KR101566083B1 - circulating culture system - Google Patents

Abstract

The present invention relates to a method for producing a cell culture comprising a plurality of double culture containers; A circulation pipe connecting the adjacent culture vessels so that the plurality of culture vessels are interconnected and connecting a pair of culture vessels disposed at both ends; A pump connected to the circulation pipe and supplying a fluid to the culture container along the circulation pipe; Pump driving means for driving the pump; A weight sensor installed in the culture container and measuring a changed weight value of the culture container during a culture process; And flow control means for selectively operating any one of the pump driving means according to a signal measured by the weight sensor. ≪ / RTI >

Description

Circulating culture system "

The present invention relates to a circulation culture system, and more particularly, to a circulation culture system in which a plurality of double culture vessels capable of three-dimensional culture can be connected to a circulation tube to set a desired circulation tube and a flow rate, .

As medical technology and biotechnology have rapidly developed, studies on cell engineering, which is a technique to observe and characterize life activity at the cellular level, are actively being carried out.

However, since the cells cultured by the conventional cell culture technique are single-layer cells spread in the two-dimensional direction, it is impossible to construct a three-dimensional tissue equivalent to that in the living body, and a specific function of the cells in the body can be maintained for a long time There is a problem that the accuracy of the simulation is not ensured.

In recent years, in order to solve such a problem, cultivation of spoloid, which is a three-dimensional tissue of cells having functions equivalent to those in vivo, has attracted attention. In particular, as the technology of stem cells develops, various methods have been attempted for application to the study of various kinds of differentiation by culturing embryonic stem cells three-dimensionally.

For example, Japanese Unexamined Patent Publication (Kokai) No. 6-327462 discloses a technique of seeding a plurality of single cells in a well having a bottom surface in a funnel shape, and coagulating and dividing the single cells on the bottom surface to cultivate the spleoid. However, the above technology does not have a place of spleen caused by the culture, and it is difficult for the spleoid to be fixed to the cell culture construct or the cell culture container. Therefore, at the time of exchanging the medium, there is a problem that it is difficult to keep the good culture environment because the spleoid disappears together with the medium or the medium exchange operation becomes considerably troublesome.

Therefore, there is a need for a specific mechanism capable of simultaneously providing a three-dimensional culture and a circulation culture while providing a culture condition similar to a living body while maintaining a good culture environment.

KR 10-1235378 (registration number)

The present invention provides a circulation culture system in which a plurality of double culture vessels capable of three-dimensional culture can be connected to a circulation tube, and a desired circulation tube and a flow rate can be set and selectively controlled to circulate the culture solution.

The present invention relates to a method for producing a cell culture comprising a plurality of double culture containers; A circulation tube connecting each of the two double culture vessels adjacent to each other so as to interconnect the plurality of double culture vessels and connecting a pair of double culture vessels disposed at both ends; A pump connected to the circulation tube and supplying the fluid to the dual culture vessel along the circulation tube; Pump driving means for driving the pump; A weight sensor installed in the dual culture vessel and measuring a changed weight value of the dual culture vessel during a culture process; And flow control means for selectively operating any one of the pump driving means according to a signal measured by the weight sensor. And a circulating culture system.

The present invention is capable of three-dimensional culture and can provide culture conditions similar to living bodies, and circulation can be performed by sequentially, selectively, and quantitatively controlling the circulation of the culture liquid.

In addition, the present invention can be applied to an apparatus for sequentially, selectively, and quantitatively controlling various types of liquid transfer in circulation apparatuses such as various laboratories, chemical production plants, wastewater treatment facilities as well as cell culture.

FIG. 1 is a block diagram showing the configuration of a circulation culture system according to an embodiment of the present invention.
FIG. 2 is a block diagram showing a configuration of a circulation culture system according to an embodiment of the present invention.
3 is a perspective view of a dual culture container according to one embodiment of the present invention.
4 is a cross-sectional view of a dual culture container according to an embodiment of the present invention.
5 is an exploded perspective view of a dual culture container according to one embodiment of the present invention.
FIG. 6 is a view showing a result of culturing various cells using a circulating culture system according to an embodiment of the present invention.

