An organ-on-a-chip with a multi-layer structure

Abstract

The present invention provides an organ-on-a-chip with a multi-layer structure, which includes an upper flow channel layer, an upper porous membrane layer, a tissue layer, a lower porous membrane layer, and a lower flow channel layer that are stacked in sequence from top to bottom. The upper flow channel layer includes a first perfusion port, a second perfusion port, a first upper flow port, and a second upper flow port; the hydrogel containing cells is perfused into the first perfusion channel or the second perfusion channel through the first perfusion port or the second perfusion port. Under the blockage of the upper porous membrane layer and the lower porous membrane layer, the hydrogel containing cells fills the tissue chamber; then, cell culture medium is injected through the first upper flow port and the second upper flow port, and permeates into the tissue chamber through the first porous membrane and the second porous membrane respectively to culture cells. The present invention can separate the perfusion channel of the hydrogel containing cells from the perfusion channel of the cell culture medium, and the perfusion of the hydrogel containing cells will not block the channel of the cell culture medium, realizing uniform circulation of the cell culture medium in the organ-on-a-chip to stimulate cells.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedical engineering and microfluidic technology, and particularly relates to an organ-on-a-chip with a multi-layer structure. Background Art

[0002] An organ-on-a-chip is a microfluidic cell culture system fabricated by microfabrication technology, which can perform three-dimensional cell culture in vitro to simulate the organ functions in the human body. The organ-on-a-chip has good application prospects in the fields of drug R & D and screening, disease model evaluation, personalized medicine, etc.

[0003] Before a drug is put on the market, it is necessary to conduct toxicological tests and safety verification of the drug. Traditional drug toxicological tests are usually carried out in two-dimensional cell models or in animals. The two-dimensional cell culture mode is difficult to simulate the complex physiological activities in human tissues and organs. Animal experiments also have disadvantages such as long cycle, high cost, and difficult observation, and there have been many ethical controversies in animal experiments in recent years. Therefore, the proposed organ-on-a-chip is expected to establish a more realistic physiological model in vitro and become a bionic, efficient, and convenient tool for physiological research and drug development.

[0004] Currently, the more common organ-on-a-chips mainly include a hydrogel perfusion channel containing cells and a culture medium perfusion channel. However, the existing organ-on-a-chips have the following defects: 1. Some organ-on-a-chips are single-layer chips, so that both the cell perfusion channel and the culture medium perfusion channel are on the same layer. In the operation of injecting the hydrogel containing cells, it is inevitable that the hydrogel blocks the culture medium channel, resulting in the cultured cells or tissues not being uniformly stimulated by the culture medium; 2. The space of the tissue chamber in some organ-on-a-chips is relatively small, making it difficult to culture relatively large-volume cells or tissues. Summary of the Invention

[0005] In order to solve the problem that the hydrogel containing cells injected into many current single-layer organ-on-a-chips blocks the culture medium channel and the problem of culturing relatively large-volume cells or tissues, the present invention provides an organ-on-a-chip with a multi-layer structure.

[0006] Based on the above object, the present invention is achieved through the following technical solutions:

[0007] An organ-on-a-chip with a multi-layer structure includes an upper flow channel layer, an upper porous membrane layer, a tissue layer, a lower porous membrane layer, and a lower flow channel layer that are sequentially stacked from top to bottom; a tissue chamber is provided on the tissue layer, a first perfusion channel and a second perfusion channel connected to the tissue chamber are provided on the tissue layer, a fifth through hole and a sixth through hole are respectively provided on both sides of the tissue chamber; a first lower flow port, a second lower flow port, a first lower flow channel, and a second lower flow channel with clearance fit are provided on the lower flow channel layer.

[0008] Preferably, a first porous membrane is provided on the upper porous membrane layer. On both sides of the first porous membrane, there are first through holes, second through holes, third through holes, and fourth through holes that are in clearance fit; on the lower porous membrane layer, there is a second porous membrane that cooperates with the first porous membrane. On both sides of the second porous membrane, there are seventh through holes and eighth through holes respectively; on the upper flow channel layer, there are a first perfusion port, a second perfusion port, a first upper flow channel, and a second upper flow channel that cooperate with each other. On the side of the first perfusion port, there are a first upper flow port and a second upper flow port that are in clearance fit with the first perfusion port. On the side of the second perfusion port, there are a third upper flow port and a fourth upper flow port that are in clearance fit with the second perfusion port.

