KR101011154B1 - Complex stimulus chamber for cell culture and cell culture apparatus in using same - Google Patents

Abstract

The present invention relates to a cell culture complex stimulation chamber and a cell culture device using the same, and the technical problem to be solved is to observe the response of the cell in response to changes in the external environment in real time by independently controlling the shear stress and the electric field received by the cell The present invention provides a useful cell culture complex stimulation chamber and a cell culture device using the same.

To this end, the present invention is connected to the culture solution inlet and the culture solution inlet, the culture medium supply unit is provided with a stepped recessed culture solution dispersion unit for inducing the dispersion of the introduced culture solution, the culture solution supply unit is coupled to cover the culture solution dispersion and introduced culture medium A flow preventing plate for blocking the flow of the flow and the flow prevention plate is integrally connected to the electrode plate is formed with an electrode insertion groove is inserted into the electrode and the outlet plate is formed integrally connected to the electrode plate and the culture solution outlet is discharged Comprising a cell cover portion and the cell support portion is coupled to the culture medium supply portion and the bottom surface of the cell cover portion and provided with a stepped recessed cell seat portion in which the culture fluid flows into the slide glass seated.

Complex stimulation chamber, shear stress, electric field, flow sensor, feedback controller

Description

Cell culture complex stimulation chamber and cell culture device using same {COMPLEX STIMULUS CHAMBER FOR CELL CULTURE AND CELL CULTURE APPARATUS IN USING SAME}

The present invention relates to a cell culture complex stimulation chamber and a cell culture device using the same, and to a cell culture complex stimulation chamber and a cell culture device using the same, which can observe the reaction of a cell according to an external environment in real time in a laboratory environment.

Cell culture requires a cell culture chamber that provides a constant space in which cells can be cultured and a culture medium that supplies nutrients to the cells.

The culture solution is a culture medium for cells and a transmission medium such as nutrients and oxygen. In other words, the culture solution supplies the cells with nutrients, oxygen, and the like and removes waste products.

Therefore, the culture solution must be injected into the cell culture chamber and used for cell culture, and then exchanged at appropriate intervals to keep the tissue fresh. Therefore, a cell culture chamber and a cell culture device using the same require a configuration capable of smoothly carrying out and discharging the continuous culture solution.

On the other hand, in studying the physiology of cells, a method of observing cellular responses to external physical stimuli is introduced.

 Related papers include "Flow effects on prostacyclin production by cultured human endothelial cells", (Frangos, JA et al., Science 227 (4693), 1477-1479, 1985), where real-time shear stress is applied to cells. This section introduces the concept of a system that can be observed. However, there is a problem in that the shape of the flow chamber in which the actual cells are subjected to the shear stress is made asymmetrical with respect to the direction of the shear stress, thereby making it impossible to create a uniform flow field.

Also, in the paper "Fluid shear-stress modulates cytosolic free calcium in vascular endothelial-cells", (Shen, J. et al., American Journal of Physiology 262: 2, C384-C390, 1992) Although there is an introduction of the designed flow chamber, there is a problem that the flow field is not uniform because it does not sufficiently disperse the flow at the inlet through which the cell culture fluid is introduced.

In addition, the paper "Endothelial cell dynamics under pulsating flows: Significance of high versus low shear stress slew rates", (Hsiai, TK et al., Annals of Biomedical Engineering 30: 5, 646-656, 2002), Although the equipment can be made, there is a problem that the microscope itself is inevitably changed due to the large size of the peripheral equipment compared to the actual cell size.

On the other hand, a paper on the study of applying electric field to cells is described in “Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo” (Song, B. et al., Nature Protocols, 2: 6, 2007) and “Movements of cultured corneal epithelial cells and stromal fibroblasts in electric fields” (Soong, HK et al., Investigative Ophthalmology and Visual Science, 31: 11, 1990). In this paper, the potential difference is given to the cells cultured in the slide glass, and the movement of the cells and the signal transmission under the influence of the electric field can be observed. However, there is a problem that can not observe the cell response in real time due to the evaporation of the culture solution, the temperature rises itself, the pH is changed due to the continuous supply of the cell culture solution. In addition, there is a problem that it is difficult to simulate the situation in the human body can not apply the shear stress to the cells.

Therefore, there is a need for a cell culture apparatus capable of effectively forming a uniform flow environment and applying electrical stimulation while maintaining cell growth conditions.

