KR101916567B1 - Intervertebral disc cell co-culture device for the study of intervertebral disc pain control mechanism - Google Patents

Abstract

The microcurrent stimulating disc cell co-culture apparatus for studying the intervertebral disc space mechanism according to an embodiment of the present invention includes: an intervertebral disc chamber in which inflammation-treated intervertebral disc cells are cultured; Neural chambers in which neurons are cultured; A connection channel connecting the nerve chamber and the disc chamber; An electrical stimulation part installed in the nerve chamber for providing electrical stimulation to the nerve cell; A piping connecting the inlet of the nerve chamber and the outlet of the intervertebral disc chamber; A circulation part installed in the piping, wherein the pain inducing substance expressed in the nerve cell flows from the nerve chamber to the intervertebral disc chamber and is operated so that the nerve stimulating substance in the intervertebral disc chamber flows into the outlet port; And a medium supply member connected to the injection port by piping to continuously supply the culture medium to the injection port during operation of the circulation unit so as to suppress the oxidation-reduction reaction of the culture medium at the injection port, wherein the inflammation-treated intervertebral disc cells and nerve cells Independently cultured, the nerve stimulating substance secreted from the inflammation-treated intervertebral disc is introduced into the nerve chamber through the connecting channel to stimulate the nerve cell, and the pain-causing substance secreted from the nerve cell by the stimulation of the nerve stimulating substance, It is preferable to observe whether the disc herniation is stimulated by stimulating the intervertebral disc cells and whether the amount of pain inducing substance is suppressed when the stimulation is applied to the nerve cell.

Description

{Intervertebral disc cell co-culture device for the study of intervertebral disc pain control mechanism}

The present invention relates to a device for co-culturing an intervertebral disc cell for the study of intervertebral disc pain, and more particularly, to a method for inhibiting the pathogenesis of backbone in the process of regeneration of the disc by molecular biological The present invention relates to a device for co-culturing an intervertebral disc cell.

The intervertebral disc consists of nucleus pulposus (Nucleus pulposus) on the inside of the tissue and annulus fibrosus on the outside. Outside one third of the fibrous ring, there is a free nerve ending that is derived from the vascular cells that can be fed and the dorsal root ganglion located in the vertebrae. Cell - cell interactions in the intervertebral disc tissue are involved in cellular signal transduction by transferring a variety of secretory proteins to the blood vessels and nerve cells present in the disc, as well as in the periphery.

The intervertebral disc is regressive of the characteristics of the retained structure due to various causes such as biomechanical loading, infection, genetic modification, and the like. In the degenerative process, the main two cells of the intervertebral disc tissue, the nucleus pulposus and fibrocyllia cells, exhibit inflammatory responses by immune cells. In the degenerative process, the intervertebral and immune cells are stimulated with various neurons such as beta-nerve growth factor (hereinafter "beta-NGF") and brain-derived neurotrophic factor And secrete a neurogenic factor.

These secreted nerve stimulating factors express various ion channels related to pain in the nerve bundles located in the spinal cord. Particularly, these receptors include acid-sensing ion channel 3 (ASIC3) and transient receptor potential cation channel subfamily V member 1 (TRPV1).

The graphs of FIGS. 5 and 6 are graphs comparing the amount of secreted protein in the conditioned medium, the amount of protein secreted from pure endothelial cells without the conditioned medium, and the amount of protein from the conditioned medium treated endothelial cells, Expression levels of secretory proteins expressed upon inflammation of the intervertebral disc can be compared.

In the graphs of Figures 5 and 6, CM is an abbreviation for a conditioned medium. In the case of ctrlHMEC-1, ctrl refers to control, and in the case of HMEC-1, it refers to endothelial cells. ctrlHMEC-1 refers to the amount of protein released from pure endothelial cells, without treatment of conditioned medium from disc cells. expHMEC-1 refers to the amount of protein from the endothelial cells treated with conditioned medium.

IL-6 (interleukin-6), IL-8 (interleukin-8) and VEGF (Vascular Endothelial Growth Factor) are inflammatory mediators of the intervertebral disc. MMP-1 (Matrix metalloproteinase-1) or MMP-3 (Matrix metalloproteinase-3) is a protein involved in tissue disruption of the intervertebral disc.

