Microfluidic chip for co-culturing multiple three-dimensional cells

Abstract

Provided is a microfluidic chip for co-culturing multiple three-dimensional cells, the microfluidic chip comprising: a central channel having a predetermined space formed therein; a pair of first channels communicating with the central channel while inclined at a first angle in two directions on one side of the central channel; a pair of second channels communicating with the central channel while inclined at a second angle in two directions on the one side of the central channel; and a third channel which is located between the pair of second channels on the one side of the central channel and can communicate with the central channel, wherein a first cell mixture flows into the first channel and moves along the central channel, a second cell mixture flows into the second channel and moves along the central channel, and a third cell mixture flows into the third channel and moves along the central channel. Each of the first cell mixture, the second cell mixture, and the third cell mixture maintains the same moving cross section while moving along the central channel, and a stem cell mixture retaining the cross sections is discharged from an outlet of the central channel.

Description

Microfluidic chip for 3D multi-cell co-culture

[0001] The present invention relates to a microfluidic chip for three-dimensional multi-cell co-culture.

[0002] All cells that make up the body are created based on embryonic cells, and the three layers of ectoderm, mesoderm, and endoderm are the most basic types of cells that organize our body.

[0003] The ectoderm forms the outer layer, the mesoderm forms the middle layer, and the endoderm forms the inner layer.

[0004] The drawing shown in Figure 3 shows the ectoderm wrapping the endoderm to form a shell, and normally, the ectoderm becomes a shell and wraps the endoderm.

[0005] The three layers are known to form their own development.

[0006] However, there is no technology to culture cells while maintaining these cross-sections intact.

[0007] That is, when culturing cells and conducting development and differentiation studies, there is a problem with accuracy if the experiment is conducted without forming a stem cell mixture that has a cross-section in which the ectoderm is located surrounding the endoderm and the mesoderm is located in the center.

[0008] Accordingly, the purpose of the present invention is to derive a stem cell mixture having a cross-section similar to that of an embryonic cell.

[0009] To this end, the purpose is to provide a technology for controlling the flow rate of each injected cell to discharge a stem cell mixture with an optimal cross-section through various experiments and fluid analysis simulations.

[0010] The purpose of the present invention is to form a stem cell mixture similar to the cross-section of embryonic cells.

[0011] The purpose of the present invention is to conduct more accurate culture, development, and differentiation research using such stem cell mixture.

[0012] A stem cell device comprising: a central channel having a predetermined space formed therein; a pair of first channels inclined at a first angle in both directions on one side of the central channel and communicating with the central channel; a pair of second channels inclined at a second angle in both directions on one side of the central channel and communicating with the central channel; and a third channel positioned between the pair of second channels on one side of the central channel and capable of communicating with the central channel, wherein a first cell mixture flows into the first channel and moves along the central channel, a second cell mixture flows into the second channel and moves along the central channel, and a third cell mixture flows into the third channel and moves along the central channel, and wherein when the first cell mixture, the second cell mixture, and the third cell mixture move along the central channel, the cross-sections along which they move are maintained as they are, and the stem cell mixture with the maintained cross-section is discharged from the outlet of the central channel.

[0013] According to the present invention having the above-described configuration, a stem cell mixture having a cross-section almost similar to that of embryonic cells can be obtained.

[0014] According to the present invention having the above-described configuration, there is an advantage in that the stem cell mixture having the above-described cross-section, i.e., morphological characteristics, is cultured in a laboratory, and the results of research on development and differentiation obtained through culture are likely to be similar to the results of development and differentiation occurring in an actual human body.

[0015] Figure 1 is a drawing of the overall configuration of the present invention;

[0016] Figure 2 is a drawing of the overall configuration of the present invention in which a separate control unit is implemented.

[0017] Figure 3 is a drawing of the shape of the ectoderm, endoderm, and mesoderm, known as embryonic cells.

[0018] The right side of Fig. 4 is a schematic diagram of an actual embryonic cell, and the left side of Fig. 4 is a diagram of a cross-section of an embryonic cell to be implemented by the present invention.

[0019] Figure 5 is a drawing of a schematic shape of a cross-sectional shape of a stem cell mixture discharged from the exit of the central channel, obtained by performing a flow analysis simulation.

