Double-chamber co-culture organ chip

Abstract

The invention provides a double-chamber co-culture organ chip, and relates to the technical field of biomedical engineering and microfluidics. The double-chamber co-culture organ chip comprises a flow channel layer and a bottom layer located at the bottom of the flow channel layer, a culture solution flow channel and a cell culture chamber are arranged at the bottom of the flow channel layer, the culture solution flow channel forms a loop, the culture solution flow channel surrounds the cell culture chamber, and the cell culture chamber is arranged in the loop. The culture solution flow channel is communicated with the cell culture chamber, and a plurality of outer cavity micro-columns are arranged at the communication part; two liquid injection holes, two glue injection holes and a through hole are formed in the runner layer, the two liquid injection holes are respectively communicated with the culture solution runner and are in central symmetry, the two glue injection holes are respectively communicated with the cell culture chamber and are in central symmetry, and the through hole is communicated with the cell culture runner. According to the invention, the growth state of the cell balls in vascularization co-culture can be observed without direct contact with cells.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and microfluidic technology, in particular to a dual-chamber co-culture organ chip. Background Art

[0002] New drug development faces challenges such as long cycles, high investment, and low success rates. Pharmaceutical companies may only achieve a new drug effective for a specific disease after more than a decade of research and development, consuming billions of dollars. During this R&D process, failure is largely due to significant differences between traditional models used for drug screening and the in vivo environment: Traditional two-dimensional culture allows cells to grow attached to a flat surface, but this limits their growth space and their morphology extends only along that plane; while traditional three-dimensional culture allows for three-dimensional cell growth, it lacks the ability to effectively control hydrogels to simulate the in vivo microenvironment; and due to the differences in species, animal models and humans are prone to significant deviations, leading to drug failure in testing.

[0003] Organ-on-a-chips are microfluidic cell culture systems. By inoculating biohydrogels into organ-on-chip devices with a variety of controllable microphysiological environments, they can simulate human physiological conditions. Fabricated through microfabrication techniques, these microfluidic cell culture systems aim to recreate the physiological and pathological characteristics of in vivo organs by creating in vitro models. These systems hold broad potential for development in life science research, disease simulation, new drug development, and precision medicine.

[0004] Constructing tumor tissue models on organ-on-chips is of great significance for the study of cancer biology, drug screening, and toxicity assessment. However, the main challenge is how to establish models that include key components such as tumors, blood vessels, and stromal cells to refine the tumor microenvironment, allowing for the observation of more complex reactions. Constructing vascular mechanisms that integrate tumors has become a cutting-edge direction in cancer treatment. The emergence of tumor spheroid vascularization chips provides researchers with an efficient in vitro tumor microenvironment platform and is of great reference value for tumor drug screening and the development of clinical treatment strategies.

[0005] Existing literature indicates that previous organ-on-a-chip vascularized co-cultures have typically involved directly mixing endothelial cells, fibroblasts, and tumor spheroids. This makes it difficult to fully explore cell interactions and migration patterns, and to observe more complex physiological conditions. Furthermore, conventional organ-on-a-chip structures can cause tumor spheroids to appear at random locations within the culture chamber, requiring researchers to spend extended time capturing experimental images, significantly impacting experimental efficiency.

[0006] In summary, it is difficult to explore the interaction between cell tumor spheres and vascularized networks and the cell migration pattern in ordinary organ chip structures. There is an urgent need for a dual-chamber co-culture organ chip to solve the above problems. Summary of the Invention

[0007] In order to solve the above problems, the dual-chamber co-culture organ chip provided by the present invention can observe the growth status of cell spheres in vascularized co-culture without direct contact with the cells.

[0008] The present invention provides a dual-chamber co-culture organ chip, comprising a flow channel layer and a bottom layer, wherein the flow channel layer is located above the bottom layer, and a culture fluid flow channel and a cell culture chamber are provided at the bottom of the flow channel layer, wherein the culture fluid flow channel forms a loop, and the culture fluid flow channel surrounds the outside of the cell culture chamber, and the culture fluid flow channel is connected to the cell culture chamber, and a plurality of outer cavity micro-columns are provided at the connection point; the flow channel layer is provided with two liquid injection holes, two glue injection holes and a through hole, wherein the two liquid injection holes are respectively connected to the culture fluid flow channel and the two liquid injection holes are symmetrical in center, the two glue injection holes are respectively connected to the cell culture chamber and the two glue injection holes are symmetrical in center, and the through hole is connected to the cell culture flow channel.

[0009] In one feasible embodiment, a cell spheroid culture chamber is further provided at the bottom of the flow channel layer, the cell spheroid culture chamber is surrounded by the cell culture chamber, the cell spheroid culture chamber is connected to the cell culture chamber and a plurality of inner cavity microcolumns are provided at the connection point, and the through hole is connected to the cell spheroid culture chamber.