5 (a), fibroblast cells (AF) secrete 40 ng / ml or more of IL-6 (interleukin-6), and nucleus pulposus cells secrete about 10 ng / ml of IL-6 . In the case of expHMEC-1, endothelial cells stimulated by endothelial cells were treated with endothelial cells by the stimulation of secretory proteins of fibricular cells and nucleus pulposus cells, (endothelial cells) secrete more than 80 ng / mL of IL-6. In the case of ctrlHMEC-1, IL-6 secreted by endothelial cells is close to 0 ng / mL.

In graph (c) of FIG. 5, fibroblast cell (AF) secretes 50 ng / ml or more of IL-8 in CM (conditioned medium) and secreting nuclei secrete IL-8 (interleukin-8) . In the case of ctrlHMEC-1, IL-8 secreted by endothelial cells is close to 0 ng / mL.

In the case of expHMEC-1, endothelial cells stimulated by endothelial cells were stimulated by the secretory protein of fibroblast cells (AF) endothelial cells secrete IL-8 at a concentration of 100 ng / ml, and endothelial cells secrete IL-8 at a concentration of more than 170 ng / ml by the stimulation of secretory proteins of the nucleus pulposus (NP).

In the graphs of FIGS. 6 (d) to 6 (f), MMP-1, MMP-3 and VEGF were also secreted in conditioned medium (CM) and expHMEC-1 but not in ctrlHMEC-1 Able to know.

In the graph of Fig. 5 (b), it can be seen that in the CM (conditioned medium), fibrous ring cells (AF) and nucleus pulposus cells (NP) do not secrete NGF. In the case of ctrlHMEC-1, NGF secreted by endothelial cells is more than 7 pg / ml in fibroblast (AF) and nucleus pulposus (NP).

In the case of expHMEC-1, endothelial cells stimulated by endothelial cells were treated by stimulation of secretory proteins of fibroblast cells (AF) Endothelial cells secrete 20 pg / m of NGF and endothelial cells secrete more than 35 pg / ml of NGF by stimulation of secretory proteins of nucleus pulposus (NP).

In the case of NGF, a small amount of NGF may be sufficient to deliver a signal, although it may seem small relative to other factors. In the graph (b), it is highly likely that cells such as fibroblasts / horses and the like do not directly express NGF, and vascular cells are inhibited by interaction between fibroblasts / endothelial cells and endothelial cells You can see the secretion.

The graph of FIG. 5 (b) shows that the secretion amount of NGF in expHMEC-1 is increased compared with the secretion amount of NGF in ctrlHMEC-1. From this, it can be deduced that the increase of secretion of NGF in the vascular cells that interact with fibroblasts and intervertebral discs promotes the growth of nerve cells. The direct factor that induces neural cell growth is NGF (nerve growth factor), and it can be seen that the origin of NGF is derived from blood vessel cells.

When the intervertebral cells are treated with an inflammatory substance, the change of the secretory protein with the change of time is as shown in Fig. IL-1beta 1 ng / mL was stimulated and observed in fibroblast cells for 72 hours, and the expression data of each protein was measured.

7 shows a concentration change with time of IL-6, FIG. 7 shows a concentration change with time of IL-8, FIG. 7 shows a change with concentration of MMP-1 with time, Shows the concentration change of MMP-3 with time.

The graph A, the graph B and the graph D shown in FIG. 7 show that IL-6, IL-8, and MMP-3 increase the concentration of the secretory protein with the lapse of time. On the other hand, through graph C, it can be seen that the concentration of MMP-1 does not increase with time but decreases after 48 hours.

FIG. 8 (a) is a photograph showing a comparison between uninfected fibroblast cells (AF) and IL-1 beta-inflammed fibrocyte cells (AF). The white dotted line is the baseline for measuring the quantified intensity in observing the translocation of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) proteins.

In FIG. 8 (a), blue is the intensity of DAPI fluorescence to stain nuclei, red is fluorescence to NF-kB p50, which is a protein to be red, and green is fluorescence intensity, which stains actin.