[0020] Figure 6 is a schematic diagram of one embodiment of the present invention in which an oil supply port is located;

[0021] Figure 7 shows a cross-section of the flow analysis simulation results in which the first cell mixture (including ectoderm), the second cell mixture (including endoderm), and the third cell mixture (including mesoderm) move along each channel and are then discharged from the outlet of the central channel, and Figure 8 is a drawing showing a cross-section of the flow analysis simulation results.

[0022] Hereinafter, a preferred embodiment of the microfluidic chip for three-dimensional multi-cell co-culture according to the present invention will be described with reference to the attached drawings.

[0023] To help understand the present invention, first, referring to Figure 3, the shapes of the ectoderm, endoderm, and mesoderm, known as embryonic cells, are examined as follows.

[0024] All cells that make up the body are created based on the embryonic cells shown in Figure 3, and the three layers of ectoderm, mesoderm, and endoderm are the most basic types of cells that make up our body.

[0025] The ectoderm forms the outer layer, the mesoderm forms the middle layer, and the endoderm forms the inner layer.

[0026] Typically, the ectoderm forms a shell and envelops the endoderm. The diagram in Figure 3 shows the ectoderm enveloping the endoderm to form a shell. It is known that each of the three layers develops independently.

[0027] It is known that the tissue organs that develop and become complete in each layer grow into the inner layer of the digestive system, liver, pancreas, and lungs in the case of the endoderm.

[0028] The mesoderm is known to develop into the circulatory system, lungs (the shell layer), skeletal system, and muscular system, while the ectoderm is known to develop into hair, nails, skin, and the nervous system.

[0029] The right side of Figure 4 is a schematic diagram of the embryonic cells described above.

[0030] The left drawing of Fig. 4 is a cross-section of an embryonic cell to be implemented by the present invention, and is a cross-section of a fluid analysis simulation that is intended to be implemented as similarly as possible to the cross-section of an actual embryonic cell.

[0031] That is, in the past, one or two types of cells were sequentially injected, that is, a cell mixture containing each of ectoderm, endoderm, and mesoderm was injected, and cells were formed three-dimensionally in the shape of a spheroid.

[0032] However, as shown in Fig. 3, embryonic cells having a shape identical or similar to the cross-section of actual embryonic cells are not formed, so there have been problems in the similarity of shape and accuracy of the characteristics of embryonic cells during cell culture, development, and differentiation research.

[0033] Accordingly, the purpose of the present invention is to simultaneously inject a cell mixture containing each of ectoderm, endoderm, and mesoderm, and to implement it so that the cross-section is as similar as possible to the cross-section of an actual embryo cell.

[0034] The purpose of this is to enable more accurate research on embryogenesis and developmental processes than before.

[0035] Figure 1 is a diagram of the overall configuration of the present invention, and a cell culture solution tank (10) containing each cell mixture, i.e., an injection port, is disclosed, and although not shown in the drawing, an injector (e.g., a syringe) into which each cell mixture can be injected is connected.

[0036] That is, as shown in Fig. 2, a first injector (11) that controls the flow rate of the first cell mixture containing the ectoderm, a second injector (12) that controls the flow rate of the second cell mixture containing the endoderm, and a third injector (13) that controls the flow rate of the third cell mixture containing the mesoderm are connected.

[0037] The flow rate of each of the first injector, second injector, and third injector is controlled by the user through a separate control unit (500).

[0038] Referring to Figure 1, the overall configuration diagram of the present invention is as follows.

[0039] As shown, a central channel (100) with a predetermined space formed inside is opened.

[0040] In the central channel (100), the first cell mixture, the second cell mixture, and the third cell mixture move while maintaining their cross-sectional shapes.

[0041] A pair of first channels (200) are opened on one side of the central channel (100) and are connected to the central channel (100) while being inclined at a first angle in both lateral directions.

[0042] The first angle (α) is 15 degrees from the reference line shown in the drawing. 0 Ideal 45 0 It is desirable to set it below, but the angle that the actual user wants to make the cross-section of the stem cell mixture is 30 0 It is desirable to do so.

[0043] At this time, the first cell mixture containing the ectoderm is injected through the first channel (200) because, as shown in Fig. 3, the ectoderm is located at the outermost part of the embryonic cells.

[0044] Next, a pair of second channels (300) are opened that are connected to the central channel (100) and are inclined at a second angle in both lateral directions on one side of the central channel (100).

[0045] As shown in the drawing, the second angle (β) is 15 based on the first channel (200). 0 Ideal 45 0 It is desirable to set it below, but the angle that the actual user wants to make the cross-section of the stem cell mixture is 30 0 It is desirable to do so.