[0010] In one feasible embodiment, a capillary bursting valve and a pressure reducing hole are further provided at the bottom of the flow channel layer. The capillary bursting valve includes a capillary bursting valve flow channel and bursting valve ports located at both ends of the capillary bursting valve flow channel. The capillary bursting valve flow channel is connected to the cell culture chamber through the bursting valve ports. The pressure reducing hole is connected to the capillary bursting valve flow channel. The capillary bursting valve flow channel is arranged around one of the injection holes.

[0011] In some feasible embodiments, the thickness of the flow channel layer is 1 to 2 mm, the length is 40 to 50 mm, and the width is 15 to 20 mm; and / or the width of the culture fluid flow channel is 50 to 150 μm, and the total length of the culture fluid flow channel is 12 to 14 mm; and / or the radius of the two liquid injection holes, the two glue injection holes and the through hole are all 500 to 600 μm; and / or the width of the outer cavity microcolumn is 80 to 120 μm, the length of the outer cavity microcolumn is 300 to 500 μm, and the gap between adjacent outer cavity microcolumns is 50 to 80 μm; and / or the flow channel height of the culture fluid flow channel and the cell culture chamber is 100 to 200 μm.

[0012] In some feasible embodiments, the radius of the cell spheroid culture chamber is 500-600 μm; and / or the width of the inner cavity microcolumn is 80-120 μm, the length of the inner cavity microcolumn is 300-500 μm, and the gap between adjacent inner cavity microcolumns is 50-80 μm.

[0013] In some feasible embodiments, the width of the bursting valve opening is greater than the gap between adjacent outer cavity microcolumns and the gap between adjacent inner cavity microcolumns, the width of the bursting valve opening is 60 μm to 100 μm, the width of the capillary bursting valve flow channel is 300 μm, and the radius of the pressure reducing hole is 500 to 600 μm.

[0014] The present invention also provides a method for preparing a dual-chamber co-culture organ chip, comprising the following steps:

[0015] Step 1) First, a mask for the flow channel layer is prepared. The mask is then photolithographically patterned into a silicon wafer having a microfluidic pattern. The microfluidic structure includes at least a culture fluid flow channel and a cell culture chamber. The silicon wafer is then affixed to a culture dish to form a mold.

[0016] Step 2) a polydimethylsiloxane prepolymer and a curing agent are mixed and poured into a flow channel layer mold to obtain a polydimethylsiloxane sheet with a microchannel structure, which is the flow channel layer;

[0017] Step 3) a polydimethylsiloxane prepolymer and a curing agent are mixed and poured into a bottom mold to obtain a polydimethylsiloxane sheet, which is the bottom layer;

[0018] Step 4) Punch the channel layer to obtain two liquid injection holes and two glue injection holes, then plasma bond the bottom of the channel layer and the bottom layer (2). After bonding, obtain the chip and heat the chip to strengthen the bonding between the chip layers.

[0019] The present invention also provides a method for using a dual-chamber co-culture organ chip, comprising the following steps:

[0020] Step 1) experimental cells are obtained, the supernatant is removed and mixed with fibrinogen to form a cell stock solution, and then the cell stock solution is obtained and mixed with thrombin to form a biohydrogel.

[0021] Step 2) injecting the biohydrogel from one of the injection holes until the biohydrogel emerges from the other injection hole, and then placing the dual-chamber co-culture organ chip in an incubator;

[0022] Step 3) The dual-chamber co-cultured organ chip is removed from the incubator in step 2) and laminin modification is injected into one of the injection holes. After the laminin modification emerges from the other injection hole, the dual-chamber co-cultured organ chip is placed in the incubator for culture;

[0023] Step 4) Take the dual-chamber co-culture organ chip out of the incubator in step 3), inject EGM-2 culture medium into one of the injection holes, and observe whether the culture medium quickly flows through the cell culture chamber and emerges from the other injection hole.

[0024] Step 5) Different amounts of EGM-2 culture medium are injected into the two injection holes respectively, and the dual-chamber co-culture organ chip is placed in an incubator until a vascular network is formed in the cell culture chamber.

[0025] In one feasible embodiment, the method for using the dual-chamber co-culture organ chip further includes any of the following technical features:

[0026] Step 1) further includes step 1-1), culturing the experimental cells until the confluence rate is above 80%, collecting the cells and counting them;

[0027] Step 1) further includes steps 1-2), wherein human umbilical vein endothelial cells or human lung fibroblasts are obtained and mixed with fibrinogen to form a cell stock solution;

[0028] Step 1) further includes steps 1-3), wherein the cell stock solution is mixed with thrombin within 10 to 20 minutes to form a biohydrogel;

[0029] Step 2) further includes step 2-1), injecting the bio-hydrogel from one of the injection holes within 10 to 30 seconds until the bio-hydrogel emerges from the other injection hole 11.1;

[0030] Step 2) further includes step 2-2), placing the dual-chamber co-culture organ chip in an incubator for 5 to 15 minutes;

[0031] Step 4-1) Inject 1-1.5 mL and 0.2-0.5 mL of EGM-2 culture medium into the two injection holes, respectively.