Figure 8 (b) is a graph of relative fluorescence intensity change versus normal distance. The graph of FIG. 8 (b) is a measurement of the intensity of each fluorescence extracted from the reference line of the white dotted line in FIG. 8 (a). For the X axis, the length of the white dotted line is the relative value. In other words, the normal distance on the X axis is the relative distance from the baseline of the white dotted line, the width of the nucleus between cytoplasm and cytoplasm.

In the graph of FIG. 8 (b), the arrows indicate whether the NF-kB p50 protein, which is the target of the target, is strongly located in the cytoplasm or strongly located in the nucleus. The graph of FIG. 8 (b) shows that when the inflammation is expressed, NF-kB shows a tendency to translocate from the cytoplasm to the nucleus.

9 and 10 are graphs of ELISA quantifying the secretory proteins secreted from fibroblast cells and nucleus pulposus cells when the fibroblast cells are treated with inflammation and when the recipient cells are treated with inflammation.

As shown in FIGS. 9 and 10, MMP-1, MMP-3, total soluble collagen, IL6 and IL-8 were almost secreted at 0 ng / ml when IL-1beta was treated with an inflammatory substance . In contrast, when treated with 0 ng / ml of IL-1 beta, fibronectin (AF) secreted 1 ng / ml of VEGF and nucleus pulposus (NP) contained less than 1 ng / ml VEGF (Vascular Endothelial Growth Factor) Secretion.

As shown in FIGS. 9 and 10, 200 ng / ml of MMP-1, 200 ng / ml of MMP-3 and less than 200 ug / ml of Total soluble in fibroblast cells treated with 10 ng / ml of IL- collagen, IL-6 at 40 ng / ml or more, IL-8 at 60 ng / ml, and VEGF at 4 ng / ml. MMP-1, 200 ng / ml MMP-3, 100 ug / ml total soluble collagen, less than 10 g / ml IL-6, and 20 ng / ml of IL-8, and 1ng / ml of VEGF (Vascular Endothelial Growth Factor).

Through the graphs shown in FIGS. 5 to 10, it can be seen that secretory proteins are secreted in the discs of the disc during inflammation of the disc, and specific proteins are secreted from the endothelial cells by stimulation of the secretory proteins. In addition, the expression level of the secretory protein changes with time during the injury of the intervertebral disc, and it can be seen that there is movement between the proteins.

In fact, there has been no previous research on how neuronal cell growth is affected by changes in the pain receptors, but it is known that nociceptive factors are already expressed in neurons that have already undergone growth. In this study, we investigated the mechanism of pain induced by molecular biology or interactions with intervertebral disc cells in a T2-echo MRI image in the absence of another nerve compression, There is a need for a device that can perform.

The present invention is based on the finding that when a pain inducing substance is expressed in a nerve cell due to degeneration or damage of an intervertebral disc, the expression of a pain inducing substance is suppressed when electrical stimulation is applied to a nerve cell, The present invention also provides an apparatus for co-culturing an intervertebral disc cell for the study of intervertebral disc pain.

The present invention provides a co-culturing apparatus for intervertebral disc cell for studying intervertebral disc mechanism capable of observing in real time the change pattern of intervertebral disc constituting cells through the application of external microcurrent to the change of nerve cells that may appear in pain induction process .