[0046] At this time, a second cell mixture containing endoderm is injected through the second channel (300). This is because, as shown in FIG. 3, the endoderm is surrounded by the ectoderm in the embryonic cells.

[0047] Furthermore, it is intended to be implemented so that the ectoderm is located on the upper side and the endoderm on the lower side based on the cross-section.

[0048] Meanwhile, a third channel (400) is located between a pair of second channels (300) described above on one side of the central channel (100) and is capable of being communicated with the central channel (100).

[0049] The third cell mixture containing mesoderm is injected through the third channel (400) because, as shown in Fig. 3, when looking at the cross-section of an actual embryo cell, the mesoderm is located at the center.

[0050] Through this configuration, the first cell mixture flows into the first channel (200) and moves along the central channel (100), the second cell mixture flows into the second channel (300) and moves along the central channel (100), and the third cell mixture flows into the third channel (400) and moves along the central channel (100).

[0051] These first cell mixture, second cell mixture, and third cell mixture do not move separately, but move simultaneously at their respective speeds set by the user.

[0052] This is because the growth of actual embryonic cells also takes into account that the ectoderm, endoderm, and mesoderm grow simultaneously rather than separately.

[0053] In the present invention, the first cell mixture, the second cell mixture, and the third cell mixture move simultaneously at respective flow rates set by the user.

[0054] Meanwhile, the first cell mixture, the second cell mixture, and the third cell mixture are characterized in that the cross-sections along which they move are maintained as they are when they move along the central channel (100), and the stem cell mixture with the cross-sections maintained is discharged from the exit of the central channel (100).

[0055] That is, FIG. 5 is a schematic diagram of the cross-sectional shape of the stem cell mixture discharged from the exit of the central channel (100) in FIGS. 1 and 2.

[0056] On the left side of the drawing is a cross-section of the flow analysis simulation results of the actual discharged ectoderm, endoderm, and mesoderm, and on the right side of the drawing is a schematic cross-section of this.

[0057] The cross-section of the stem cell mixture of Fig. 5 is ultimately similar to the cross-section of the embryonic cells of Fig. 3 described above, and a stem cell mixture with such a cross-section must be produced to enable more accurate research on embryo formation and development than before.

[0058] It is characterized in that, inside the central channel (100), the first cell mixture, the second cell mixture, and the third cell mixture are discharged from the central channel (100) while moving without mixing with each other and forming a virtual wall (V) between them.

[0059] That is, as illustrated in FIG. 5, according to the present invention, the first cell mixture, the second cell mixture, and the third cell mixture pass through the central channel (100) while forming a virtual wall (V) between them.

[0060] And, they move without being mixed with each other, and when this cross-section is maintained, a virtual wall (V) is formed, and after the cross-section where the virtual wall (V) is formed is maintained as is, it is discharged from the central channel (100).

[0061] Meanwhile, the cross-section of the stem cell mixture is shown in Fig. 5, with the first cell mixture positioned on the upper side and the second cell mixture positioned on the lower side.

[0062] It is characterized in that a third cell mixture is located at the center, and the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation.

[0063] 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.4 ~ 1.6 : 0.4 ~ 0.6 : 0.7 ~ 0.9

[0064] That is, such speed control can be achieved by controlling the flow rate by the control unit illustrated in Fig. 2. Through various experiments, the relative speeds implemented so that the first cell mixture, the second cell mixture, and the third cell mixture do not mix with each other and a virtual wall (V) is formed, and are similar to the cross-section of an actual embryo cell, are as above.

[0065] Furthermore, in the present invention, as a result of several flow analysis simulation experiments, the relative speed that maintains the optimal cross-section was derived as follows.

[0066] 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.5 : 0.5 : 0.8

[0067] For reference, the basic unit of speed in the test of the present invention is mm / s, and in this unit, first cell mixture: second cell mixture: third cell mixture = 1.5 mm / s: 0.5 mm / s: 0.8 mm / s.

[0068] Meanwhile, as illustrated in Fig. 6, the stem cell mixture having a rectangular cross-section is connected to a separate oil supply port (600) through which oil is supplied on one side of the central channel (100) so as to be similar to the cross-section of an actual embryonic cell.

[0069] It is characterized in that the stem cell mixture is cut into a predetermined size of circular cross-section by oil introduced through the oil supply port (600).