The present invention relates to a circulation culture system, comprising: a plurality of dual culture vessels; A circulation tube connecting each of the two double culture vessels adjacent to each other so as to interconnect the plurality of double culture vessels and connecting a pair of double culture vessels disposed at both ends; A pump connected to the circulation tube and supplying the fluid to the dual culture vessel along the circulation tube; Pump driving means for driving the pump; A weight sensor installed in the dual culture vessel and measuring a changed weight value of the dual culture vessel during a culture process; And flow control means for selectively operating any one of the pump driving means according to a signal measured by the weight sensor. .

Here, a pair of dual culture vessels disposed at both ends means a pair of double culture vessels disposed outermost among a plurality of double culture vessels.

More specifically, the present invention relates to a circulation culture system capable of selectively controlling a desired circulation tube among a plurality of circulation tubes.

The dual culture container may further include an inlet formed at one side of the dual culture container for introducing the fluid into the inside thereof and an outlet formed at the other side of the dual culture container for discharging the fluid introduced into the dual culture container, Is connected to the inlet of the dual culture container and the other side is connected to the outlet of the neighboring dual culture container.

In addition, the circulation pipe connecting the pair of dual culture vessels disposed at both ends may include at least one fluid shutoff valve, and the circulation pipe may further include a flow sensor.

Also, the dual culture container of the present invention means a cell culture unit capable of culturing cells, cells, tissues or microorganisms of human or copper / plant.

Particularly, the dual culture container has an outer container opened on its upper surface and capable of containing a culture liquid; A container cover coupled to an upper end of the outer container to prevent infiltration of microorganisms; And an inner container which is located inside the outer container and is capable of holding a culture liquid, the inner container being detachable from the outer container; Wherein the inner container may include a bottom surface and a mesh-shaped side where cell attachment is possible, and the outer container and the inner container may be made of different materials, respectively.

Further, it may further include a power supply means for supplying power to the pump driving means, the weight sensor, and the flow rate control means.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, It is to be understood that equivalents and modifications are possible.

FIG. 1 is a configuration diagram of a circulation culture system according to an embodiment of the present invention. FIG. 2 is a configuration diagram of a circulation culture system according to an embodiment of the present invention. FIG. FIG. 5 is an exploded perspective view of a dual culture container according to an embodiment of the present invention, and FIG. 6 is an exploded perspective view of a dual culture container according to an embodiment of the present invention. FIG. 4 is a view showing the result of culturing various cells using a circulation culture system according to one embodiment. FIG. The configuration and function of the circulation culture system of the present invention will be described in detail with reference to FIGS. 1 to 6. FIG.

The present invention relates to a circulation culture system (10) capable of circulating a culture liquid by selectively controlling a desired circulation tube (200).

Referring to FIGS. 1 and 2, the present invention includes a plurality of dual culture vessels 100, a plurality of double culture vessels 100 adjacent to each other such that the plurality of dual culture vessels 100 are interconnected, A circulation tube 200 connected to the pair of culture vessels 100 arranged in the circulation tube 200 and connected to the circulation tube 200 so that the fluid is supplied to the double culture vessel 100 along the circulation tube 200 A pump driving means 310 for driving the pump 300 and a variable weighing device installed in the dual culture container 100 and measuring a changed weight value of the dual culture container 100 during the culturing process And a flow rate control unit 500 for selectively operating any one of the pump driving units 310 according to the signals measured by the weight sensor 400 and the weight sensor 400.

In the present invention, the dual culture container 100 may be a cell culture container having a dual structure and may be a culture container capable of culturing cells, tissues or microorganisms of a human or a plant / plant, and is not limited to any one .

In particular, in the present invention, the dual culture container 100, the circulation pipe 200, the pump 300, the pump driving means 310, and the weight sensor 400 are formed in plurality, and the circulation pipe 200, 300, pump driving means 310 and weight sensor 400 correspond to the number of the dual culture vessels 100.