[0032] Step 4-2) Place the dual-chamber co-cultured organ chip in an incubator for 0.5-1.5 hours, then observe whether bubbles form at the two injection holes. If bubbles form, clear the two injection holes from the bottom with a pipette tip.

[0033] Step 4-3) Observe the two injection holes every 2 to 6 hours. If bubbles are generated, treat them using the method in step 4-2);

[0034] Step 4-4) The volume of the medium is rotated and replaced every 20 to 28 hours until a vascular network is formed in the cell culture chamber.

[0035] The present invention also provides a use of a dual-chamber co-culture organ chip for organ vascularization.

[0036] The dual-chamber co-culture organ chip provided by the present invention has the following beneficial effects:

[0037] 1) The dual-chamber co-culture organ chip provided by the present invention includes a flow channel layer and a bottom layer, which is used to observe the growth status of cell spheroids in vascularized co-culture without direct contact with the cells. Furthermore, the method of placing or removing the cell spheroids is relatively convenient.

[0038] 2) Furthermore, the cell spheres were placed in a cell sphere culture chamber with a radius of 500 μm, so that the cell tumor spheres could be directly observed under a microscope more easily.

[0039] 3) Existing literature indicates that previous vascularized co-cultures have simply mixed endothelial cells, fibroblasts, and spheroids together, making it difficult to distinguish the interactions and migration patterns between spheroids and the vascular network. The dual-chamber co-culture organ-on-a-chip provided by the present invention allows spheroids to be placed in independent spheroid culture chambers, allowing for separate spheroid culture. Microscopic observation of interactions between spheroids and blood vessels, such as tumor cell migration into blood vessels and drug delivery to kill tumors, is possible.

[0040] 4) Furthermore, the circular structure of the cell spheroid culture chamber of the present invention can better explore the growth and migration direction of cells (radially or in other directions) compared to cell chambers of other shapes.

[0041] 5) The present invention has a simple structure, only two layers, convenient operation, easy chip production, and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the separation of the flow channel layer and the bottom layer in the present invention.

[0043] Figure 2 This is a bonding diagram of the flow channel layer and the bottom layer in the present invention.

[0044] Figure 3 It is a top view of the flow channel layer in the present invention.

[0045] Figure 4 It is a schematic diagram of the overall appearance of the present invention.

[0046] Reference numerals

[0047] Runner layer 1

[0048] Culture medium flow channel 11

[0049] Filling hole 11.1

[0050] Cell culture chamber 12

[0051] Glue injection hole 12.1

[0052] Spheroid culture chamber 13

[0053] Through hole 13.1

[0054] Capillary burst valve 14

[0055] Capillary burst valve flow channel 14.1

[0056] Burst valve port 14.2

[0057] Pressure relief hole 14.3

[0058] Bottom Floor 2

[0059] External cavity microcolumn 3

[0060] Inner cavity microcolumn 4

[0061] Glass tube 5 DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be noted that the orientation or position relationship indicated by the terms "left side", "right side", "upper side", "lower side", "above", "below", etc. is based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0063] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0064] Furthermore, in the description of the present invention, unless otherwise specified, “plurality” means two or more.

[0065] The present invention provides a dual-chamber co-culture organ chip, see Figure 3 , which can be used as an auxiliary reference Figure 1 and Figure 2 , comprising a flow channel layer 1 and a bottom layer 2, wherein the flow channel layer 1 is located above the bottom layer 2, and a culture fluid flow channel 11 and a cell culture chamber 12 are provided at the bottom of the flow channel layer 1, and the bottom layer 2 is used to seal the culture fluid flow channel 11 and the cell culture chamber 12 and make them a closed structure. The flow channel layer 1 and the bottom layer 2 can usually be connected by ionic bonding. For example, the culture fluid flow channel 11 is used to pass the culture fluid and laminin, and the cell culture chamber 12 is used to pass the biohydrogel. Figure 3 The culture fluid flow channel 11 forms a loop, and the culture fluid flow channel 11 surrounds the outside of the cell culture chamber 12. The culture fluid flow channel 11 is connected to the cell culture chamber 12 and a plurality of external cavity micro-pillars 3 are provided at the connection point. The function of the external cavity micro-pillars 3 is to block the cell gel in the culture fluid flowing through the culture fluid flow channel 11 from passing through, but allow small molecules in the culture fluid to pass through and exchange material information. The principle is that the cell gel will form a gel interface due to surface tension, so that it cannot pass through the gap between adjacent micro-pillars. Figure 1 and Figure 2 , which can be used as an auxiliary reference Figure 3 The flow channel layer 1 is provided with two liquid injection holes 11.1, two glue injection holes 12.1 and a through hole 13.1. The two liquid injection holes 11.1 are respectively connected to the flow channel layer 1 and the two liquid injection holes 11.1 are symmetrical about the center. The two glue injection holes 12.1 are respectively connected to the cell culture chamber 12 and the two glue injection holes 12.1 are symmetrical about the center. The through hole 13.1 is connected to the cell spheroid culture chamber 13. The function of the liquid injection holes 11.1 is to perfuse the biological hydrogel into the culture fluid flow channel 11, the function of the glue injection holes 12.1 is to perfuse the culture fluid into the cell culture chamber 12, and the function of the through hole 13.1 is to place cultured material into the cell culture chamber 12 or the cell spheroid culture chamber 13 in other embodiments, such as perfusing tumor spheroids or organoids.

