CN113862154A - Organ chip for three-dimensional culture of organ tissues and culture method of organ tissues - Google Patents

Organ chip for three-dimensional culture of organ tissues and culture method of organ tissues Download PDF

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CN113862154A
CN113862154A CN202111460739.7A CN202111460739A CN113862154A CN 113862154 A CN113862154 A CN 113862154A CN 202111460739 A CN202111460739 A CN 202111460739A CN 113862154 A CN113862154 A CN 113862154A
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culture
flow channel
layer
chip
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顾忠泽
陈早早
欧阳珺
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Southeast University Suzhou Medical Device Research Institute
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Southeast University Suzhou Medical Device Research Institute
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Abstract

The invention provides an organ chip for three-dimensional culture of organ tissues and a culture method of the organ tissues. The chip comprises a sealing layer, a culture layer and a connecting layer, wherein the culture layer comprises a first sub-culture layer, a separation layer and a second sub-culture layer, the culture layer is provided with a culture chamber, an inlet flow channel, a flow guide flow channel and an outlet flow channel, the sealing layer is provided with a first bubble removing flow channel, the connecting layer is provided with an inlet, a discharge port and a second bubble removing flow channel, the inlet flow channel of the first sub-culture layer is respectively connected with the inlet and the first bubble removing flow channel, the flow guide flow channel is respectively connected with the first bubble removing flow channel and the culture chamber, and the outlet flow channel is respectively connected with the culture chamber and the discharge port; the inlet flow channel of the second sub-culture layer is respectively connected with the inlet and the second bubble removing flow channel, the flow guide flow channel is respectively connected with the second bubble removing flow channel and the culture chamber, and the outlet flow channel is respectively connected with the culture chamber and the outlet. Provides an organ culture chip capable of simulating the absorption process of a human body.

Description

Organ chip for three-dimensional culture of organ tissues and culture method of organ tissues
Technical Field
The invention belongs to the technical field of biological tissue engineering and biomedicine, and particularly relates to an organ chip for three-dimensional culture of organ tissues and a method for culturing the organ tissues.
Background
An organ chip, also called a micro-physiological system, is a new technology for realizing the simulation of the functions of human organs by three-dimensional culture of cells in an in vitro chip. The organ chip has wide application prospect in the fields of new drug research and development, disease models, personalized medicine, aerospace medicine and the like. In 2016, organ chips were listed as "ten new technologies" by the Darwos economic forum.
One of the most challenging links in drug development is how to test the effectiveness and safety of drugs. Cell and animal experiments are two experiment platforms widely used in the current drug research and development and evaluation processes, the former is difficult to simulate the human physiological microenvironment, and the latter is complex, expensive, time-consuming and has ethical debate. Therefore, economical and efficient animal and even clinical alternative experiments are necessary. Organ chips are a new experimental method for drug evaluation which has been proposed in recent years.
The organ chip is a three-dimensional cell culture system based on a microfluid chip, and simulates the microstructure, microenvironment and physiological function of a specific organ of a human body in a bionic manner. The multi-organ chip comprises a plurality of cell culture subareas simulating human tissue and organ environments, and cells cultured in three dimensions in each subarea are connected through a bionic circulating system. Provides a new system model which is closer to the real environment of the human body for the fields of new drug research and development, disease research models, personalized medicine and the like. Provides a complete, real and real brand-new system capable of monitoring in real time for the effect, the action mechanism, the cytotoxicity, the cell invasion in the research of disease models and the like in the research and development of new drugs.
One of the newly emerging new drug research and development and disease models can construct three dimensions of organ tissues and provide a new model for drug research and development, but still is a single-organ static research, and cannot simulate the real environment of human body for realizing nutrition and drug absorption or human body tumor metastasis and invasion through functional circulation such as blood circulation, vascular filtration and the like.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art and provides an organ chip for three-dimensional culture of organ tissues and a method for culturing organ tissues.