[0009] Preferably, the first upper flow port and the fourth upper flow port are connected through the first upper flow channel and the second upper flow channel; the first lower flow port and the second lower flow port are connected through the first lower flow channel and the second lower flow channel; the first perfusion port is in communication with the first through hole and the first perfusion channel; the second perfusion port is in communication with the second through hole and the second perfusion channel; the second upper flow port is in communication with the third through hole, the fifth through hole, and the seventh through hole; the third upper flow port is in communication with the fourth through hole, the sixth through hole, and the eighth through hole.

[0010] Preferably, the first perfusion channel and the second perfusion channel are respectively arranged at two diagonal ends of the tissue chamber.

[0011] Preferably, the first perfusion port and the second perfusion port are in clearance fit with the vertical projection of the first porous membrane in the vertical direction.

[0012] Preferably, both the first upper flow channel and the second upper flow channel are arranged on the side surface of the upper flow channel layer facing the upper porous membrane layer; both the first lower flow channel and the second lower flow channel are arranged on the side surface of the lower flow channel layer facing the lower porous membrane layer.

[0013] Preferably, the connection parts of the first upper flow port, the fourth upper flow port with the first upper flow channel and the second upper flow channel are set as rhombuses; the connection parts of the first lower flow port, the second lower flow port with the first lower flow channel and the second lower flow channel are set as rhombuses; the first upper flow channel and the second upper flow channel are wound and interlaced with each other in a spiral winding manner; the first lower flow channel and the second lower flow channel are wound and interlaced with each other in a spiral winding manner.

[0014] Preferably, the upper flow channel area formed by the first upper flow channel and the second upper flow channel covers the first porous membrane; the lower flow channel area formed by the first lower flow channel and the second lower flow channel covers the second porous membrane.

[0015] Preferably, the thickness of the tissue layer is preferably 100 - 1000 μm; the thickness of the upper flow channel layer and the lower flow channel layer is preferably 2 - 10 mm, 5 mm; the thickness of the first upper flow channel, the second upper flow channel, the first lower flow channel, and the second lower flow channel is preferably 100 - 600 μm, 100 μm; the thickness of the upper porous membrane layer and the lower porous membrane layer is 30 - 150 μm, 50 μm.

[0016] Preferably, the shape of the tissue chamber can be rectangular so as to have a relatively large planar area.

[0017] Preferably, a plurality of liquid storage pools respectively connected to the first perfusion port and the second perfusion port are arranged above the upper flow channel layer; the height of the first liquid storage pool is equal to the height of the second liquid storage pool, the liquid levels of the third liquid storage pool and the fourth liquid storage pool are equal, and the liquid levels of the culture solution in the first liquid storage pool and the second liquid storage pool are higher than those in the third liquid storage pool and the fourth liquid storage pool.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] (1) The tissue layer for cell culture is separately arranged from the flow channel layer for the culture solution, and the upper and lower porous membrane layers are arranged. The injection of the gel for cells will not affect the subsequent full infiltration of the culture solution into the tissue chamber, so that the cultured cells or tissues can be uniformly stimulated by the culture solution.

[0020] (2) The planar shape of the tissue chamber and the thickness of the tissue layer can be adjusted within a preferred range so as to culture cells or tissues with a relatively large volume.

[0021] In summary, by separately arranging the tissue layer and the flow channel layer, the present invention can separately perfuse the process of perfusion of the hydrogel containing cells and the cell culture solution. The hydrogel containing cells is perfused into the first perfusion channel or the second perfusion channel through the first perfusion port or the second perfusion port. Under the blockage of the upper porous membrane layer and the lower porous membrane layer, the hydrogel containing cells fills the tissue chamber; then, the cell culture solution is injected through the first upper flow port and the second upper flow port, and respectively penetrates into the tissue chamber through the first porous membrane and the second porous membrane to culture cells, realizing the circulation of the cell culture solution in the organ chip. During this process, due to the separation of the perfusion channel of the hydrogel containing cells from the perfusion channel of the cell culture solution, the perfusion of the hydrogel containing cells will not block the channel of the cell culture solution. Description of the Drawings