The microcurrent stimulating disc cell co-culture apparatus for studying the intervertebral disc space mechanism according to an embodiment of the present invention includes: an intervertebral disc chamber in which inflammation-treated intervertebral disc cells are cultured; Neural chambers in which neurons are cultured; A connection channel connecting the nerve chamber and the disc chamber; An electrical stimulation part installed in the nerve chamber for providing electrical stimulation to the nerve cell; A piping connecting the inlet of the nerve chamber and the outlet of the intervertebral disc chamber; A circulation part installed in the piping, wherein the pain inducing substance expressed in the nerve cell flows from the nerve chamber to the intervertebral disc chamber and is operated so that the nerve stimulating substance in the intervertebral disc chamber flows into the outlet port; And a medium supply member connected to the injection port by piping to continuously supply the culture medium to the injection port during operation of the circulation unit so as to suppress the oxidation-reduction reaction of the culture medium at the injection port, wherein the inflammation-treated intervertebral disc cells and nerve cells Independently cultured, the nerve stimulating substance secreted from the inflammation-treated intervertebral disc is introduced into the nerve chamber through the connection channel to stimulate the nerve cell, and the pain inducing substance secreted from the nerve cell by the stimulation of the nerve stimulating substance, It is preferable to observe whether the disc herniation is stimulated by stimulating the intervertebral disc cells and whether the amount of pain inducing substance is suppressed when the stimulation is applied to the nerve cell.
Also, the microcurrent stimulating disc cell co-culture apparatus for studying the intervertebral disc pain mechanism comprises: a nerve chamber in which nerve cells are cultured; An intervertebral disc culture chamber connected to a central chamber and a central chamber connected to the nerve chamber and in which at least one of a nucleus cell, a fibricular cell or a vascular cell constituting an intervertebral disc cell is cultured, and the nucleus pulposus cell, An intervertebral disc in which nerve stimulating material secreted from at least one of the cells flows from an intervertebral culture chamber to a central chamber; A connection channel connecting the nerve chamber and the central chamber such that the nerve stimulating material flows from the central chamber to the nerve chamber; And an electric stimulation unit provided in the nerve chamber for providing electric stimulation to the nerve cell, wherein the inflammation-treated intervertebral disc cell and the nerve cell are independently cultured, and the nerve stimulating substance secreted from the inflammation- In addition, the pain of the intervertebral disc caused by stimulation of the intervertebral disc by the flow of the pain stimulating substance secreted from the nerve cell into the intervertebral disc chamber through the connection channel is simulated by stimulation of the nerve stimulating substance, It is desirable to be able to observe whether or not the expression amount of the pain inducing substance is suppressed when the electric stimulation is applied to the nerve cell.

In one embodiment of the present invention, the stimulation control unit further includes a stimulation control unit connected to the electric stimulation unit to control the intensity of the electric stimulation, .

In one embodiment of the present invention, the electric stimulation unit includes: an ITO glass installed on the bottom surface of the nerve chamber; And a conductive electrode patterned in ITO glass.

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In one embodiment of the present invention, it is preferable that the apparatus further includes a byproduct storage member connected to the outlet by the piping and storing the nerve stimulating substance and the pain causing substance inhaled from the outlet at the operation of the circulating unit.

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In one embodiment of the present invention, the connection channel is preferably a microchannel structure that blocks movement of nerve cells and intervertebral disc cells, and permits movement between nerve stimulating substances and pain inducers.

The present invention relates to a method for modulating the intensity of electrical stimulation provided to a nerve cell, which is based on the reactivity of the nerve cell to the effect of the micro current flowing to the human body, or by applying an external electric current to the nerve cell stimulated by the intervertebral disc inflammation process Based studies of pain-inducing receptors and factors can be performed simultaneously.

In addition, the present invention enables molecular biology testing by collecting continuous reaction products from the outlet through the circulation, thus providing convenience in the study of the mechanism of basal and electrochemical changes related to back pain.

The present invention relates to a method of co-culturing intervertebral disc cells and nerve cells in a single co-culture chip to examine the pathogenesis of back pain in the process of regression of the intervertebral discs in a molecular biologic Changes can be observed in real time.

The present invention is based on the discovery that pain and various types of penotypic and genotypic changes in the disc regression process are responsible for various neuronal cell mediated secretory proteins known to be responsible for various intercellular interactions and pain of intervertebral disc cells And to enable a basic study of the responsiveness of neurons to electrical signals.

In addition, the present invention can be used to identify interactions by paracrine signaling to study the clinical mechanisms that cause pain in the absence of nerve compression.

The present invention is based on the observation that the cause of the pathogenesis of back pain in the process of degeneration of the human body is the intercellular interactions between the spinal cord-derived nerve cells and intervertebral disc tissue, and the nerve cell stimulating substances secreted from the intervertebral disc cells It is possible to confirm what kind of effect it has on the cells.