In one aspect of the invention, a universal organ chip for three-dimensional culture of multiple organ tissues is provided, the chip comprises a sealing layer, a culture layer and a connecting layer which are sequentially stacked, the culture layer comprises a first sub-culture layer, a separation layer and a second sub-culture layer which are sequentially arranged on the sealing layer, the first sub-culture layer is provided with a first culture chamber, a first inlet flow channel, a first flow guide flow channel and a first outlet flow channel, the second sub-culture layer is provided with a second culture chamber, a second inlet flow channel, a second flow guide flow channel and a second outlet flow channel, the sealing layer is provided with a first bubble removing flow channel, and the connecting layer is provided with an inlet, an outlet and a second bubble removing flow channel; wherein,
the first inlet flow channel is respectively connected with the inlet and the first bubble removing flow channel, the first flow guide flow channel is respectively connected with the first bubble removing flow channel and the first culture chamber, and the first outlet flow channel is respectively connected with the first culture chamber and the outlet;
the second inlet flow channel is respectively connected with the introducing port and the second bubble removing flow channel, the second flow guide flow channel is respectively connected with the second bubble removing flow channel and the second culture chamber, and the second outlet flow channel is respectively connected with the second culture chamber and the discharge port.
Optionally, the first inlet flow channel and the first outlet flow channel are both arranged on the side of the first sub-culture layer facing the sealing layer; and/or the presence of a gas in the gas,
the second inlet flow channel and the second outlet flow channel are both arranged on one side, away from the sealing layer, of the second sub-culture layer.
Optionally, the first sub-culture layer is provided with a first hollowed square culture groove penetrating through the first sub-culture layer in the thickness direction, and the first hollowed square culture groove forms the first culture chamber; and/or the presence of a gas in the gas,
the second sub-culture layer is provided with a second hollowed square culture groove penetrating through the second sub-culture layer in the thickness direction, and the second hollowed square culture groove forms the second culture chamber.
Optionally, the introducing port comprises a first introducing port and a second introducing port, the discharging port comprises a first discharging port and a second discharging port, and the second sub-culture layer is further provided with a first introducing through hole and a first discharging through hole penetrating through the thickness thereof; wherein,
the second inlet port is connected to the second inlet flow passage, and the second outlet port is connected to the second outlet flow passage;
the first introduction port is connected to the first inlet flow path through the first introduction through hole, and the first discharge port is connected to the first outlet flow path through the first discharge through hole.
Optionally, the connecting layer is further provided with an observation groove penetrating through the thickness of the connecting layer, and the position of the observation groove corresponds to the position of the culture chamber;
the chip further comprises an observation window cover, and the observation window cover is arranged on the observation groove in a sealing mode.
Optionally, a perfusion port and a vent port are further arranged on the connecting layer, a first perfusion channel and a first ventilation channel are further arranged on the first sub-culture layer, and a second perfusion channel and a second ventilation channel are further arranged on the second sub-culture layer; wherein,
the first perfusion flow channel is respectively connected with the perfusion port and the first culture chamber, and the first ventilation flow channel is respectively connected with the first culture chamber and the ventilation port;
the second perfusion channel is respectively connected with the perfusion opening and the second culture chamber, and the second ventilation channel is respectively connected with the second culture chamber and the ventilation opening.
Optionally, the perfusion ports include a first perfusion port and a second perfusion port, the vent port includes a first vent port and a second vent port, and the second sub-culture layer is further provided with a second introduction through hole and a second discharge through hole penetrating through the thickness of the second sub-culture layer; wherein,
the second perfusion opening is connected with the second perfusion flow channel, and the second vent opening is connected with the second vent flow channel;
the first filling port is connected with the first filling flow channel through the second introducing through hole, and the first vent port is connected with the first ventilating flow channel through the second discharging through hole.
Optionally, the first perfusion flow channel and the first ventilation flow channel are both disposed on a side of the first sub-culture layer facing away from the sealing layer; and/or the presence of a gas in the gas,
the second perfusion channel and the second ventilation channel are both arranged on one side, facing the sealing layer, of the second sub-culture layer.
Optionally, the separator is a porous membrane.