[0022] Figure 1 is a schematic structural diagram of the present invention in Embodiment 1;

[0023] Figure 2 is a schematic distribution structural diagram of the liquid storage pools on the top surface of the upper flow channel layer in Embodiment 1;

[0024] Figure 3 is a schematic structural diagram of the upper flow channel layer in Embodiment 1;

[0025] Figure 4 is in Embodiment 1 Figure 3 Schematic diagram of the structure of part A;

[0026] Figure 5It is a schematic structural diagram of the upper porous membrane layer in Example 1;

[0027] Figure 6 It is in Example 1 Figure 5 Schematic structural diagram of part B;

[0028] Figure 7 It is a schematic structural diagram of the tissue layer in Example 1;

[0029] Figure 8 It is a schematic structural diagram of the lower porous membrane layer in Example 1;

[0030] Figure 9 It is in Example 1 Figure 8 Schematic structural diagram of part C;

[0031] Figure 10 It is a schematic structural diagram of the downstream channel layer in Example 1;

[0032] Figure 11 It is in Example 1 Figure 10 Schematic structural diagram of part D.

[0033] In the figure, 1 is the upstream channel layer; 11 is the first perfusion port; 12 is the first upstream channel port; 13 is the second upstream channel port; 14 is the second perfusion port; 15 is the third upstream channel port; 16 is the fourth upstream channel port; 17 is the first upstream channel; 18 is the second upstream channel; 2 is the upper porous membrane layer; 21 is the first through hole; 22 is the second through hole; 23 is the third through hole; 24 is the fourth through hole; 25 is the first porous membrane; 3 is the tissue layer; 31 is the first perfusion channel; 32 is the second perfusion channel; 33 is the tissue chamber; 34 is the fifth through hole; 35 is the sixth through hole; 4 is the lower porous membrane layer; 41 is the seventh through hole; 42 is the eighth through hole; 43 is the second porous membrane; 5 is the downstream channel layer; 51 is the first downstream channel port; 52 is the second downstream channel port; 53 is the first downstream channel; 54 is the second downstream channel; 61 is the first liquid storage pool; 62 is the second liquid storage pool; 63 is the third liquid storage pool; 64 is the fourth liquid storage pool. Detailed implementation manners

[0034] The present invention will be further described in detail below through specific examples, but the scope of the present invention is not limited.

[0035] Example 1:

[0036] An organ chip with a multi-layer structure, whose structure is as Figures 1-11As shown in the figure, it includes an upper flow channel layer 1, an upper porous membrane layer 2, a tissue layer 3, a lower porous membrane layer 4, and a lower flow channel layer 5 that are stacked in sequence from top to bottom; a tissue chamber 33 is provided on the tissue layer 3, and a first perfusion channel 31 and a second perfusion channel 32 connected to the tissue chamber 33 are provided on the tissue layer 3. A fifth through hole 34 and a sixth through hole 35 are respectively provided on both sides of the tissue chamber 33; a first lower flow port 51, a second lower flow port 52, a first lower flow channel 53, and a second lower flow channel 54 with clearance fit are provided on the lower flow channel layer 5.

[0037] A first porous membrane 25 is provided on the upper porous membrane layer 2, and a first through hole 21, a second through hole 22, a third through hole 23, and a fourth through hole 24 with clearance fit are respectively provided on both sides of the first porous membrane 25; a second porous membrane 43 that cooperates with the first porous membrane 25 is provided on the lower porous membrane layer 4, and a seventh through hole 41 and an eighth through hole 42 are respectively provided on both sides of the second porous membrane 43; a first perfusion port 11, a second perfusion port 14, a first upper flow channel 17, and a second upper flow channel 18 that cooperate with each other are provided on the upper flow channel layer 1. A first upper flow port 12 and a second upper flow port 13 with clearance fit with the first perfusion port 11 are provided on the side of the first perfusion port 11, and a third upper flow port 15 and a fourth upper flow port 16 with clearance fit with the second perfusion port 14 are provided on the side of the second perfusion port 14.