Here, the dual culture container 100 includes an inlet 111 and an outlet 112, and the inlet 111 and the outlet 112 may be installed to face each other. For example, an inlet 111 for introducing fluid into one side of the dual culture vessel 100, and an outlet 112 for discharging the introduced fluid to the other side of the dual culture vessel 100 can do.

As shown in FIG. 1, for example, the dual culture container 100 may include three, and for convenience, a first culture container 101, a second culture container 102, a third culture container 103 do.

The first culture vessel is connected to the second culture vessel 102 by the circulation tube 200 and is connected to the circulation tube 200 for connecting the first culture vessel 101 and the second culture vessel 102 for convenience. The circulation pipe 200 connecting the second culture container 102 and the third culture container 103 is referred to as a first circulation pipe 201 and the second circulation pipe 202 and the third culture container 103 And the first culture container 101 are referred to as a third circulation pipe 203. [

Particularly, one side of the circulation pipe 200 is connected to the inlet port 111 of the culture container, and the other side of the circulation pipe 200 can be connected to the outlet port 112 of the neighboring culture container. The first circulation pipe 201 may be connected to the inlet 111 of the first culture vessel 101 and the outlet 112 of the second culture vessel 102.

In addition, the circulation pipe 200 may further include a flow rate sensor. The flow rate sensor is for maintaining the flow rate and the flow rate of the circulating culture liquid constant when the culture liquid circulates in the culture container. For example, the flow rate sensor may be a pressure resistive milliliter flow sensor.

Also, the circulation tube 200 connecting the pair of dual culture vessels 100 disposed at both ends may include at least one fluid shutoff valve 210. For example, the third circulation pipe 203.

Here, the fluid shutoff valve 210 is for blocking the circulation of the fluid, and may be a conventional stop valve.

In addition, a pump 300 and a pump driving means (not shown) for driving the pump 300 are provided between the plurality of dual culture vessels 100 to supply the fluid to the dual culture vessel 100 along the circulation pipe 200 310 and may be connected to the circulation pipe 200. The first circulation pipe 201 may be connected to the first pump 301 and the second circulation pipe 202 may be connected to the second pump 302 and the third circulation pipe 203 may be connected to the third pump 303, And may include a first pump driving means 311 for driving the first pump 301 and a second pump driving means 312 for driving the second pump 302. [ And a third pump driving means for driving the third pump 303. [

As an example, the pump 300 used in the present invention may be a piezo pump. Here, the piezoelectric pump can convert electric energy into mechanical energy using a piezoelectric phenomenon so that the fluid can flow. That is, the piezoelectric pump can pressurize the fluid using a piezoelectric transducer.

When a voltage is applied to the piezoelectric transducer, the electrostriction effect of distorting the piezoelectric crystal constituting the piezoelectric transducer occurs. The piezoelectric pump can move the fluid air in a certain direction by using the mechanical energy generated due to the change of the position of the piezoelectric crystal due to the electrostriction effect.

The piezoelectric transducer means the pump driving means 310 in the present invention.

Particularly, the pump 300 is driven by the pump driving means 310, and the pump driving means 310 operates according to a signal measured and received by the weight sensor 400 installed in the dual culture container 100 So that the pump 300 can be driven.

The weight sensor 400 is installed in the dual culture container 100 and converts the weight measured by the sensor for measuring the weight of the fluid in the dual culture container 100 into an electric signal to generate the electric signal, To the signal processing unit. The weight sensor 400 installed in the first culture vessel 101 is connected to the first weight sensor 401 and the weight sensor 400 installed in the second culture vessel 102 is connected to the second weight sensor 402, The weight sensor 400 provided in the culture container 103 is referred to as a third weight sensor 403. [ In the present invention, the weight measurement range of the weight sensor 400 may be 0.2 to 130 g.

In particular, the present invention may include a flow control means 500 to selectively operate the pump driving means 310 among a plurality of pump driving means 310 according to a signal measured by the weight sensor 400.