The present invention has been made to solve the above problems, cell culture complex stimulation useful for observing the response of the cell in real time by changing the external stress and the electric field independently received by the cell in vivo The object is to provide a chamber and a cell culture apparatus using the same.

In addition, by effectively dispersing the culture medium to form a uniform flow field, by circulating the culture medium by itself can provide a cell culture complex stimulation chamber and cell culture apparatus that can check the effect of electrical stimulation while maintaining cell growth conditions have.

The cell culture complex stimulation chamber according to the present invention for achieving the object as described above is connected to the culture solution inlet and the culture solution inlet, the culture medium supply unit is provided with a stepped recessed culture solution dispersion to induce the dispersion of the introduced culture solution, It is coupled to the culture medium supply unit and covers the culture medium dispersion unit and the flow prevention plate for blocking the flow of the culture medium introduced and the electrode plate and the electrode insertion groove is formed integrally connected with the flow prevention plate and the electrode is inserted integrally with the electrode plate Cells are connected to the cell cover portion consisting of an outlet plate formed with a plurality of culture outlets for outflow of the culture solution and the stepped recessed cell seating portion is coupled to the bottom of the culture supply and the cell cover portion and the culture solution flows into the seated slide glass It is characterized by including a support .

In addition, the culture medium supply unit may be provided with a spill prevention packing unit formed to step around the culture medium dispersion unit to block the outflow of the culture solution.

In addition, the leakage preventing packing portion may be packed with silicon.

In addition, the electrode inserted into the electrode insertion groove may be made of a wire electrode.

In addition, a conductive material may be inserted into the electrode insertion groove to protect cells from electrolysis by-products of the electrode.

In addition, the conductive material may be formed of an agar gel.

In addition, the cell support may be provided with a spill prevention packing portion formed to step around the cell seat to block the outflow of the culture solution. At this time, the leakage preventing packing portion may be packed with silicon.

In addition, the cell seating portion may be a gasket of the same thickness as the slide glass is seated around the slide glass.

In addition, the gasket may be made of a silicon material.

In addition, the culture solution supply portion, the cell cover portion and the cell support portion may be made of a polycarbonate material.

In addition, the culture solution supply portion, the cell cover portion and the cell support portion may be fastened with bolts and nuts, respectively.

In addition, the cell culture apparatus using the cell culture complex stimulation chamber according to the present invention, the cell culture complex stimulation chamber according to the present invention, and the peristaltic pump for generating a flow of the culture solution to supply the culture solution into the cell culture complex stimulation chamber, Relieve the pulsation of the culture fluid flowing by the peristaltic pump is a culture medium buffer and the culture medium flowing out of the cell culture complex stimulation chamber and uniform culture medium flowing into the complex stimulation chamber is stored and the culture medium is supplied by the peristaltic pump It can be configured to include a culture reservoir.

In addition, the cell culture device may be configured to include a flow rate sensor installed between the culture medium buffer and the cell culture complex stimulation chamber to measure the flow of the culture medium.

In addition, the cell culture device is installed between the flow sensor and the peristaltic pump to monitor the flow sensor but compares the set value and the flow value of the flow sensor to set the flow rate of the peristaltic pump if the flow value is greater than the set value It can be configured to include a feedback controller to reduce the.

In addition, the preset value set in the feedback controller may have a volume flow rate of 50ml to 500ml per minute.

In addition, the cell culture device may be configured to include a constant temperature bath that is the culture medium buffer and the culture medium reservoir is seated to maintain a constant temperature of the culture medium.

In addition, the culture medium may be maintained at 37 ℃ by the constant temperature bath.

In addition, the cell culture apparatus may be configured to include a pH regulator connected to the culture medium reservoir to maintain a constant pH of the culture medium.

In addition, the pH of the culture solution can be kept constant by the carbon dioxide concentration of 5% moistened by the pH regulator.

According to the cell culture complex stimulation chamber and the cell culture device using the same according to the present invention as described above by controlling the electric field due to the shear stress and the potential difference that the cells are subjected to in vivo in the laboratory environment, it appears as the external environment changes It has a useful effect in observing cell responses.

In addition, by effectively dispersing the culture medium to form a uniform flow field, by circulating the culture medium itself to maintain the growth conditions of the cells, it is possible to study the reaction by various physical stimulus complex.

In addition, in performing the actual experiment, the experimenter can easily observe in real time on a microscope, so that the same cells can be tracked while observing the change or quantifying the change of the pattern over time. Contribute to the research and development of

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. First, it should be noted that the same components or parts in the drawings represent the same reference numerals as much as possible. In describing the present invention, detailed descriptions of related well-known functions or configurations are omitted in order not to obscure the gist of the present invention.