[0038] The first upper flow port 12 and the fourth upper flow port 16 are connected and communicated through the first upper flow channel 17 and the second upper flow channel 18; the first lower flow port 51 and the second lower flow port 52 are connected and communicated through the first lower flow channel 53 and the second lower flow channel 54; the first perfusion port 11 is in through communication with the first through hole 21 and the first perfusion channel 31; the second perfusion port 14 is in through communication with the second through hole 22 and the second perfusion channel 32; the second upper flow port 13 is in through communication with the third through hole 23, the fifth through hole 34, and the seventh through hole 41; the third upper flow port 15 is in through communication with the fourth through hole 24, the sixth through hole 35, and the eighth through hole 42.

[0039] The first perfusion channel 31 and the second perfusion channel 32 are respectively arranged at two diagonal ends of the tissue chamber 33. The first perfusion port 11 and the second perfusion port 14 are in clearance fit with the vertical projection of the first porous membrane 25 in the vertical direction. The first upper flow channel 17 and the second upper flow channel 18 are both arranged on the side surface of the upper flow channel layer 1 facing the upper porous membrane layer 2; the first lower flow channel 53 and the second lower flow channel 54 are both arranged on the side surface of the lower flow channel layer 5 facing the lower porous membrane layer 4. The connection parts of the first upper flow port 12, the fourth upper flow port 16 with the first upper flow channel 17 and the second upper flow channel 18 are arranged in a rhombus shape; the connection parts of the first lower flow port 51, the second lower flow port 52 with the first lower flow channel 53 and the second lower flow channel 54 are arranged in a rhombus shape.

[0040] The upper flow channel region formed by the first upper flow channel 17 and the second upper flow channel 18 covers the first porous membrane 25; the area of the lower flow channel region formed by the first lower flow channel 53 and the second lower flow channel 54 covers the second porous membrane 43.

[0041] The thickness of the tissue layer 3 is preferably 100 - 1000 μm; the thickness of the upper flow channel layer 1 and the lower flow channel layer 5 is preferably 2 - 10 mm and 5 mm respectively; the thickness of the first upper flow channel 17, the second upper flow channel 18, the first lower flow channel 53, and the second lower flow channel 54 is preferably 100 - 600 μm and 100 μm respectively; the thickness of the upper porous membrane layer 2 and the lower porous membrane layer 4 is 30 - 150 μm and 50 μm respectively; the tissue chamber 33 is a rectangle with a relatively large planar area.

[0042] Above the upper flow channel layer 1, there are multiple liquid storage pools respectively connected to the first perfusion port 11 and the second perfusion port 14.

[0043] The process of assembling this organ chip and culturing cells is as follows:

[0044] Step 1: After assembling the upper flow channel layer 1, the upper porous membrane layer 2, the tissue layer 3, the lower porous membrane layer 4, and the lower flow channel layer 5 of the organ chip in a certain order, seal the connection edges of each layer. If the material of each layer is PDMS (polydimethylsiloxane), bonding can be achieved by plasma.

[0045] Step 2: The hydrogel containing cells is perfused through the first perfusion port 11 or the second perfusion port 14. The hydrogel containing cells at the first perfusion port 11 flows into the first perfusion channel 31 through the first through-hole 21, and the hydrogel containing cells at the second perfusion port 14 flows into the second perfusion channel 32 through the second through-hole 22. Due to the blocking of the upper porous membrane layer 2 and the lower porous membrane layer 4, the hydrogel containing cells will gradually fill the entire tissue chamber 33 through the first perfusion channel 31 or the second perfusion channel 32.

[0046] Step 3: The cell culture medium is injected from the first upper flow channel port 12 and the second upper flow channel port 13. The cell culture medium at the first upper flow channel port 12 fills the entire upper flow channel region through the first upper flow channel 17 and the second upper flow channel 18, and then circulates through the fourth upper flow channel port 16; the cell culture medium at the second upper flow channel port 13 flows to the first lower flow channel port 51 through the third through-hole 23, the fifth through-hole 34, and the seventh through-hole 41, then fills the entire lower flow channel region through the first lower flow channel 53 and the second lower flow channel 54, and then flows to the third upper flow channel port 15 through the second lower flow channel port 52, the eighth through-hole 42, the sixth through-hole 35, and the fourth through-hole 24, realizing the circulation of the cell culture medium in the organ chip. The cell culture medium in the upper flow channel region and the lower flow channel region penetrates into the tissue chamber 33 through the first porous membrane 25 and the second porous membrane 43 to culture cells, realizing the circulation of the cell culture medium in the organ chip.