Figure 1 is an anatomical schematic view of a normal disc.
FIG. 2 is a schematic plan view of an intervertebral disc co-culture apparatus for studying intervertebral disc pain according to an embodiment of the present invention.
3 schematically shows a top view of a culture chip according to an embodiment of the present invention.
4 shows a cross-sectional view taken along line AA of Fig.
Figures 5 and 6 are graphs comparing the amount of secreted protein in conditioned medium, the amount of protein secreted in pure endothelial cells without conditioned medium, and the amount of protein from conditioned medium treated endothelial cells.
7 shows the concentration change with time of IL-6, FIG. 7 shows the concentration change with time of IL-8, FIG. 7 shows the concentration change with time of MMP-1, Shows the concentration change of MMP-3 with time.
FIG. 8 (a) is a photograph showing a comparison between uninfected fibroblast cells (AF) and IL-1beta inflammation treated fibroblast cells (AF). Figure 8 (b) is a graph of relative fluorescence intensity change versus normal distance.
FIGS. 9 and 10 are graphs of ELISA quantifying secretory proteins secreted by fibroblast cells and nucleus pulposus cells when the fibroblast cells are treated with inflammation and when the recipient cells are treated with inflammation.

Hereinafter, an apparatus for co-culturing an intervertebral disc cell for studying intervertebral disc pain according to a preferred embodiment of the present invention will be described with reference to the accompanying drawings.

The present invention simulates the pain of the intervertebral disc to compare the amount of pain inducing substance of the neural cell before electric stimulation with the amount of pain inducing substance after electric stimulation to determine whether the electric stimulation affects the pain causing substance of the intervertebral disc It is the device used to study the pain control mechanism of what effect it has.

2, the intervertebral disc co-culture apparatus 100 for studying intervertebral discs according to an embodiment of the present invention includes a culture chip 110, an intervertebral disc chamber 120, a nerve chamber 130, An electric stimulation unit 117, a stimulation control unit 140, and a circulation unit 150.

The culture chip 110 is embedded in the housing 111. The culture chip 110 is a member in which an intervertebral disc as a body tissue is simulated. The culture chip 110 is preferably made of a PDMS (polydimethylsiloxane stamp) material.

2, the culture chip 110 is provided with an intervertebral disc chamber 120, a nerve chamber 130, an inlet 115a, an outlet 115b, a connection channel 117, a first channel 116, Two channels 118 are provided in the same plane by photolithography.

In the culture chip 110, the intervertebral disc cells and nerve cells are co-cultured in an independent space. The intervertebral disc may be a nuclear receptive cell, a fibricular cell, or a vascular cell.

In the present invention, the intervertebral disc structure can be simulated by independently culturing the xenogeneic cells forming the intervertebral discs in the disc chamber 120 and the nerve chamber 130. In addition, the present invention can be used to simulate damage and / or degeneration of an intervertebral disc by treating the disc cells with inflammation.

The intervertebral disc chamber 120 comprises a central chamber 121 and a plurality of intervertebral disc culture chambers. As shown in FIG. 3, the central chamber 121 is connected to the nerve chamber 130 by a connection channel 117.

And, the central chamber 121 is connected to the outlet 115b by the second channel 118. A plurality of intervertebral culture chambers are connected to the central chamber 121. The central chamber 121 is a space into which a variety of secretory proteins including nerve stimulating substances secreted from inflammation-treated intervertebral disc cells are introduced into a plurality of intervertebral disc culture chambers. The central chamber 121 is partitioned by a plurality of partition members 126 to limit movement between the intervertebral disc cells.

The plurality of intervertebral disc culture chambers are connected to the central chamber 121 and are spaces where at least one of the nucleus pulposus cells, the fibricular cells or the vascular cells constituting the intervertebral disc cells are respectively cultured. In this embodiment, for convenience of explanation, the first intervertebral disc culture chamber 122, the second intervertebral disc culture chamber 123, the third intervertebral disc culture chamber 124, and the fourth intervertebral disc culture chamber 125). ≪ / RTI > However, it should be understood that the number of the intervertebral disc culture chambers connected to the central chamber 121 may vary according to the purpose of the experiment.

In order to simulate the inflammation of the intervertebral disc during the injury or degeneration of the intervertebral disc, the first intervertebral culture chamber 122 to the fourth intervertebral disc culture chamber 125 may be cultured with nuclear receptors, fibrocytic cells, vascular cells, and microwave fibrils.