1 is an exploded perspective view of a cell culture complex stimulation chamber according to an embodiment of the present invention, Figure 2 is a combined perspective view, Figure 3 is a view showing a microscope observation region. In addition, Figure 4 is a schematic diagram of a cell culture apparatus according to an embodiment of the present invention, Figure 5a is a diagram showing a state in which shear stress acts on monolayer cells, Figure 5b is a state in which the electric field acts on the monolayer cells It is a figure which shows.

The cell culture complex stimulation chamber according to an embodiment of the present invention includes a culture solution supply part, a cell cover part, and a cell support part.

1 and 2, the culture solution supply unit is provided with a culture solution inlet 111 and a culture solution dispersion unit 112. At this time, the culture solution supply unit 110 may be made of a polycarbonate material.

The culture solution inlet 111 penetrates from one side of the culture solution supply unit 110 to the culture solution dispersion unit 112 formed on the other side of the culture solution supply unit 110.

The culture solution dispersion unit 112 is connected to the culture solution inlet 111, but is formed in a stepped shape to induce the dispersion of the introduced culture solution. At this time, the step depth of the culture dispersion portion 112 may be formed to 0.5mm.

That is, the culture solution introduced through the culture solution inlet 111 loses its original flow rate as the flow direction is vertically changed by the flow preventing plate 121 of the cell cover part 120 to be described later, and the culture solution dispersion unit The culture medium dispersed by the induction of 112 is moved to the cell support 130 by the flow pressure.

In addition, the culture medium supply unit 110 is formed stepped around the culture medium dispersion unit 112 to block the culture medium dispersed by the induction of the culture medium dispersion unit 112 flows out from the culture medium supply unit 110 to the outside. Outflow prevention packing unit 113 may be provided. At this time, the leakage preventing packing portion 113 may be packed with silicon.

The cell cover part 120 is provided with a flow preventing plate 121, an electrode plate 122 and the outlet plate 123. In this case, the cell cover part 120 may be made of a polycarbonate material.

The flow preventing plate 121 is coupled to the culture medium supply unit 110 provided with the culture medium dispersion unit 112 to cover the culture medium dispersion unit 112 and at the same time, the culture medium flowing through the culture medium inlet 111 To block flow.

The electrode plate 122 is integrally connected to the flow preventing plate 121, the electrode insertion groove 122a is formed on the upper surface is inserted into the electrode.

The electrode insertion groove 122a may be formed of two parallel grooves, and the electrode inserted into the electrode insertion groove 122a may be a wire electrode.

In addition, a conductive material may be inserted into the electrode insertion groove 122a to protect cells from electrolysis by-products of the electrode after the electrode is inserted. In this case, the conductive material may be formed of an agar gel.

The wire electrode may be seated in the electrode insertion groove 122a through the electrode inlet 122c and the electrode outlet 122d formed at both sides of the electrode insertion groove 122a.

The wire electrode may be connected to a power supply or a wave generator, and may apply electrical stimulation to monolayer cells by applying a direct current or alternating voltage to form a potential difference of a desired shape. That is, the slide glass 131a in which the cells are cultured may be seated at a corresponding position of the cell seating portion 131 of the cell support 130 to be described later corresponding to the wire electrodes so that a constant electric field may be applied to the cells. . At this time, the observation region of the microscope is between the positive electrode 122b to which the electrical stimulation is applied, as shown in FIG.

The outlet plate 123 is integrally connected to the electrode plate 122, and a plurality of culture solution outlets 123a through which the culture solution flows out are formed through the top and bottom of the outlet plate 123. The culture liquid outlet 123a is composed of four, so the width of the flow change is not large compared to when the culture liquid outlet is one, it is possible to secure a relatively wider and even flow field.

The cell support unit 130 is coupled to the bottom surface of the culture medium supply unit 110 and the cell cover unit 120 and is provided with a cell seating unit 131 therein. In this case, the cell support 130 may be made of a polycarbonate material.

The cell support unit 130 blocks the flow of the culture solution moving through the culture solution dispersing unit 112, but changes the flow direction of the culture solution to supply the cells with a uniform flow.

The cell seat 131 has a recessed shape formed stepped to a depth of 0.5 mm, and the slide glass 131a in which the cells are cultured is seated. In this case, the slide glass 131a may have a thickness of 1 mm, and two slide glasses 131a may be seated on the cell seat 131.