In another aspect of the present invention, there is provided a method for culturing organ tissues, using the chip described above, the method specifically comprising:
using human-derived multifunctional stem cells to promote the differentiation into mature human-derived endothelial cells and human-derived myocardial cells respectively;
inoculating the chip into a culture system, and sterilizing the chip and the culture system by adopting ethylene oxide, ultraviolet rays or alcohol;
respectively injecting 70-90 ul of collagen solution into the first sub-culture chamber and the second sub-culture chamber in an aseptic culture environment, refrigerating and standing at 3-4 ℃ for 1.5-2 h, and performing cell adsorption treatment on the surface of the porous membrane in the culture chambers;
sucking out collagen in the culture, putting the collagen in a sterile oven, and drying for 1-1.5 h;
70 ul-80 ul with the concentration of 0.5x105~1x105Injecting a human endothelial cell solution per ml into the first sub-culture chamber, inverting the chip, placing the chip into a sterile incubator, and standing for 2-3 hours to ensure that endothelial cells are firmly attached to the lower surface of the porous membrane in the culture chamber;
taking out the chip from the incubator, and adding 70-80 ul of the chip with the concentration of 0.5x105~1x105Injecting the human cardiac cell solution per ml into a second culture chamber, placing the chip into an aseptic culture box and standing for 2-3 hours, so that the cardiac cells are firmly attached to the upper surface of the porous membrane in the culture chamber;
injecting 10 ml-15 ml of culture medium into each culture bottle, starting a culture system, and starting continuous perfusion culture of cells in the culture chamber;
putting the culture system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days until endothelial cells and heart cells show functional characteristics;
taking out the chip from the incubator, respectively loading sterile platinum electrodes on the chip in a sterile environment, and electrifying weak square wave current with the frequency of 0.5 Hz-1 Hz to start a perfusion culture system;
putting the system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days to stimulate the upper layer heart tissue in the culture chamber to have regular pulsation consistent with the electrifying frequency;
when the power is cut off and the current is switched on, the heart tissue can grow well and form spontaneous regular pulsation;
changing the culture medium corresponding to the second sub-culture layer to a concentration of 1 x10 in a sterile environment-6mol/L~1*10-6A mol/L culture medium of noradrenaline;
and starting a culture system, and automatically culturing the culture system and a 35-37 ℃ incubator of the chip house for 1-2 days to obtain the organ tissues.
The invention provides an organ culture chip capable of simulating the human body absorption process and provides a brand-new model for drug screening and disease research, and the chip is suitable for mixed culture of various organ tissues such as skin, heart, liver, kidney, intestinal tract, spleen, pancreas and the like. The chip of the invention can realize the real process of simulating the transportation and absorption of medicines, nutrition and the like in vivo through blood vessels in vitro, and can replace animal experiments to be used as an important platform for medicine screening and medicine toxicity experiments.
Drawings
FIG. 1 is an exploded view of a chip according to an embodiment of the invention;
FIG. 2 is a schematic structural diagram of a connection layer in a chip according to an embodiment of the invention;
FIG. 3 is a schematic view showing the structure of a second sub-culture layer in a culture layer according to an embodiment of the present invention;
FIG. 4 is a schematic structural view of a first sub-culture layer in a culture layer according to an embodiment of the present invention;
FIG. 5 is a schematic structural diagram of a sealing layer according to an embodiment of the present invention
FIG. 6 is a schematic structural view of a culture system according to an embodiment of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
As shown in FIG. 1, the universal organ chip for three-dimensional culture of multiple organ tissues comprises a sealing layer A1, a culture layer and a connecting layer A5 which are sequentially stacked, wherein the culture layer comprises a first sub-culture layer A2, a separation layer A3 and a second sub-culture layer A4 which are sequentially arranged on the sealing layer A1. The sealing layer A1, the first sub-culture layer A2, the second sub-culture layer A4 and the connecting layer A5 can be made of high transparent polymer materials such as PMMA, PC, PS, COC and the like, and can be formed by die casting or injection molding, or can be formed by processing materials such as glass and the like. The separator A3 may have a porous membrane structure or a similar porous membrane structure, and for example, the separator A3 may have a porous membrane made of a material such as PC, PET, PTFE, NC or the like. Of course, besides, those skilled in the art may select other materials to form the sealing layer a1, the first sub-culture layer a2, the separation layer A3, the second sub-culture layer a4 and the connection layer a5 according to actual needs, which is not limited in this embodiment.
It will be appreciated that the culture layer may comprise, in addition to two sub-culture layers and one separation layer, also a plurality of sub-culture layers, for example, the culture layer may comprise three sub-culture layers and two separation layers. Of course, in addition to this, a larger number of sub-culture layers may be included, and one (or more) separation layers may be provided between two adjacent sub-culture layers.
It should be noted that, how the sealing layer a1, the culture layer, and the connecting layer a5 are connected together is not limited. Illustratively, the upper surface of the second sub-culture layer A4 and the lower surface of the connecting layer A5 can be connected together by double-sided adhesive, biological adhesive, thermocompression bonding, ultrasonic bonding, laser bonding, and the like. The first sub-culture layer A2, the partition layer A3 and the second culture layer A4 can be connected together by double-sided adhesive, biological adhesive, thermocompression bonding, ultrasonic bonding, laser bonding and the like, and the partition layer A3 divides the culture chamber into a first culture chamber D6 and a second culture chamber C6. The upper surface of the sealing layer A1 and the lower surface of the first sub-culture layer A2 can be connected together by double-sided adhesive, biological adhesive, thermocompression bonding, ultrasonic bonding, laser bonding, and the like. Of course, besides, those skilled in the art may also adopt other connection manners to connect the sealing layer a1, the culture layer and the connection layer a5 together according to actual needs, and this embodiment is not particularly limited thereto.