[0066] The dual-chamber co-culture organ chip provided in the embodiment of the present invention is described in detail in Figure 3 , which can be used as an auxiliary reference Figure 1 and Figure 2The bottom of the flow channel layer 1 is also provided with a spheroid culture chamber 13. The spheroid culture chamber 13 is surrounded by the cell culture chamber 12. The spheroid culture chamber 13 is connected to the cell culture chamber 12 and a plurality of inner cavity micro-pillars 4 are provided at the connection point. The through hole 13.1 is connected to the spheroid culture chamber 13. The function of the inner cavity micro-pillars 4 is to block the passage of cell gel in the culture fluid flowing through the culture fluid flow channel 11, but allow small molecules in the culture fluid to pass through and exchange material information. The principle is that the cell gel forms a gel interface due to surface tension, which prevents it from passing through the gaps between adjacent micro-pillars.

[0067] The dual-chamber co-culture organ chip provided in the embodiment of the present invention is combined with reference to Figure 1 and Figure 2 , which can be used as an auxiliary reference Figure 3 The bottom of the flow channel layer 1 is also provided with a capillary bursting valve 14 and a pressure reducing hole 14.3. The capillary bursting valve 14 includes a capillary bursting valve flow channel 14.1 and bursting valve ports 14.2 located at both ends of the capillary bursting valve flow channel 14.1. The capillary bursting valve flow channel 14.1 is connected to the cell culture chamber 12 through the bursting valve port 14.2. The pressure reducing hole 14.3 is connected to the capillary bursting valve flow channel 14.1. The function of the pressure reducing hole 14.3 is to release the pressure generated in the cell culture chamber 12 when the cell gel is injected. The capillary bursting valve flow channel 14.1 is arranged around one of the injection holes 12.1. For illustration, the injection hole 12.1 surrounded by the capillary burst valve channel 14.1 can be referred to as the gel injection hole, and the other injection hole 12.1 can be referred to as the gel outlet hole. In the embodiment equipped with the capillary burst valve 14, the gel injection hole always serves as the injection hole for the bio-hydrogel. As the bio-hydrogel flows from the gel injection hole into the cell culture chamber 12, the capillary burst valve channel 14.1 releases the pressure generated within the channel in a direction opposite to the bio-hydrogel, ultimately releasing the pressure through the pressure relief hole 14.3.

[0068] In a specific embodiment, referring to Figures 1 to 3 The culture fluid flow channel 11 and the cell culture chamber 12 are both centrally symmetrical structures. The cell spheroid culture chamber 13 is circular and located in the center of the cell culture chamber 12. The culture fluid flow channel 11 is dumbbell-shaped and centrally symmetrical. The two injection holes 11.1 are respectively connected to the hammer-shaped parts on both sides of the culture fluid flow channel 11, and the cross-sectional shape of the part of the culture fluid flow channel 11 connected to the injection holes 11.1 is diamond-shaped. The part of the culture fluid flow channel 11 in the hammer-shaped part is a serpentine with multiple bends. The top of the flow channel layer 1 can also be connected to the external glass tube 5. The glass tube 5 can refer to Figure 4The glass tube 5 is concentrically arranged with the injection hole 11.1 and is used to inject the culture fluid into the culture fluid flow channel 11. The cell culture chamber 12 is elongated in shape. The central area of the cell culture chamber 12, that is, the area surrounded by the outer cavity micropillars 3, is circular. The two edge areas of the cell culture chamber 12 are teardrop-shaped. The two injection holes 12.1 are respectively connected to the two edge areas. The flow channel width of the edge area of the cell culture chamber 12 gradually decreases from the injection holes 12.1 to the cell culture chamber 12, then maintains this width to form a straight flow channel, and then gradually increases to form the central area of the cell culture chamber 12. The central area of the cell culture chamber 12 may be provided with a cell spheroid culture chamber 13. The capillary bursting valve channel 14.1 is in the shape of a U-shaped capillary tube, and the capillary bursting valve 14 is surrounded by the hammer-shaped portion of the culture fluid channel 11. The bursting valve port 14.2 is connected to the direct current channel of the cell culture chamber 12. The width of the direct current channel is 1000-1400 μm.

[0069] In addition, the flow channel layer 1 has a thickness of 1 to 2 mm, a length of 40 to 50 mm, and a width of 15 to 20 mm. Preferably, the flow channel layer 1 has a thickness of 1 mm, a length of 40 mm, and a width of 15 mm.