[0047] Among them, the height of the first liquid storage tank 61 is kept equal to the height of the second liquid storage tank 62, the liquid levels of the third liquid storage tank 63 and the fourth liquid storage tank 64 are equal, and the liquid levels of the cell culture solution in the first liquid storage tank 61 and the second liquid storage tank 62 are higher than those in the third liquid storage tank 63 and the fourth liquid storage tank 64; the first liquid storage tank 61 is communicated with the first upper flow port 12, the second liquid storage tank 62 is communicated with the second upper flow port 13, the third liquid storage tank 63 is communicated with the third upper flow port 15, and the fourth liquid storage tank 64 is communicated with the fourth upper flow port 16.

[0048] The thickness of the upper flow channel layer 1 and the lower flow channel layer 5 can be 5 mm, the thickness of the flow channel can be 100 μm, the thickness of the upper porous membrane layer 2 and the lower porous membrane layer 4 can be 50 μm, and the thickness of the tissue layer 3 can be adjusted within a preferred range of 100 - 1000 μm. The shape of the tissue chamber 33 can be rectangular so as to have a relatively large planar area.

[0049] In addition, those skilled in the art can also design other local dimensions such as the shape, inlet, and flow channel of the tissue chamber 33 according to actual needs, and this embodiment does not specifically limit this.

[0050] In summary, the present invention provides a multi-layer organ chip, which solves the problem that the culture solution channels are blocked by injecting hydrogels containing cells in many current single-layer organ chips; the thickness of the tissue layer 3 and the shape of the tissue chamber 33 of the organ chip can be adjusted within a preferred range so as to culture relatively large volumes of cells or tissues.

[0051] Example 2:

[0052] A multi-layer organ chip, different from Example 1 in that: the thickness of the upper flow channel layer 1 and the lower flow channel layer 5 is preferably 5 mm.

[0053] Example 3:

[0054] A multi-layer organ chip, different from Example 1 in that: the thickness of the upper porous membrane layer 2 and the lower porous membrane layer 4 is preferably 50 μm.

[0055] Example 4:

[0056] A multi-layer organ chip, different from Example 1 in that: the thickness of the flow channel is preferably 100 μm.

[0057] Example 5:

[0058] A culture method for a multi-layer organ chip, the steps including:

[0059] Step 1: After assembling the upstream channel layer 1, upper porous membrane layer 2, tissue layer 3, lower porous membrane layer 4, and downstream channel layer 5 of the organ-on-a-chip in a certain order, seal the connection edges of each layer. If the material of each layer is PDMS (polydimethylsiloxane), plasma bonding can be used.

[0060] Step 2: The hydrogel containing cells is perfused through the first perfusion port 11 and the second perfusion port 14. The hydrogel containing cells at the first perfusion port 11 flows into the first perfusion channel 31 through the first through-hole 21, and the hydrogel containing cells at the second perfusion port 14 flows into the second perfusion channel 32 through the second through-hole 22. Due to the blockage of the upper porous membrane layer 2 and the lower porous membrane layer 4, the hydrogel containing cells will gradually fill the entire tissue chamber 33 through the first perfusion channel 31 and the second perfusion channel 32.

[0061] Step 3: The cell culture medium is injected from the first upstream port 12, the fourth upstream port 16, the second upstream port 13, and the third upstream port 15. The cell culture medium at the first upstream port 12 and the fourth upstream port 16 fills the entire upstream channel area through the first upstream channel 17 and the second upstream channel 18. The cell culture medium at the second upstream port 13 flows to the first downstream port 51 through the third through-hole 23, the fifth through-hole 34, and the seventh through-hole 41, and then fills the entire downstream channel area through the first downstream channel 53 and the second downstream channel 54. The cell culture medium at the third upstream port 15 flows to the second downstream port 52 through the fourth through-hole 24, the sixth through-hole 35, and the eighth through-hole 42, and then fills the entire downstream channel area through the first downstream channel 53 and the second downstream channel 54. The cell culture medium penetrates into the tissue chamber 33 through the first porous membrane 25 and the second porous membrane 43 to culture the cells, realizing the circulation of the cell culture medium in the organ-on-a-chip;

[0062] Among them, the height of the first reservoir 61 is kept equal to the height of the second reservoir 62, the liquid levels of the third reservoir 63 and the fourth reservoir 64 are equal, and the liquid levels of the cell culture medium in the first reservoir 61 and the second reservoir 62 are higher than those in the third reservoir 63 and the fourth reservoir 64.