In addition, the cell seat 131 may have a gasket having the same thickness as the slide glass 131a around the slide glass 131a to secure an even laminar flow surface, wherein the gasket is made of silicon. Can be done.

In addition, the cell support 130 is a spill prevention step formed around the cell seat 131 to prevent the culture fluid flowing in the cell seat 131 flows out from the cell support 130 to the outside The packing part 132 may be provided. At this time, the leakage preventing packing portion 132 may be packed with silicon.

On the other hand, the culture medium supply unit 110, the cell cover portion 120 and the cell support portion 130 may be coupled by a fastening member, respectively. That is, the corresponding side surface of the cell cover unit 120 is coupled to the culture medium supply unit 110, and the cell support unit 130 is coupled to the bottom surface of the culture medium supply unit 110 and the cell cover unit 120 coupled thereto. . At this time, the fastening member may be made of a bolt and a nut.

Hereinafter, with reference to the accompanying drawings will be described in detail a cell culture apparatus using a cell culture complex stimulation chamber according to an embodiment of the present invention.

Cell culture apparatus according to an embodiment of the present invention, as shown in Figure 4, the cell culture complex stimulation chamber 100, the peristaltic pump 200, the culture medium buffer 300 according to an embodiment of the present invention And, it comprises a culture medium reservoir (400).

The cell culture complex stimulation chamber 100 is the same as the configuration of the cell culture complex stimulation chamber according to an embodiment of the present invention described above.

The peristaltic pump 200 is connected to the culture medium reservoir 400 to generate a flow of the culture solution stored in the culture medium reservoir 400, as shown in Figure 1 and 2, the cell culture complex stimulation chamber 100 Culture medium is supplied to the cells in the cell seat 131 in the). Therefore, the cells in the cell culture complex stimulation chamber 100 are subjected to shear stress proportional to the flow rate of the culture medium while maintaining appropriate growth conditions.

The culture medium buffer 300 is connected to the peristaltic pump 200, as shown in Figure 4, to relax and equalize the pulsation of the culture fluid flowing by the peristaltic pump 200 and the cell culture complex stimulation chamber 100 The moderated culture is supplied into

The culture medium reservoir 400 is stored with the culture solution flowed out from the cell culture complex stimulation chamber 100, and supplies the culture solution back into the cell culture complex stimulation chamber 100 by the peristaltic pump 200.

On the other hand, the cell culture device may be configured to include a flow sensor 500 for measuring the flow of the culture fluid.

The flow sensor 500 is installed between the peristaltic pump 200 and the cell culture complex stimulation chamber 100, and can measure the volume flow rate.

In addition, the cell culture device may be configured to include a feedback controller 600 is installed between the flow sensor 500 and the peristaltic pump 200.

The feedback controller 600 is composed of a Proportional-Integral Derivative Controller (PID controller), each coefficient of which can be optimized by the Ziegler-Nichols method.

The feedback controller 600 monitors the flow rate sensor 500, and automatically controls the speed of the peristaltic pump 200 by comparing the set value with the flow value of the flow rate sensor 500.

That is, when the flow value of the flow sensor 500 is greater than the set value of the feedback controller 600, the speed of the peristaltic pump 200 is decreased, and the flow value of the flow sensor 500 is the feedback controller 600. If less than the set value of) increases the speed of the peristaltic pump 200. In this case, the preset value set in the feedback controller 600 may be a volume flow rate of 50ml to 500ml per minute, the shear stress received by the actual cell in proportion to the volumetric flow rate may be 3dyne to 30dyne per cm 2. . Therefore, during the experiment, the specific shear stress can be kept constant within the range of the volume flow rate, or a specific type of shear stress pattern can be applied according to a preset input value.

In addition, the cell culture device may be configured to include a constant temperature water tank 700 to maintain a constant temperature of the culture.

The constant temperature water tank 700 may maintain the temperature of the culture solution at 37 ° C. in order to form an appropriate cell culture environment in which the culture medium buffer 300 and the culture medium storage tank 400 are seated.

In addition, the cell culture device may be configured to include a pH regulator 800 to maintain a constant pH of the culture.

The pH regulator 800 may be connected to the culture medium reservoir 400 may be made of a humidifier, it is possible to maintain the pH of the culture medium by carbon dioxide of 5% concentration moistened by the pH controller 800.

Hereinafter, with reference to the accompanying drawings will be described in detail the operation of the cell culture complex stimulation chamber and cell culture apparatus using the same according to an embodiment of the present invention.