Illustratively, as shown in FIGS. 1 and 4, the first sub-culture layer A2 is provided with a first culture chamber D6, a first inlet flow channel D4-1, a first guide flow channel D4-4 and a first outlet flow channel D3-1. The first inlet flow channel D4-1 is provided with an inlet D4 and an outlet D4-3, the first diversion flow channel D4-4 is provided with an inlet D4-3, and the first outlet flow channel D3-1 is provided with an outlet D3. As shown in FIGS. 1 and 3, the second sub-culture layer A4 is provided with a second culture chamber C6, a second inlet flow channel C1-1, a second guide flow channel C1-4 and a second outlet flow channel C2-1. The second inlet flow channel C1-1 is provided with an inlet C1 and an outlet C1-2, the second guide flow channel C1-4 is provided with an inlet C1-3, and the second outlet flow channel C2-1 is provided with an outlet C2. As shown in fig. 1 and 5, a first bubble removing flow passage E9-2 is provided in the sealing layer a 1. The first bubble removal flow path E9-2 is shown to have an inlet E9-3 and an outlet E9-1. As shown in fig. 1 and 2, the connection layer a5 is provided with an inlet, an outlet, and a second bubble removing flow path B9-2. The introduction port may include a first introduction port B4 and a second introduction port B1, and the discharge port may include a first discharge port B3 and a second discharge port B2. The second bubble removal flow passage B9-2 has an inlet B9-3 and an outlet B9-1.
Specifically, as shown in fig. 2, 4 and 5, an inlet D4 of the first inlet flow channel D4-1 is connected to the first inlet B4, an outlet D4-3 of the first inlet flow channel D4-1 is connected to an inlet E9-3 of the first debubbling flow channel E9-2, an inlet D4-3 of the first guide flow channel D4-4 is connected to an outlet E9-1 of the first debubbling flow channel E9-2, an outlet of the first guide flow channel D4-4 is connected to the first culture chamber D6, an inlet of the first outlet flow channel D3-1 is connected to the first culture chamber D6, and an outlet D3 of the first outlet flow channel D3-1 is connected to the first outlet B3.
As shown in FIGS. 2 and 3, an inlet C1 of the second inlet flow channel C1-1 is connected to the second introduction port B1, an outlet C1-2 of the second inlet flow channel C1-1 is connected to an inlet B9-3 of the second bubble removal flow channel B9-2, an inlet C1-3 of the second guide flow channel C1-4 is connected to an outlet B9-1 of the second bubble removal flow channel B9-2, an outlet of the second guide flow channel C1-4 is connected to the second culture chamber C6, an inlet of the second outlet flow channel C2-1 is connected to the second culture chamber C6, and an outlet C2 of the second outlet flow channel C2-1 is connected to the second discharge port B2.
When organ tissue culture is performed using the chip of this embodiment, specifically, when culture is performed using the second sub-culture layer a4, the culture medium (or culture solution) that enters the second introduction port B1 of the connection layer a5 enters the second inlet channel C1-1 through the inlet C1 of the second inlet channel C1-1, then enters the inlet B9-3 of the second bubble-removing channel B9-2 of the connection layer a5 through the outlet C1-2 of the second inlet channel C1-1, flows into the second bubble-removing channel B9-2, then enters the inlet C1-3 of the second diversion channel C1-4 through the outlet B9-1 of the second bubble-removing channel B9-2, and then enters the second culture chamber C6 through the second diversion channel C1-4. The culture medium passes through the second culture chamber C6 and then enters the second outlet flow channel C2-1, then enters the second outlet opening B2 on the connecting layer A5 through the outlet C2 of the second outlet flow channel C2-1, and returns to the culture medium bottle through an external pipeline to form a circulating flow channel of the second culture chamber C6, so that nutrition is provided for tissue cells in the second culture chamber C6.