In the first intervertebral disc culture chamber 122, a nucleus pulposus cell is cultured. Fibricular cells are cultured in the second intervertebral disc culture chamber 123. In the third intervertebral disc culture chamber 124, blood vessel cells are cultured. In the fourth intervertebral disc culture chamber 125, a microwave paper can be cultured.

The nerve stimulating material secreted from at least one of the inflammatory nucleus pulposus cells, the fibrocystic cells or the vascular cells is introduced into the central chamber 121 and then supplied to the nerve chamber 130 through the connection channel 117.

As the inflammatory substance, cytokines such as Interleukin-1beta (IL-1b) or Tumor Necrosis Factor-alpha (TNF-alpha) may be used. However, it is an exemplary material, and it is needless to say that various inflammation-treating substances can be used within a range that is obvious to a person skilled in the art.

The connection channel 117 is a passage for connecting the nerve chamber 130 and the intervertebral disc chamber 120. The connection channel 117 has a microchannel structure that blocks movement between nerve cells and intervertebral disc cells and permits only movement between nerve stimulating substances and pain inducers. The connection channel 117 has a microchannel structure in which a passage interval is divided by micrometers by a plurality of partitions. Preferably, the channel spacing of the connection channel 117 is such that the dendrites of the nerve cell can move.

The nerve chamber 130 is a space where nerve cells are cultured. The nerve chamber 130 is connected to the central chamber 121 by a connection channel 117. The nerve chamber 130 is connected to the injection port 115a by a first channel 116. [

As shown in FIG. 4, the nerve chamber 130 is provided with an electrical stimulation portion. The electrical stimulation part provides electrical stimulation to the nerve cell. The electric stimulation portion is composed of the ITO glass 131 and the conductive electrode 133. The ITO glass 131 is installed on the bottom surface of the nerve chamber 130. The conductive electrode 133 is patterned on the ITO glass 131 and connected to the stimulation control unit 140. The intensity of the electrical stimulation is controlled by the stimulation control unit 140. [

When an electric stimulus is applied to a nerve cell, a current exceeding a threshold value flows to the nerve cell. On the other hand, if no electric stimulation is given to the nerve cell, the current value is in a resting state.

The nerve stimulating material secreted from the inflammation-treated intervertebral disc is introduced into the nerve chamber 130 through the connection channel 117, and the neuron expresses the pain inducing substance by the stimulation of the nerve stimulating substance, To grow into the disc chamber 120, and the expression of the pain-inducing substance in the electric stimulation can be suppressed.

In the present invention, when the intensity of an electric stimulus for stimulating a nerve cell is adjusted by the stimulation controller 140, electric stimulation is applied to the intervertebral disc from the outside through the change of the nerve cell according to the intensity of the electric stimulus, In the study of pain control mechanisms. That is, the experimenter uses the stimulation control unit 140 to variously control the intensity of the electric stimulus applied to the nerve cell, thereby studying the pain control mechanism according to the change of the nerve cell and the intervertebral disc cell according to the intensity of the electric stimulus .

The circulation part 150 is provided in order to solve the problem that the continuous progression of the continuous interaction between the nerve stimulating substance secreted from the intervertebral disc and the pain inducing substance secreted from the nerve cell proceeds very slowly over a long period of time .

The present invention allows a pain-causing substance secreted from a neuron to flow from the neural chamber 130 to the disc chamber 120 during operation of the circulation unit 150, thereby preventing pain during disc regeneration for a long period of time Can be simulated for a short period of time (approximately 10 to 20 days).

The circulation unit 150 is connected to the inlet 115a and the outlet 115b by a pipe 154 in a circulating structure. The inlet 115a and the outlet 115b are provided in the culture chip 110. [

The injection port 115a is connected to the nerve chamber 130 by the first channel 116. The injection port 115a is filled with a culture medium. A feedstock supply member 151 is connected to the injection port 115a.

The outlet 115b is connected to the central chamber 121 by a second channel 118. The outlet 115b is a portion where the secretory protein of the central chamber 121 is accommodated. Here, the secretory protein refers to various kinds of proteins including nerve stimulating substances secreted from inflammation-treated intervertebral disc cells.