[0063] The above are only the preferred embodiments of the present invention, but are not limited to the above examples. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A multi-layered organ-on-a-chip, characterized in that, It includes an upper flow channel layer, an upper porous membrane layer, a tissue layer, a lower porous membrane layer, and a lower flow channel layer that are stacked in sequence from top to bottom; a tissue chamber is provided on the tissue layer, and a first perfusion channel and a second perfusion channel connected to the tissue chamber are provided on the tissue layer. A fifth through hole and a sixth through hole are respectively provided on both sides of the tissue chamber; a first lower flow port, a second lower flow port, a first lower flow channel, and a second lower flow channel with clearance fit are provided on the lower flow channel layer. A first porous membrane is provided on the upper porous membrane layer, and a first through hole, a second through hole, a third through hole, and a fourth through hole with clearance fit are respectively provided on both sides of the first porous membrane; a second porous membrane that cooperates with the first porous membrane is provided on the lower porous membrane layer, and a seventh through hole and an eighth through hole are respectively provided on both sides of the second porous membrane; a first perfusion port, a second perfusion port, a first upper flow channel, and a second upper flow channel that cooperate with each other are provided on the upper flow channel layer. A first upper flow port and a second upper flow port with clearance fit with the first perfusion port are provided on the side of the first perfusion port, and a third upper flow port and a fourth upper flow port with clearance fit with the second perfusion port are provided on the side of the second perfusion port. The first upper flow port and the fourth upper flow port are connected and communicated through the first upper flow channel and the second upper flow channel; the first lower flow port and the second lower flow port are connected and communicated through the first lower flow channel and the second lower flow channel; the first perfusion port is in through connection with the first through hole and the first perfusion channel; the second perfusion port is in through connection with the second through hole and the second perfusion channel; the second upper flow port is in through connection with the third through hole, the fifth through hole, and the seventh through hole; the third upper flow port is in through connection with the fourth through hole, the sixth through hole, and the eighth through hole. The first perfusion channel and the second perfusion channel are respectively arranged at two diagonal ends of the tissue chamber. The first upper flow channel and the second upper flow channel are both arranged on the side surface of the upper flow channel layer facing the upper porous membrane layer; the first lower flow channel and the second lower flow channel are both arranged on the side surface of the lower flow channel layer facing the lower porous membrane layer. The connection parts of the first upper flow port, the fourth upper flow port with the first upper flow channel and the second upper flow channel are set as diamonds; the connection parts of the first lower flow port, the second lower flow port with the first lower flow channel and the second lower flow channel are set as diamonds; the first upper flow channel and the second upper flow channel are wound and interlaced with each other in a spiral winding manner; the first lower flow channel and the second lower flow channel are wound and interlaced with each other in a spiral winding manner.

2. The multi-layered organ-on-a-chip according to claim 1, characterized in that, The first perfusion port and the second perfusion port are in clearance fit with the vertical projection of the first porous membrane in the vertical direction.

3. The multi-layered organ-on-a-chip according to claim 2, characterized in that, The upper flow channel area formed by the first upper flow channel and the second upper flow channel covers the first porous membrane; the lower flow channel area formed by the first lower flow channel and the second lower flow channel covers the second porous membrane.

4. The multi-layered organ-on-a-chip according to claim 3, characterized in that, The thickness of the tissue layer is 100 - 1000 μm; the thickness of the upper flow channel layer and the lower flow channel layer is 2 - 10 mm; the thickness of the first upper flow channel, the second upper flow channel, the first lower flow channel, and the second lower flow channel is 100 - 600 μm; the thickness of the upper porous membrane layer and the lower porous membrane layer is 30 - 150 μm.

5. The multi-layered organ-on-a-chip according to claim 1 or 4, characterized in that, A plurality of liquid storage pools respectively connected to the first perfusion port and the second perfusion port are provided above the upper flow channel layer.

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