[0070] The first cell mixture is characterized by containing ectoderm cells, nutrients, culture medium, and ECM, the second cell mixture is characterized by containing endoderm cells, nutrients, culture medium, and ECM, and the third cell mixture is characterized by containing mesoderm cells, nutrients, culture medium, and ECM. The experimental sequence for actually implementing the present invention is as follows.

[0071] -Example-

[0072] 1. Prepare cells (mixture of stem cells + culture medium + nutrients + antibiotics + extracellular matrix (ECM) etc.).

[0073] The above stem cells refer to ectoderm cells, endoderm cells, and mesoderm cells.

[0074] 2. Prepare a culture medium by mixing nutrients and antibiotics.

[0075] 3. The extracellular matrix is ​​neutralized with HEPES solution, stored in the refrigerator, and then prepared by mixing with cells and culture medium.

[0076] 4. After mixing everything, store in a 37 degree incubator for about 20 minutes before use.

[0077] 5. Tubing is connected to the first inlet (11) of the first channel (200), the second inlet (12) of the second channel (300), and the third inlet (13) of the third channel (400), the tubing is connected to a syringe, and the syringe is connected to a syringe pump, respectively, and the first cell mixture is injected into the first channel (200), the second cell mixture into the second channel (300), and the third cell mixture into the third channel (400).

[0078] 6. Cells entering from two places in the first channel (200) move while forming a virtual wall (V) in the central channel (100), but are regarded as one shape even if they are not mixed with the same cell mixture.

[0079] 7. Similarly, cells entering from two places in the second channel (300) move while forming a virtual wall (V) in the central channel (100), but are regarded as one shape even if they are not mixed with the same cell mixture.

[0080] 8. Operate the syringe pump to transfer the solution through the tubing to the central channel (100) of the chip.

[0081] 9. Oil that has no effect on the stem cell mixture is injected at a constant rate from a separate channel connected at a 90-degree angle from the outlet of the central channel (100) to separate the stem cell mixture at constant intervals and discharge it.

[0082] 10. A stem cell mixture with a circular cross-section, more precisely a certain size, is cultured in a certain space.

[0083] Meanwhile, FIG. 7 shows a cross-section in which the first cell mixture (including ectoderm), the second cell mixture (including endoderm), and the third cell mixture (including mesoderm) move along each channel and are discharged from the outlet of the central channel (100), and FIG. 8 is a drawing showing this cross-section.

[0084] A cell culture method using a microfluidic chip for three-dimensional multi-cell co-culture with the above configuration is disclosed.

[0085] To this end, the present invention includes a step of preparing a first cell mixture, a second cell mixture, and a third cell mixture, a step of allowing the first cell mixture to flow into a first channel (200) and move along a central channel (100), a step of allowing the second cell mixture to flow into a second channel (300) and move along the central channel (100), a step of allowing the third cell mixture to flow into a third channel (400) and move along the central channel (100), and a step of allowing the first cell mixture, the second cell mixture, and the third cell mixture to simultaneously move into the first channel (200), the second channel (300), and the third channel (400), respectively, and then a step of allowing a stem cell mixture in which a virtual wall (V) is formed to move along the central channel (100) while maintaining a cross-section through which the stem cell mixture is discharged from an outlet of the central channel (100).

[0086] The stem cell mixture is characterized in that the cross-section has the first cell mixture positioned at the upper side, the second cell mixture positioned at the lower side, and the third cell mixture positioned at the center, and the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation.

[0087] 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.4 ~ 1.6 : 0.4 ~ 0.6 : 0.7 ~ 0.9

[0088] The relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture are characterized by satisfying the following equation.

[0089] 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.5 : 0.5 : 0.8

[0090] The specific steps have already been explained, so they are omitted here.

[0091] Meanwhile, the specific fluid analysis simulation method of the present invention is described as follows.

[0092] -Computer-based fluid analysis simulation-

[0093] The present applicant manufactured a modeling shape of a cell culture microfluidic chip with a five-channel inlet to explore flow conditions for co-culturing three-dimensional multi-cells in a desired shape.

[0094] Using the above modeling shape, the distribution of the 1, 2, and 3 stem cell mixtures injected at each inlet was evaluated under various flow conditions as follows.

[0095] Under the presented flow conditions, the results of the flow analysis simulation were derived with a shape similar to that of the existing embryonic cell.

[0096] The results are shown in Figures 7 and 8.