Two slide glasses cultured with monolayer cells are fixed to the cell seating portion 131 of the cell support 130, as shown in FIG. As shown in Figure 4, the culture medium supplied to maintain the appropriate growth conditions of the cell is flowed by the peristaltic pump 200, and has a uniform flow without pulsation while passing through the culture medium buffer 300, Thereafter, it is supplied into the cell culture complex stimulation chamber (100).

At this time, the flow rate supplied into the cell culture complex stimulation chamber 100 is measured by a flow sensor 500 connected in front of the cell culture complex stimulation chamber 100. The flow value of the flow sensor 500 is compared with the set value of the feedback controller 600 for monitoring the flow sensor 500 and the speed of the pump is automatically controlled. That is, the feedback controller 600 reduces the speed of the peristaltic pump 200 when the flow value is greater than the set value through an optimized control method, and, conversely, when the flow value is smaller than the set value, the peristaltic pump 200 is controlled. To increase the speed. Therefore, the culture medium having a specific flow amount or the culture medium having a specific flow pattern can be supplied into the cell culture complex stimulation chamber 100.

1 and 2, the culture solution is introduced into the culture solution inlet 111 provided in the culture solution supply unit 110, the flow is blocked by the flow prevention plate 121 of the cell cover portion 120 As the flow direction is changed vertically, the original flow rate is lost, and is dispersed by the induction of the culture solution dispersion unit 112 provided in the culture solution supply unit 110. In addition, the culture fluid flowing through the culture medium dispersion 112 by the flow pressure is again blocked by the cell support 130, the flow direction is changed vertically, so that the culture medium having a uniform flow is the cell seat Cells fixed at 131 are supplied.

In order to block the outflow of the culture medium and to supply the culture medium, a flow preventing packing unit is provided around the culture dispersion unit 112 and the cell support unit 130, and a uniform fluidized bed between the cell cover unit 120 and the cell support unit 130. Gaskets having the same thickness as the slide glass 131a are provided in the cell seat 131 to form a stream.

Since the culture solution flows out through the plurality of culture solution outlets 123a formed in the cell cover part 120, the width of the flow change is not large compared to when the culture solution outlet is one. Thus, a relatively wider and even flow field can be secured.

As shown in 5a, a culture medium flowing between the cell cover part 120 and the cell support part 130 has a velocity gradient formed by friction between the cell cover part 120 and the cell support part 130, Shear stress is applied to the cells fixed to the cell seat 131.

At the same time, by applying a direct current or alternating voltage through an electrode inserted into the cell cover portion 120 to form a potential difference of a desired shape, as shown in FIG. 5B, a constant electric field is applied to the cells.

Observed in real time under a microscope, but by independently controlling the electric field caused by the shear stress and potential difference received by the cells, the difference in shape and mobility of cells, generation and differentiation of cells, expression level of specific proteins and genetic information Various aspects of change, such as activation changes, can be quantified.

As shown in FIG. 4, the culture solution discharged from the cell culture complex stimulation chamber 100 is stored in the culture solution reservoir 400 and circulated again by the peristaltic pump 200.

On the other hand, the temperature is kept constant by the constant temperature water tank 700 in order to form an appropriate cell culture environment, the pH is controlled by carbon dioxide moistened by the pH controller 800.

As described above with reference to the drawings illustrating a cell culture complex stimulation chamber and a cell culture device using the same according to the present invention, the present invention is not limited by the embodiments and drawings disclosed herein, the present invention Of course, various modifications may be made by those skilled in the art within the scope of the technical idea.

1 is an exploded perspective view of a cell culture complex stimulation chamber according to an embodiment of the present invention.

Figure 2 is a perspective view of the combined cell culture complex stimulation chamber according to an embodiment of the present invention.

3 is a view showing a microscope observation region of the cell culture complex stimulation chamber according to an embodiment of the present invention.

Figure 4 is a schematic diagram of a cell culture apparatus according to an embodiment of the present invention.

Figure 5a is a diagram showing the action of the shear stress on the monolayer cells.

Figure 5b is a diagram showing the action of the electric field on the monolayer cells.