Here, the flow rate control unit 500 may process the electric signal transmitted from the weight sensor 400 to control the circulation / stoppage of the fluid and the amount of fluid circulated.

In addition, the dual culture container 100 of the present invention may be a cell culture container having a dual structure, as shown in Figs. 3 to 5. More specifically, the dual culture container 100 of the present invention is opened and comprises an outer container 110 capable of containing a culture medium; A container cover 120 coupled to an upper end of the outer container 110 to prevent infiltration of microorganisms; And an inner container (130) located inside the outer container (110) and capable of containing the culture liquid, the inner container being detachable from the outer container (110); As shown in FIG.

The outer container 110 is configured such that an upper surface thereof is opened so as to contain the culture liquid, and the container cover 120 coupled to the upper end of the outer container 110 can prevent the infiltration of microorganisms. Also, the outer container 110 and the container cover 120 may be made by injection molding or blow molding of synthetic resin which is light-transmitting and elastic material.

In this case, the culture medium may be a cell line of an animal (human), a plant, or a medium suitable for the purpose of microbial cultivation, but is not limited thereto. For example, when an animal (human) cell line is cultured, DMEM (Dulbecco's Modified Eagle's Medium) may be used.

In addition, the dual culture container 100 of the present invention enables three-dimensional culture.

More specifically, as shown in FIGS. 4 and 5, the inner container 130 of the present invention may include a cell-attachable bottom surface and a mesh-shaped side surface. Since the side surface of the inner container 130 of the present invention is formed in a mesh shape, the cell culture can be performed on the side surface, and three-dimensional cell culture can be performed. In particular, the present invention is capable of culturing in a three-dimensional manner, thereby creating an in vivo mimivative atmosphere and reducing the difference between in vitro and in vivo. Further, the present invention can be presented as a method for reducing animal experiments.

In addition, the inner container 130 is present inside the outer container 110, and the effect of preventing the outer container 110 from being mixed at the time of culturing the heterogeneous cell can be exerted.

Further, the mesh shape is a mesh-like shape and is not particularly limited as long as it is a form for preventing mixing of cells cultured in the outer container 110 and the inner container 130.

Also according to a particular embodiment, the inner container 130 of the present invention includes a bottom surface and a side surface capable of cell attachment, and both the bottom surface and the side surface may be mesh-shaped. In this case, fibronectin may be coated on the outer bottom of the inner container 130, and cells may be mixed with methylcellulose in the inside. Such a structure is effective in observing the infiltration and migration force of cells. It is also possible to observe whether it is influenced by signaling factors exiting to the outside, and it is possible to observe the infiltration and migration force of a cell under a microscope without any other operation.

Here, fibronectin is a glycoprotein present on the surface of normal fibroblasts or endothelial cells, the basal membrane of the intestinal epithelial cells, and various other cells. Although fibronectin has a variety of functions, the present invention is useful for promoting adhesion and expansion of cultured cells on Petri dishes.

In addition, the mesh-shaped inner container 130 according to the present invention allows cells to be separated from each other and a cell capable of forming a colony, which is characterized in that cells cohere with each other. .

The material of the outer container 110 and the inner container 130 of the present invention may be any one of PS (polystyrene), PP (polypropylene), and acryl Lt; / RTI >

In particular, the outer container 110 and the inner container 130 may be made of different materials. This allows simultaneous incubation of adherent cells and nonadherent cells or heterologous nonadherent cells.

As an example of the present invention, PP (Polypropylene) is used for the outer container 110 and PS (Polystyrene) is used for the inner container 130. At this time, the non-adherent cells were cultured in the outer container 110 and the adherent cells were cultured in the inner container 130. In addition, the cell signaling factors can be secreted into the culture medium and the interaction can be observed.

Here, the cell signal transduction factor may be a growth factor, cytokine, or angiogenic factor (VEGF), and it is natural that the cell signal transduction factor that achieves such a purpose falls within the scope of the present invention .