[0097] -Flow analysis program used: Commercial computational fluid dynamics (CFD) program

[0098] -Speed ​​0.2 mm / s ~ 2.0 mm / s

[0099] - Height, width, and length of the 5-channel shaped input: 0.5 mm, 0.4 mm, and 14 mm

[0100] -Height and width of the shaped central channel: 1.0 mm and 1.5 mm

[0101] -1,2,3 Cell mixture density: 1010 kg / m 3

[0102] -1,2,3 Cell mixture viscosity: 1000 cP ~ 4000 cP

[0103] -Result measurement location: End section of the central channel

[0104] As can be seen in Figures 7 and 8, the injected 1, 2, and 3 stem cell mixtures formed virtual walls with each other, and were positioned in the upper part (ectoderm), lower part (endoderm), and middle part (mesoderm), showing a structure almost similar to that of existing embryonic cells.

Claims

1. A central channel with a predetermined space formed inside; A pair of first channels connected to the central channel and inclined at a first angle in both lateral directions on one side of the central channel; A pair of second channels connected to the central channel and inclined at a second angle in both lateral directions on one side of the central channel; A third channel located between the pair of second channels on one side of the central channel and capable of being communicated with the central channel; It is characterized in that a first cell mixture flows into the first channel and moves along the central channel, a second cell mixture flows into the second channel and moves along the central channel, and a third cell mixture flows into the third channel and moves along the central channel. A three-dimensional multi-cell co-culture microfluidic chip characterized in that the cross-sections along which the first cell mixture, the second cell mixture, and the third cell mixture move along the central channel are maintained as is, and the stem cell mixture with the maintained cross-sections is discharged from the outlet of the central channel.

2. In claim 1, A three-dimensional multi-cell co-culture microfluidic chip characterized in that the first cell mixture, the second cell mixture, and the third cell mixture are discharged from the central channel while moving without mixing with each other and forming a virtual wall between them.

3. In claim 2, The above cross section is, It is characterized in that the first cell mixture is located at the top, the second cell mixture is located at the bottom, and the third cell mixture is located at the center. A microfluidic chip for three-dimensional multi-cell co-culture, characterized in that the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation. 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.4 ~ 1.6 : 0.4 ~ 0.6 : 0.7 ~ 0.9 4. In claim 3, A microfluidic chip for three-dimensional multi-cell co-culture, characterized in that the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation. 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.5 : 0.5 : 0.8 5. In claim 4, A three-dimensional multi-cell co-culture microfluidic chip characterized in that a separate oil supply port for supplying oil is connected to one side of the central channel exit, and the stem cell mixture is cut into a predetermined circular cross-section size by the oil introduced through the oil supply port.

6. In claim 5, The above first cell mixture is characterized by containing ectoderm cells, nutrients, culture medium, and ECM, The second cell mixture is characterized by containing endoderm cells, nutrients, culture medium, and ECM. A microfluidic chip for three-dimensional multi-cell co-culture, characterized in that the third cell mixture includes mesodermal cells, nutrients, culture medium, and ECM.

7. Step of preparing the first cell mixture, the second cell mixture, and the third cell mixture; A step in which the first cell mixture is introduced into the first channel and moves along the central channel; A step in which a second cell mixture is introduced into the second channel and moves along the central channel; A step in which a third cell mixture is introduced into the third channel and moves along the central channel; and A step in which the first cell mixture, the second cell mixture, and the third cell mixture move simultaneously to the first channel, the second channel, and the third channel, respectively, and then the stem cell mixture in which a virtual wall is formed is discharged from the exit of the central channel while maintaining a cross-section along the central channel; A cell culture method using a microfluidic chip for three-dimensional multi-cell co-culture, including:

8. In claim 7, The above stem cell mixture is arranged such that the first cell mixture is positioned at the top, the second cell mixture is positioned at the bottom, and the third cell mixture is positioned at the center. A cell culture method using a microfluidic chip for three-dimensional multi-cell co-culture, characterized in that the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation. 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.4 ~ 1.6 : 0.4 ~ 0.6 : 0.7 ~ 0.9 9. In claim 8, A cell culture method using a microfluidic chip for three-dimensional multi-cell co-culture, characterized in that the relative speeds of the first cell mixture, the second cell mixture, and the third cell mixture satisfy the following equation. 1st cell mixture : 2nd cell mixture : 3rd cell mixture = 1.5 : 0.5 : 0.8

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