[0070] In addition, the bottom layer 2 has a thickness of 1 to 1.5 mm, a length of 40 to 50 mm, and a width of 15 to 20 mm. Preferably, the bottom layer 2 has a thickness of 1 mm, a length of 40 mm, and a width of 15 mm.

[0071] In addition, the width of the culture fluid flow channel 11 is 50 to 150 μm, the total length of the serpentine flow channel in the culture fluid flow channel 11 is 7 to 8 mm, the total length of the culture fluid flow channel 11 is 12 to 14 mm, and the flow channel height of the culture fluid flow channel 11 and the cell culture chamber 12 is 80 to 120 μm. Preferably, the length of the bottom layer 2 is 40 mm and the width is 15 mm, and the flow channel height of the culture fluid flow channel 11 and the cell culture chamber 12 is 100 or 200 μm.

[0072] Additionally, the middle portion of the cell culture chamber 12 is circular, and its radius is 1000-1200 μm.

[0073] As a supplement, the radius of the two liquid injection holes 11.1, the two glue injection holes 12.1 and the through hole 13.1 are all 500-600 μm. If the external glass tube 5 is connected, the diameter of the glass tube 5 is 8 mm and the height is 15 mm.

[0074] In addition, the width of the outer cavity microcolumn 3 is 80 to 120 μm, the length of the outer cavity microcolumn 3 is 300 to 500 μm, and the gap between adjacent outer cavity microcolumns 3 is 50 to 80 μm. The width of the inner cavity microcolumn 4 is 80 to 120 μm, the length of the inner cavity microcolumn 4 is 300 to 500 μm, and the gap between adjacent inner cavity microcolumns 4 is 50 to 80 μm. Both the outer cavity microcolumn 3 and the inner cavity microcolumn 4 are complete rings before molding, and then the rings are cut into several parts to form outer cavity microcolumns 3 or inner cavity microcolumns 4. Therefore, the width of the outer cavity microcolumn 3 and the inner cavity microcolumn 4 can be understood as the width of the ring, and the length can be understood as the arc length of the outer cavity microcolumn 3 and the inner cavity microcolumn 4.

[0075] Additionally, the radius of the cell spheroid culture chamber 13 is 500-600 μm.

[0076] In addition, the width of the bursting valve opening 14.2 is greater than the gap between adjacent outer cavity micropillars 3 and the gap between adjacent inner cavity micropillars 4. The width of the bursting valve opening 14.2 is 60 to 100 μm, the width of the capillary bursting valve channel 14.1 is 300 μm, and the radius of the pressure reducing hole 14.3 is 500 to 600 μm.

[0077] Example 1: Preparation Method of a Dual-Chamber Co-culture Organ Chip

[0078] This embodiment provides a method for preparing a dual-chamber co-culture organ chip, comprising the following steps:

[0079] Step 1) First, a mask for the channel layer 1 is made, and then the mask is photolithographically etched into a silicon wafer with a microchannel pattern. The microchannel structure includes at least a culture fluid channel 11 and a cell culture chamber 12. The silicon wafer is then pasted into a culture dish to make a mold.

[0080] In step 1), a drawing of the flow channel layer 1 can be drawn using drawing software, and a mask plate of the flow channel layer 1 can be made according to the drawing.

[0081] In step 1), the height of the culture fluid flow channel 11 and the cell culture chamber 12 is 100 μm.

[0082] In step 1), the micro-channel pattern may further include a capillary burst valve 14 .

[0083] Step 2) A polydimethylsiloxane prepolymer and a curing agent are mixed and poured into a flow channel layer mold to obtain a polydimethylsiloxane sheet with a microchannel structure. The polydimethylsiloxane sheet is the flow channel layer 1.

[0084] In step 2), the ratio of polydimethylsiloxane prepolymer to curing agent is 10:1.

[0085] In step 2), after the polydimethylsiloxane prepolymer and the curing agent are mixed and poured into the runner layer mold, the steps of vacuuming, blowing bubbles, drying and peeling are also included.

[0086] Step 3) A polydimethylsiloxane prepolymer and a curing agent are mixed and poured into a bottom layer mold to obtain a polydimethylsiloxane sheet, which is the bottom layer.

[0087] In step 3), the size of the culture dish is 100 mm 2 , the mass of the polydimethylsiloxane prepolymer is 4.5 to 5 g.

[0088] In step 3), after the polydimethylsiloxane prepolymer and the curing agent are mixed and poured into the runner layer mold, the steps of vacuuming, blowing bubbles, drying and peeling are also included.

[0089] Step 4) Punch the channel layer 1 to obtain two liquid injection holes 11.1 and two glue injection holes 12.1, then plasma bond the bottom of the channel layer 1 and the bottom layer 2. After bonding, obtain the chip and heat the chip to strengthen the bonding between the chip layers.

[0090] In step 4), the channel layer 1 is punched to obtain a through hole 13.1 and a capillary burst valve 14 hole.