In addition, when the outer container 110 of the present invention is made of polypropylene (PP), unlike a conventional cell culture dish, the strength of the incubator is increased. In addition, since it can be reused, not only the cost reduction effect but also the non-adherent cell culture can be made easier than the adherent cell. However, in the case of PP (polypropylene) material, adhesion of adhering cells to the container can be achieved by a simple polyglycine coating, so that it is possible to co-culture different adherent cells in the outer container 110 and the inner container 130, Adherent cells can also be cultured.

Also, the inner container 130 of the present invention can be easily separated from the outer container 110. Therefore, it is convenient to separate only the inner container 130 and move it under different culture conditions, and it is easy to collect the cells of the inner container 130 and the outer container 110, respectively.

In addition, the dual culture container 100 of the present invention may be formed to have a size of 1 to 3 cm, which is 5 to 20 cm in diameter, so as to facilitate pouch culture, It is not limited to the size. As an example, a double culture container having a diameter of 5 to 6 cm and a height of 1 to 9 cm can be used. The accommodating portion 700 is for accommodating the dual culture container 100 and is determined depending on the size of the dual culture container 100. The size of the accommodating portion 700 may be limited to any size that can accommodate the dual culture container 100 It does not.

In a specific aspect, the dual culture container 100 of the present invention may further include a gas supply unit 121. The gas supply unit 121 is a passage configured to allow air required for culture to enter and exit, and a filter 122 may be provided therein. The filter 122 allows the microorganisms contained in the air to be caught inside the air passage so that only the air can flow in and out of the dual culture container 100.

Here, the gas supplied through the gas supply unit 121 may be oxygen or carbon dioxide. More specifically, oxygen is required for animal cells, and carbon dioxide for plant cells. Of course, nitrogen or other gases can also be supplied.

Further, the gas supply unit according to the present invention can be opened and closed. More specifically, when the culture liquid is supplied to and discharged from the inlet port 111 and the outlet port 112 according to the present invention, gas is supplied through the gas supply port, and gas is supplied to and discharged from the inlet port 111 and the outlet port 112 The cell can be cultured by blocking the gas supply part 121. [ As an example, a conventional tape can be used to block the gas supply part 121.

For example, when cells and plants or microorganisms are cultured under special conditions such as hypoxic conditions, the gas supply unit 121 provided in the container cover 120 is shut off and the culture solution inlet and outlet 112) can be used to inject and discharge a gas mixture of low oxygen. In this case, the inner container 130 may be placed in a general cell culture section and filled with a culture medium, and then configured in the form of a culture container of a triple-holding container provided with the outer container 110 provided by the present invention.

Also, a culture liquid supply device and a gas supply device may be connected to the outside of the cell culture part, and the culture liquid and gas may be supplied into the dual culture container 100. At this time, the gas supply device may be a conventional gas regulator, for example, a Herison gas regulator may be used.

Further, the circulating culture system 10 of the present invention circulates the culture liquid through the circulation unit and has a temperature sensor in the cell culture unit, so that a temperature suitable for culturing cells or tissues can be set and maintained.

For example, 30 to 50 DEG C or 40 DEG C to maintain the temperature of the culture liquid.

In addition, the circulating culture system 10 of the present invention further includes a receiving portion 700 capable of receiving the dual culture container 100, and the receiving portion 700, as shown in FIG. 2, And is easily configured to stack a plurality of the dual culture containers 100.

At this time, the receptacle 700 may be formed in a shelf shape so as to easily accommodate a plurality of cell culture units, and a hole may be formed at a position corresponding to the inlet 111 and the outlet 112 of the cell culture unit . And a plurality of cell culture units may be connected to the circulation tube 200 through the holes.

In addition, at least one dual culture container 100 of the plurality of dual culture vessels 100 may further include a fluid supply unit capable of supplying fluid into the incubator, and the pump driving unit 310, the weight sensor 400 And power supply means 600 for supplying power to the flow rate control means 500. [

In addition, the flow rate control means 500 of the present invention may further include a control time setting unit to set the control time. When the operation of the circulation culture system 10 is completed including the operation state unit, The pump 300, or the weight sensor 400 is faulty.