The culture medium supply member 151 is connected to the injection port 115a by the pipe 154. [ The culture medium supply member 151 supplies the culture medium to the injection port 115a when the circulation unit 150 operates. When the initial inflammation model is formed in the intervertebral disc chamber 120 and the neural chamber 130, the circulation unit 150 can flow the culture medium to the neuron to simulate the electrophysiological mechanism by the neuron . At this time, at the time of operation of the circulation unit 150, the culture medium supply member 151 can supply a fresh culture medium to the injection port 115a to suppress the redox reaction of the culture medium.

The byproduct storage member 153 is connected to the outlet 115b by a pipe 154. [ The byproduct storage member 153 is a space in which secreted proteins inhaled from the outlet 115b are stored during operation of the circulation unit 150. [ Here, secretory proteins inhaled at the outlet 115b include nerve stimulating substances and / or pain inducing substances. The present invention can be used in molecular biological tests to subsequently secrete secreted proteins stored in the byproduct storage member 153.

Hereinafter, the pain generation process of the disc will be described.

When the intervertebral disc cells are cultured with the inflammation-treating substance in the intervertebral disc chamber 120, a nerve stimulating substance is formed through the reaction between fibricular cells and vascular cells or macrophages.

The nerve stimulating substance is introduced into the nerve chamber 130 through the connection channel 117 via the central chamber 121. [ This is because the ratio of the nerve stimulating material flowing into the central chamber 121 is high, and the nerve stimulating material flows from the central chamber 121 toward the nerve chamber 130.

The nerve stimulating material flowing into the nerve chamber 130 is bound to receptors such as ASIC-3 (Acid sensing ion channel 3) and transient receptor potential vanilloid (TRPV1) expressed in nerve cells. Basically, tissues show pain in the painful condition of the nucleus, which is expected to involve certain proteins.

The present invention is used when it is intended to examine how a secretory protein due to the interaction of nucleus pulposus cells (fibroblasts, vascular cells, macrophages) stimulates nerve cells to cause internal growth of nerve cells and furthermore, do. The secretion of neurotic pain-causing substances can be detected by ELISA or Western blot.

The present invention relates to a method for comparing the amount of a pain-causing substance secreted when an electrical stimulus is applied to a neural cell after the inflammation and pain of the disc is simulated in the above manner, . ≪ / RTI > The amount of expression of the pain-causing substance after electric stimulation can be measured by ELISA or Western blot. That is, the present invention is used to study the inhibition of the expression of pain-inducing substances upon electrical stimulation to neurons.

Through the above-described structure, the present invention (100) actually stops the circulation of the culture medium for a certain period of time in an experiment of intercellular reactivity, induces microcurrent stimulation and inflammation of intervertebral disc cells, The culture medium for the challenge can be circulated again.

The present invention relates to a method for inducing the pathogenesis of back pain in a disc regression process by co-culturing intervertebral disc cells and nerve cells in a co-culture chip (110) and applying micro current stimulation to the neurons, And other vascular cell interactions can be observed in real time.

In the present invention (100), the pain and various types of penotypic and genotypic changes in the disc regression process are known to be the main cause of intercellular interactions and pain of intervertebral disc cells, It is possible to observe the effect on the protein and enable a basic study on the reactivity of neurons to electrical signals.

In addition, the present invention (100) can confirm the interaction by the paracrine signal transmission without direct intercellular contact with the clinical mechanism causing the pain in the state without neural compression in the disc regression.

In the present invention (100), it is possible to observe, in real time, the change pattern of intervertebral disk-shaped cells which are manifested through the application of an external micro current to the change of nerve cells that may appear in pain induction process.

According to the present invention (100), the microfluidic microcurrent stimulation cell co-culturing apparatus is characterized in that the cause of the pathogenesis of back pain in the process of degeneration of the human body is a nerve cell derived from the spinal cord, And it is possible to confirm how the nerve cell stimulating substance secreted from the intervertebral disc cell actually affects the nerve cell.