[0091] In step 4), the dual-chamber co-culture organ chip is placed on a hot plate and baked for 5 minutes to strengthen the bonding between the chip layers.

[0092] The process may further include step 5) using polydimethylsiloxane to adhere multiple glass tubes 5 to the top of the flow channel layer 1 and connect them to the liquid injection hole 11.1 and the glue injection hole 12.1 respectively, and then curing the chip to obtain a dual-chamber co-culture organ chip with glass tubes 5.

[0093] In step 5), the dual-chamber co-culture organ chip is placed in an oven for curing. The curing time is 2 hours and the curing temperature is 70°C.

[0094] As a supplementary note, the dual-chamber co-culture organ chip needs to be sterilized at high temperature before the experiment, placed in a clean bench for UV sterilization for 30 minutes, and stored properly.

[0095] Example 2: A method for preparing a dual-chamber co-culture organ chip using photosensitive dry film technology

[0096] Step 1) First prepare a mask for the flow channel layer 1, then stick a photosensitive polymer film to the surface of the mask and hot-press to obtain a pre-silicon wafer.

[0097] In step 1-1), a drawing of the flow channel layer 1 can be drawn using drawing software, and then the drawing can be processed into a film mask.

[0098] Step 1-2) Cut the SAF100 photopolymer film (each layer is 100 μm thick) into a square to cover the entire chip, remove the PET film on the surface of the photopolymer film and stick it to the chip surface, and use scissors to cut the excess polymer film around the chip.

[0099] Step 1-3) Place the sample in a hot press at 95°C and perform two rounds of hot pressing to consolidate the sample.

[0100] Step 2) The pre-silicon wafer prepared in step 1) is exposed and then developed to obtain a silicon wafer with a microchannel pattern, which is then cast to form a channel layer 1.

[0101] Step 2-1) Place the pre-silicon wafer on the corresponding wafer stage of the photolithography machine to expose it, then bake it and develop it with 2% sodium carbonate solution to obtain a silicon wafer with a microchannel pattern after hardening.

[0102] Step 2-2) Polydimethylsiloxane is cast to form a flow channel layer 1.

[0103] Example 2: A method for using a dual-chamber co-culture organ chip

[0104] This embodiment provides a method for using a dual-chamber co-culture organ chip, comprising the following steps:

[0105] Step 1) experimental cells are obtained, the supernatant is removed, and the resultant solution is mixed with fibrinogen to form a cell stock solution, and the cell stock solution is then obtained and mixed with thrombin to form a biohydrogel.

[0106] Step 1-1) The experimental cells were cultured until the confluence rate was above 80%, and the cells were collected and counted to ensure that the cell concentration met the experimental requirements.

[0107] Step 1-2) Human umbilical vein endothelial cells (HUVEC) or human lung fibroblasts (NHLF) are obtained and mixed with fibrinogen to form a cell stock solution.

[0108] Steps 1-3) The cell stock solution was mixed with thrombin within 15 minutes to form a biohydrogel.

[0109] In step 1-3), the ratio between the cell stock solution and thrombin is 10:1.

[0110] Step 2) injecting biohydrogel from one of the injection holes 12.1 until the biohydrogel emerges from the other injection hole 12.1, and then placing the dual-chamber co-culture organ chip into an incubator;

[0111] Step 2-1) Inject the bio-hydrogel from one of the injection holes 12.1 within 20 seconds until the bio-hydrogel emerges from the other injection hole 11.1.

[0112] In step 2-1), if a capillary bursting valve 14 is provided, the bio-hydrogel is injected from the injection hole 12.1 surrounded by the capillary bursting valve flow channel 14.1, that is, the bio-hydrogel is injected from the gel injection hole.

[0113] Step 2-2), place the dual-chamber co-culture organ chip in an incubator for 10 minutes.

[0114] In step 2), the bio-hydrogel will fill the entire cell culture chamber 12 , but due to the effect of the inner cavity micro-pillars 4 , the cells will not leak into the culture fluid channel 11 .

[0115] Step 3) Remove the dual-chamber co-culture organ chip from the incubator in step 2) and inject the laminin modification into one of the injection holes 11.1. After the laminin modification emerges from the other injection hole 11.1, place the dual-chamber co-culture organ chip in the incubator for culture.

[0116] In step 3), the injection volume of the modified laminin is 8 to 12 μL, preferably 10 μL.

[0117] In step 3), the dual-chamber co-cultured organ chip is placed in an incubator and cultured for 10 to 20 minutes, preferably 10 minutes. In step 4), the dual-chamber co-cultured organ chip is removed from the incubator in step 3), and EGM-2 culture medium is injected into one of the injection holes 11.1. Observe whether the culture medium quickly flows through the cell culture chamber 12 and emerges from the other injection hole 11.1.

[0118] Step 5) Different amounts of EGM-2 culture medium are injected into the two injection holes 11.1 respectively, and the dual-chamber co-culture organ chip is placed in an incubator until a vascular network is formed in the cell culture chamber 12.

