can be manufactured in various sizes, so that a PDMS device of various lengths can be prepared (FIG. 3).

Then, a multichannel peristaltic pump (Gilson's MINIPULS ^® 3) (FIG. 4A) was connected to the upper part of the PDMS device in which the nerve conduit was inserted using a silicone tube, and a 25 ml T- (Fig. 4B and Fig. 5) prepared by modifying the flask of the present invention were connected to prepare a nerve cell in vitro culture apparatus. A 25 ml T-flask is punched through the top and a needle and tube are inserted for inlet and outlet and the air flow is maintained using a T-flask with a filter cap So that contamination can be prevented. The in vitro culture system of the prepared nerve cell was supplied to the PDMS device in which the culture medium in the culture medium supply container was inserted into the nerve conduit using a pump.

< Example  3> DRG Eating out Seeding (seeding)

In order to seed the explant of the dorsal root ganglion (DRG) inside the nerve conduit, DRG (Fig. 6 and Fig. 7) excreted from the 15th day embryo of the rat was papain- Treated for 1 minute in an aqueous solution and then sufficiently washed with a culture medium. DRG was mixed in a culture medium of 1 ml or less and the DRG meal piece was seeded at the upper part of the nerve conduit at a rate of 50 μl (μl / min) using a peristaltic pump (FIG. 8). At seeding, 2 to 3 DRGs were appropriate for nerve conduits of 1.6-1.7 mm in diameter and 16 mm in length. Because the diameter of the nerve conduit is 1.6 to 1.7 mm and the diameter of the microchannel is 10 to 20 μm, the DRG exodeviation with a diameter of about 300 to 500 μm is located at the top of the nerve conduit without entering the nerve conduit.

< Example  4> DRG Eating out  culture

After the DRG meal was seeded, a culture medium containing a factor or medicament related to nerve regeneration was introduced into the medium supply container, and the peristaltic pump was operated so that the culture medium flowed from the upper part of the nerve conduit to the lower part. At this time, the inflow rate of the culture medium was kept at 10 μl / μl / min or less so that the DRG meal did not enter the inside of the nerve conduit. The culture medium was continuously flown at a rate of 10 μl / min in a CO ₂ incubator and cultured for 1 to 3 days (FIG. 10).

< Example  5> cultured DRG From eating out  adult Axon (axon) confirmation

After 3 days of incubation, the DRG meal was frozen and the sections were axon stained with SMI-312 antibody.

As a result, it was confirmed that the DRG exoskeleton remained at the top of the nerve conduit and the axons of the DRG were oriented along the microchannels inside the nerve conduit (Fig. 11).

Claims (14)

(A) weight / volume% (w / v%) of PLGA and TG, which means the weight (g) of the polylactic acid-glycolic acid copolymer (PLGA) which is a hydrophobic polymer which dissolves in 1 L of tetraglycol (TG) A 10% to 40% PLGA-TG solution is prepared, and the central portion of the capillary tube is heated to form a bottleneck point to form an upper channel and a lower channel which are inclined at discontinuous angles. The diameter of the lower channel is smaller A plurality of water-soluble glass fibers having a diameter of 10 to 20 占 퐉 are inserted axially densely in the upper channel of the glass tube and the lower channel of the glass tube is immersed in the PLGA-TG solution, The PLGA-TG solution is allowed to penetrate into the gaps between the glass fibers, and the glass fiber infiltrated with the PLGA-TG solution is separated from the glass tube and is completely immersed in the ultra-pure water DW, The PLGA is cured in a space in which the glass fiber is dissolved to form a plurality of microchannels having a diameter of 10 to 20 탆 and a process in which the PLGA-TG solution is immersed in the DW The TG reacts with the DW to form micropores in the microchannels while leaving the microchannels. The microchannels formed with the micropores are frozen with liquid nitrogen and then cut and molded to prepare nerve conduits having a diameter of 1.6 to 1.7 mm A nerve conduit preparation step;
(B) inserting the nerve conduit into a PDMS chamber made of polydimethylsiloxane (PDMS), allowing the agarose completely dissolved in water to cool, and then penetrating into the space between the PDMS chamber and the nerve conduit so that the agarose is cured Preparing a PDMS device, preparing a multi-channel peristaltic pump on the PDMS device, connecting the PDMS device to a peristaltic pump with a tube, and connecting a media supply container to a lower portion of the PDMS device;
(C) injecting neuron into a culture medium in the medium supply container;
(D) supplying a culture medium in the culture medium supply container to the PDMS apparatus having the nerve conduit inserted thereto at a flow rate of 30 to 60 μl / min using the peristaltic pump to transfer neurons having a diameter of 300 to 500 μm to the nerve conduit A neuronal cell seeding step of seeding at the top; And
(E) a factor or medicament related to nerve regeneration is mixed with the culture medium in the medium supply container using the peristaltic pump, and then the mixed culture medium is added to the PDMS device in which the neuron is seeded to a concentration of 5 to 20 μl / min And culturing the neuron so that the axon of the neuron grows downward by flowing the culture medium from the upper end to the lower end of the nerve conduit by gravity, Cell culture method in vitro.
The method according to claim 1,
Wherein the culture medium supply container comprises a T-flask provided with a filter cap to maintain circulation of air but prevent contamination from the outside.
The method according to claim 1,
Wherein the factor relating to nerve regeneration is any one or more selected from the group consisting of nerve growth factor (NGF), neurotrophic factor (NTF), and the like. delete delete delete delete delete delete delete delete delete delete delete

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