<Description of Symbols for Main Parts of Drawings>

100: cell culture complex stimulation chamber 110: culture medium supply unit

111: culture liquid inlet 112: culture liquid dispersion unit

113,132: spill prevention packing part 120: cell cover part

121: flow prevention plate 122: electrode plate

122a: electrode insertion groove 122b: electrode

122c: electrode inlet 122d: electrode outlet

123: outlet plate 123a: culture liquid outlet

130: cell support 131: cell seat

131a: slide glass 200: interlocking pump

300: culture medium buffer 400: culture medium reservoir

500: flow sensor 600: feedback controller

700: constant temperature water bath 800: pH controller

900: monolayer cell

Claims (20)

A culture solution supply unit having a culture solution inlet and a stepped recessed culture solution dispersion unit connected to the culture solution inlet to induce dispersion of the introduced culture solution; An electrode plate coupled to the culture solution supply unit to cover the culture solution dispersion unit and blocking the flow of the introduced culture solution, an electrode plate having an electrode insertion groove which is integrally connected to the flow prevention plate and into which the electrode is inserted, and the electrode plate A cell cover part which is integrally connected with the cell and comprises an outlet plate having a plurality of culture fluid outlets through which the culture fluid flows out; And Cell culture complex stimulation chamber is coupled to the culture solution supply and the bottom surface of the cell cover portion, characterized in that it comprises a cell support having a stepped recessed cell seat portion in which the culture fluid flows into the slide glass seated. The method of claim 1, Cell culture complex stimulation chamber, characterized in that the culture medium supply unit is formed stepped around the culture medium dispersion unit is provided with an outflow prevention packing to block the outflow of the culture medium. 3. The method of claim 2, The leakage preventing packing portion cell culture complex stimulation chamber, characterized in that the packing. The method of claim 1, The cell culture complex stimulation chamber, characterized in that the electrode is inserted into the electrode insertion groove made of a wire electrode. The method of claim 1, The electrode insertion groove cell culture complex stimulation chamber, characterized in that the conductive material is inserted to protect the cells from the electrolysis by-products of the electrode. The method of claim 5, The conductive material is a cell culture complex stimulation chamber, characterized in that consisting of agar gel (ager gel). The method of claim 1, The cell support part is formed in a step around the cell mounting portion cell culture complex stimulation chamber, characterized in that the leakage prevention packing portion is provided to block the outflow of the culture medium. The method of claim 7, wherein The leakage preventing packing portion cell culture complex stimulation chamber, characterized in that the packing. The method of claim 1, The cell seating cell culture complex stimulation chamber, characterized in that the gasket of the same thickness as the slide glass is seated around the slide glass. The method of claim 9, The gasket is a cell culture complex stimulation chamber, characterized in that made of a silicon material. The method of claim 1, Cell culture complex stimulation chamber, characterized in that the culture medium supply portion, the cell cover portion and the cell support portion made of a polycarbonate material. The method of claim 1, The culture medium supply stimulation chamber, characterized in that the culture medium supply portion, the cell cover and the cell support is fastened with a bolt and a nut, respectively. The cell culture complex stimulation chamber according to any one of claims 1 to 12; A peristaltic pump generating a flow of the culture solution to supply the culture solution into the cell culture complex stimulation chamber; A culture medium buffer for introducing a uniform culture into the cell culture complex stimulation chamber by relieving the pulsation of the culture fluid flowing by the peristaltic pump; And Cell culture apparatus characterized in that it comprises a culture medium storage tank for storing the culture solution flowing out of the cell culture complex stimulation chamber and supply the culture solution by the peristaltic pump. The method of claim 13, And a flow rate sensor installed between the culture medium buffer and the cell culture complex stimulation chamber to measure a flow rate of the culture solution. 15. The method of claim 14, A feedback controller installed between the flow sensor and the peristaltic pump to monitor the flow sensor, and comparing the set value with the flow value of the flow sensor to reduce the speed of the peristaltic pump if the flow value is greater than the set value. Cell culture apparatus, characterized in that configured by. The method of claim 15, The preset value set in the feedback controller is a cell culture device, characterized in that the volume flow rate is 50ml to 500ml per minute. The method of claim 13, The culture medium buffer device and the culture medium reservoir, the cell culture device characterized in that it comprises a constant temperature bath for maintaining a constant temperature of the culture medium. The method of claim 17, Cell culture apparatus characterized in that the culture medium is maintained at 37 ℃ by the constant temperature water bath. The method of claim 13, Cell culture apparatus characterized in that it comprises a pH regulator connected to the culture reservoir to maintain a constant pH of the culture. The method of claim 19, Cell culture apparatus characterized in that the pH of the culture solution is kept constant by the carbon dioxide concentration of 5% moistened by the pH regulator.

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