In addition, the present invention (100) is based on the reactivity of neurons to the influence of microcurrents flowing in the human body, and based on a study on pain-induced receptors and factors in neurons stimulated by the disc inflammation process by applying an external current Can be performed simultaneously.

In addition, the present invention (100) enables the molecular biological test by collecting continuous reaction products from the outlet (115b) through the circulation part (150), thereby providing convenience for the mechanism study on the basal and electrochemical change patterns related to back pain .

Although several embodiments of the present invention have been shown and described, those skilled in the art will appreciate that various modifications may be made without departing from the principles and spirit of the invention . The scope of the invention will be determined by the appended claims and their equivalents.

100: Intervertebral disc co-culture device
110: culture chip 120: disc chamber
130: Neural chamber 140: Stimulation control unit
150: circulation part

Claims (8)

An intervertebral disc chamber in which inflammation-treated intervertebral disc cells are cultured;
Neural chambers in which neurons are cultured;
A connection channel connecting the nerve chamber and the disc chamber;
An electric stimulation unit installed in the nerve chamber for providing electric stimulation to the nerve cell;
A pipe connecting the inlet of the nerve chamber and the outlet of the disc chamber;
A circulation unit installed in the pipeline, wherein the pain inducing substance expressed in the nerve cell flows from the nerve chamber to the intervertebral disc chamber, and the nerve stimulating substance in the intervertebral disc chamber is operated to flow into the outlet; And
And a medium supply member connected to the injection port by the piping to continuously supply the culture medium to the injection port during operation of the circulation unit so as to suppress the redox reaction of the culture medium at the injection port,
The irritated intervertebral disc cells and the nerve cells are independently cultured to cause the nerve stimulating material secreted from the inflammation-treated intervertebral disc cells to flow into the nerve chamber through the connection channel to stimulate the nerve cells, The pain caused by stimulation of the intervertebral disc by the influx of the pain stimulating substance secreted from the nerve cell into the intervertebral disc chamber through the connection channel is simulated by stimulation of the stimulating substance,
Wherein the inhibition of the expression amount of the pain-inducing substance during the electrical stimulation to the nerve cell can be observed.
Neural chambers in which neurons are cultured;
An intervertebral disc culture chamber in which a central chamber connected to the neural chamber and at least one of a nucleus pulposus cell, a fibrocystic cell or a vascular cell, which is connected to the central chamber and constitutes intervertebral disc cells, is cultured, An intervertebral disc in which a nerve stimulating substance secreted from at least one of the fibrous ring cell or the vascular cell flows from the intervertebral disc culture chamber to the central chamber;
A connection channel connecting the nerve chamber and the center chamber; And
And an electrical stimulation portion provided in the nerve chamber for providing electrical stimulation to the nerve cell,
The irritated intervertebral disc cells and the nerve cells are independently cultured to cause the nerve stimulating material to flow into the nerve chamber through the connection channel to stimulate the nerve cells, Induced pain is injected into the disc chamber through the connection channel to simulate the disc pain caused by irritating the disc cells,
Wherein the inhibition of the expression amount of the pain-inducing substance during the electrical stimulation to the nerve cell can be observed. 3. The method according to claim 1 or 2,
Further comprising a stimulation control part connected to the electric stimulation part and controlling the intensity of the electric stimulation to observe a degree of suppression of the amount of the pain inducing substance depending on the intensity of the electric stimulation, Microcurrent stimulating intervertebral disc co-culture device for pain mechanism research.
The magnetic resonance imaging apparatus according to claim 1 or 2,
An ITO glass installed on a bottom surface of the nerve chamber; And
And a conductive electrode patterned on the ITO glass. The microcurrent stimulating disc cell co-culture apparatus for studying the intervertebral disc pain mechanism.
The method according to claim 1,
Further comprising a byproduct storage member connected to the outflow port by the pipe to store the nerve stimulating substance sucked from the outlet at the operation of the circulation unit and the pain inducing substance. Disc cell co-culture apparatus.
3. The method according to claim 1 or 2,
Wherein the connection channel is a microchannel structure that blocks movement of the nerve cell and the intervertebral disc and permits movement between the nerve stimulating substance and the pain inducing substance. Device.




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