[0119] Step 5-1) Inject 1.2 mL and 0.2 mL of EGM-2 culture medium into the two injection holes 11.1, respectively.

[0120] Step 5-2) Place the dual-chamber co-culture organ chip in the incubator for 1 hour, then observe whether bubbles form at the two injection holes 11.1; if bubbles form, insert a pipette tip into the two injection holes 11.1 and clear them; after clearing, place the dual-chamber co-culture organ chip back into the incubator.

[0121] Step 5-3) Observe the two injection holes 11.1 every 4 hours. If bubbles are generated, treat them using the method in step 4-2).

[0122] Step 5-4) The volume of the medium is rotated and replaced every 24 hours until a vascular network is formed in the cell culture chamber 12 .

[0123] In step 5), the different injection volumes of EGM-2 culture medium in the glass tube 5 corresponding to the two injection holes 11.1 create a difference in the liquid level, thereby mechanically stimulating the cells in the cell culture chamber 12. Furthermore, the difference in the liquid level in the glass tube 5 corresponding to the two injection holes 11.1 is required to be equal to 1 mL.

[0124] The method may further include step 6) selecting different co-culture methods according to the structural differences of the dual-chamber co-culture organ chips.

[0125] In step 6), if the inner cavity microcolumns 4 are not provided in the cell culture chamber 12, the space reserved columns are removed after the vascular network is formed, and a vascular network with a cylindrical cavity in the center will be formed in the cell culture chamber 12. The culture to be vascularized is then placed through the through hole 13.1 into the cell culture chamber 12 with the formed vascular network for co-culture.

[0126] In step 6), if the cell culture chamber 12 is provided with an inner cavity microcolumn 4, after the vascular network is formed, the pre-vascularized tumor spheres or organoids are placed in the cell sphere culture chamber 13 for pre-vascularization culture.

[0127] Furthermore, the method may further include step 7) recording and analyzing the data so as to perform subsequent experimental operations.

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and substitutions can be made without departing from the technical principles of the present invention. These improvements and substitutions should also be regarded as the scope of protection of the present invention.

Claims

1. A dual-chamber co-culture organ chip, characterized by: The invention comprises a flow channel layer (1) and a bottom layer (2), wherein the flow channel layer (1) is located above the bottom layer (2), and a culture fluid flow channel (11) and a cell culture chamber (12) are provided at the bottom of the flow channel layer (1), wherein the culture fluid flow channel (11) forms a loop, and the culture fluid flow channel (11) surrounds the outside of the cell culture chamber (12), and the culture fluid flow channel (11) is connected to the cell culture chamber (12), and a plurality of external cavity microcolumns (3) are provided at the connection point; The flow channel layer (1) is provided with two liquid injection holes (11.1), two glue injection holes (12.1) and a through hole (13.1); the two liquid injection holes (11.1) are respectively connected to the culture fluid flow channel (11) and the two liquid injection holes (11.1) are centrally symmetrical; the two glue injection holes (12.1) are respectively connected to the cell culture chamber (12) and the two glue injection holes (12.1) are centrally symmetrical; the through hole (13.1) is connected to the cell culture flow channel.

2. The dual-chamber co-culture organ chip according to claim 1, characterized in that: A cell spheroid culture chamber (13) is further provided at the bottom of the flow channel layer (1), the cell spheroid culture chamber (13) is surrounded by the cell culture chamber (12), the cell spheroid culture chamber (13) is connected to the cell culture chamber (12), and a plurality of inner cavity micro-columns (4) are provided at the connection point, and the through hole (13.1) is connected to the cell spheroid culture chamber (13).

3. The dual-chamber co-culture organ chip according to claim 2, characterized in that: A capillary bursting valve (14) and a pressure reducing hole (14.3) are further provided at the bottom of the flow channel layer (1). The capillary bursting valve (14) comprises a capillary bursting valve flow channel (14.1) and bursting valve ports (14.2) located at both ends of the capillary bursting valve flow channel (14.1). The capillary bursting valve flow channel (14.1) is connected to the cell culture chamber (12) via the bursting valve ports (14.2). The pressure reducing hole (14.3) is connected to the capillary bursting valve flow channel (14.1). The capillary bursting valve flow channel (14.1) is arranged around one of the injection holes (12.1).

4. The dual-chamber co-culture organ chip according to any one of claims 1 to 3, characterized in that: The thickness of the flow channel layer (1) is 1 to 2 mm, the length is 40 to 50 mm, and the width is 15 to 20 mm; and / or the width of the culture fluid flow channel (11) is 50 to 150 μm, and the total length of the culture fluid flow channel (11) is 12 to 14 mm; and / or the radii of the two liquid injection holes (11.1), the two glue injection holes (12.1) and the through hole (13.1) are all 500 to 600 μm; and / or the width of the outer cavity microcolumn (3) is 80 to 120 μm, the length of the outer cavity microcolumn (3) is 300 to 500 μm, and the gap between adjacent outer cavity microcolumns (3) is 50 to 80 μm; and / or the flow channel height of the culture fluid flow channel (11) and the cell culture chamber (12) is 100 to 200 μm.