Hereinafter, the effects of the present invention will be described in detail with reference to experimental examples.

< Experimental Example >

Experimental Example  1. Using the circulating culture system of the present invention, various other cell cultures

In the present inventor's example, four kinds of cells were cultured in order to observe the three-dimensional culture.

Four types of cells, the mouse normal human fibroblast cell NIH 3T3 cell line (ATCC), lipid precursor 3T3-L1 (ATCC), human kidney cancer cell 786-o (ATCC) and human hepatoma cell hepG2 Two-dimensional culture was carried out on the culture dish of the invention.

Next, they were treated with trypsin (Gibco, USA) to separate into single cells and then mixed with methylcellulose (1.4%, R & D, USA) so as to have a concentration of 5 × 10 ⁴ cells / ml. And cultured in a dual culture container (100) as a culture container. Specifically, cultivation was prepared by planting in the inner container 130 of the dual culture container 100, respectively.

Each of the four dual culture vessels 100 was then accommodated in the receptacle 700 of the circulation culture system 10 and mounted thereon and incubated at 37 DEG C for 72 hours while circulating the culture at a rate of 7 mL / min. Then, cell viability and culture conditions were stained with hematoxylin (Sigma, USA) to observe the cultured cells.

As a result, as shown in FIG. 6, it was observed that the cells were cultured three-dimensionally on the side surface of the inner container 130.

In addition, the cell culture system of the present invention was used to circulate the culture solution sequentially, selectively or quantitatively, so that three-dimensional cell culture could be performed smoothly

10: Circulating culture system
100: Dual culture container 101: First culture container
102: Second culture container 103: Third culture container
110: outer vessel 111: inlet
112: Outlet
120: container cover
121: gas supply unit 122: filter
130: inner container
200: circulation pipe 201: first circulation pipe
202: second circulation pipe 203: third circulation pipe
210: fluid shutoff valve
300: Pump 301: First pump
302: second pump 303: third pump
310: pump driving means 311: second pump driving means 312:
312: second pump driving means 313: third pump driving means
400: weight sensor 401: first weight sensor
402: second weight sensor 403: third weight sensor
500: flow rate control means
600: Power supply
700:

Claims (9)

A plurality of double culture vessels;
A circulation pipe connecting each of the two dual culture vessels so that the plurality of double culture vessels are interconnected and connecting a pair of double culture vessels disposed at both ends of the plurality of double culture vessels;
A pump connected to the circulation tube and supplying the fluid to the dual culture vessel along the circulation tube;
Pump driving means for driving the pump;
A weight sensor installed in the dual culture vessel and measuring a changed weight value of the dual culture vessel during a culture process; And
Flow rate control means for selectively operating any one of the pump driving means according to a signal measured by the weight sensor; / RTI &gt;
The dual culture vessel
A container cover which is opened on an upper surface and can contain a culture liquid, a container cover coupled to an upper end of the outer container to prevent infiltration of microorganisms, and a bottom cover And a mesh-shaped side, said inner vessel being detachable from said outer vessel. The method according to claim 1,
An inlet formed at one side of the double culture container and introducing the fluid into the inside; And
An outlet formed on the other side of the dual culture container and discharging the fluid introduced therein; &Lt; / RTI &gt; 3. The method of claim 2,
Wherein one end of the circulation tube is connected to the inlet of the dual culture vessel and the other side is connected to the outlet of the adjacent dual culture vessel. The method according to claim 1,
Wherein the circulation tube connecting the pair of dual culture vessels disposed at both end of the end includes at least one fluid shutoff valve. The method according to claim 1,
Wherein the circulation tube further comprises a flow rate sensor. delete delete The method according to claim 1,
Wherein the outer container and the inner container are made of different materials. The method according to claim 1,
Further comprising power supply means for supplying power to the pump driving means, the weight sensor and the flow rate control means.

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