5. The dual-chamber co-culture organ chip according to claim 2, characterized in that: The radius of the cell spheroid culture chamber (13) is 500-600 μm; and / or the width of the inner cavity microcolumn (4) is 80-120 μm, the length of the inner cavity microcolumn (4) is 300-500 μm, and the gap between adjacent inner cavity microcolumns (4) is 50-80 μm.

6. The dual-chamber co-culture organ chip according to claim 3, characterized in that: The width of the bursting valve opening (14.2) is greater than the gap between adjacent outer cavity microcolumns (3) and the gap between adjacent inner cavity microcolumns (4). The width of the bursting valve opening (14.2) is 60 μm to 100 μm, the width of the capillary bursting valve flow channel (14.1) is 200 to 300 μm, and the radius of the pressure reducing hole (14.3) is 500 to 600 μm.

7. A method for preparing a dual-chamber co-culture organ chip according to any one of claims 1 to 6, comprising the following steps: Step 1) First, a mask for the flow channel layer (1) is prepared, and then the mask is photoetched into a silicon wafer with a microchannel pattern, wherein the microchannel structure at least includes a culture fluid flow channel (11) and a cell culture chamber (12), and then the silicon wafer is pasted into a culture dish to form a flow channel layer mold; Step 2) a polydimethylsiloxane prepolymer and a curing agent are mixed and poured into a flow channel layer mold to obtain a polydimethylsiloxane sheet with a microchannel structure, which is the flow channel layer (1); Step 3) a polydimethylsiloxane prepolymer is selected and mixed with a curing agent and poured into a bottom layer mold to obtain a polydimethylsiloxane sheet, which is the bottom layer (2); Step 4) The flow channel layer (1) is punched to obtain two liquid injection holes (11.1) and two glue injection holes (12.1), and then the bottom of the flow channel layer (1) and the bottom layer (2) are plasma bonded. After the bonding is completed, the chip is obtained and the chip is heated to strengthen the bonding between the chip layers.

8. A method for using the dual-chamber co-culture organ chip according to any one of claims 1 to 6, comprising the following steps: Step 1) taking experimental cells, removing the supernatant, and mixing with fibrinogen to form a cell stock solution, and then taking the cell stock solution and mixing it with thrombin to form a biohydrogel; Step 2) Inject the biohydrogel from one of the injection holes (11.1) until the biohydrogel emerges from the other injection hole (11.1), and then place the dual-chamber co-culture organ chip in an incubator; Step 3) Remove the dual-chamber co-cultured organ chip from the incubator in step 2) and inject the laminin modification into one of the injection holes (11.1). After the laminin modification emerges from the other injection hole (11.1), place the dual-chamber co-cultured organ chip in the incubator for culture; Step 4) Take the dual-chamber co-culture organ chip out of the incubator in step 3) and inject EGM-2 culture medium into one of the injection holes (11.1), and observe whether the culture medium quickly flows through the cell culture chamber (12) to emerge from the other injection hole (11.1); Step 5) Different amounts of EGM-2 culture medium are injected into the two injection holes (11.1) respectively, and the dual-chamber co-culture organ chip is placed in an incubator until a vascular network is formed in the cell culture chamber (12).

9. The method for using the dual-chamber co-culture organ chip according to claim 8, further comprising any one of the following technical features: Step 1) further includes step 1-1), culturing the experimental cells until the confluence rate is above 80%, collecting the cells and counting them; Step 1) further includes steps 1-2), wherein human umbilical vein endothelial cells or human lung fibroblasts are obtained and mixed with fibrinogen to form a cell stock solution; Step 1) further includes steps 1-3), wherein the cell stock solution is mixed with thrombin within 10 to 20 minutes to form a biohydrogel; Step 2) further includes step 2-1), injecting the bio-hydrogel from one of the injection holes (11.1) within 10 to 30 seconds until the bio-hydrogel emerges from the other injection hole (11.1); Step 2) further includes step 2-2), placing the dual-chamber co-culture organ chip in an incubator for 5 to 15 minutes; Step 5-1) Inject 1-1.5 mL and 0.2-0.5 mL of EGM-2 culture medium into the two injection holes (11.1), respectively; Step 5-2) Place the dual-chamber co-culture organ chip in an incubator for 0.5-1.5 hours, then observe whether bubbles form at the two injection holes (11.1); if bubbles form, clear the two injection holes (11.1) from the bottom with a pipette tip; Step 5-3) Observe the two injection holes (11.1) every 2 to 6 hours. If bubbles are generated, treat them using the method in step 4-2). Step 5-4) The volume of the medium is rotated and replaced every 20 to 28 hours until a vascular network is formed in the cell culture chamber (12).

10. Use of the dual-chamber co-culture organ chip according to any one of claims 1 to 6 for microfluidic cell culture.

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