In addition, when the culture is performed using the first sub-culture layer A2, the external medium enters the first sub-culture layer A2 through the connecting layer A5 and the second sub-culture layer A4. In order to simplify the fluid introduction path of the medium from the connection layer a5 to the first sub-culture layer a2, thereby simplifying the chip structure, illustratively, as shown in fig. 3, the second sub-culture layer a4 is further provided with a first introduction through-hole C4 and a first discharge through-hole C3 throughout the thickness thereof. In this embodiment, the introduction port of the connection layer a5 includes a first introduction port B1 and a second introduction port B3, and the discharge port includes a first discharge port B2 and a second discharge port B4, the second introduction port B3 is connected to the inlet C1 of the second inlet flow passage C1-1, and the second discharge port B2 is connected to the outlet C2 of the second outlet flow passage C2-1. The first introduction port B4 is connected to an inlet D4 of the first inlet flow passage D4-1 through the first introduction through hole C4, and the first discharge port B3 is connected to an outlet D3 of the first outlet flow passage D3-1 through the first discharge through hole C3. Thus, the culture medium entering from the first inlet B4 of the connecting layer A5 enters the first inlet channel D4-1 through the first inlet through hole C4 and the inlet D4 of the first inlet channel D4-1, then enters the inlet E9-3 of the first bubble removing channel E9-2 of the sealing layer A1 through the outlet D4-2 of the first inlet channel D4-1, flows into the first bubble removing channel E9-2, then enters the inlet D4-3 of the first diversion channel D4-4 through the outlet E9-1 of the first bubble removing channel E9-2, and enters the first culture chamber D6 through the first diversion channel D4-4. The culture medium passes through the first culture chamber D6 and then enters the first outlet flow channel D3-1, then enters the first discharge through hole C3 through the outlet D3 of the first outlet flow channel D3-1, then enters the first discharge hole B3 on the connecting layer A5, and returns to the culture medium bottle through an external pipeline to form a circulating flow channel of the first culture chamber D6, so that nutrition is provided for tissue cells in the first culture chamber D6.
It should be understood that the first sub-culture layer A2 and the second sub-culture layer A4 can also be designed with a larger number of culture chambers according to actual needs, each culture chamber can also correspond to a plurality of inlet flow channels, a plurality of outlet flow channels, and the like, and can be determined according to actual needs, and the embodiment is not particularly limited thereto.
The chip of the embodiment provides an organ culture chip capable of simulating the human body absorption process, provides a brand-new model for drug screening and disease research, and is suitable for mixed culture of various organ tissues such as skin, heart, liver, kidney, intestinal tract, spleen, pancreas and the like. The chip of the embodiment can realize the real process of simulating the transportation and absorption of medicines, nutrients and the like in vivo through blood vessels in vitro, and can replace animal experiments to be used as an important platform for medicine screening and medicine toxicity experiments.
Illustratively, as shown in FIG. 4, the first inlet flow channel D4-1 and the first outlet flow channel D3-1 may be both provided on the side of the first sub-culture layer A2 facing the sealant A1. The second inlet flow channel C1-1 and the second outlet flow channel C2-1 are both disposed on the side of the second sub-culture layer A4 facing away from the sealant A1. Of course, those skilled in the art can design other distribution positions of the inlet flow channel and the outlet flow channel according to actual needs.
Illustratively, as shown in FIGS. 1 and 4, the first sub-culture layer A2 is provided with a first hollow square culture groove penetrating therethrough in the thickness direction, the first hollow square culture groove forming the first culture chamber D6. As shown in FIGS. 1 and 3, the second sub-culture layer A4 is provided with a second hollow square culture groove penetrating therethrough in the thickness direction, and the second hollow square culture groove forms the second culture chamber C6.
Illustratively, as shown in fig. 1 and 2, the connecting layer a5 is further provided with an observation groove B7 and a culture chamber groove B6 penetrating the thickness thereof, and the position of the observation groove B7 corresponds to the position of the culture chamber. The chip also comprises an observation window cover A6, wherein the observation window cover A6 is sealed and arranged on the observation groove B7. The observation window cover A6 is openably covered on the observation groove B7, so that the observation window cover A6 can be used not only for examining and observing the organ cultured in the culture chamber by using an optical instrument, but also for applying water-insoluble paste or powder drugs and reagents such as cosmetics to the cultured good tissue, and partially taking out the cultured good tissue for destructive testing, and the rest tissue in the chip can continue to grow.
Illustratively, as shown in fig. 1 and 2, the connecting layer a5 is further provided with a first filling port B5, a second filling port B11, a first vent port B10 and a second vent port B8. As shown in FIGS. 1 and 4, the first sub-culture layer A2 is further provided with a first perfusion flow channel D5-1 and a first ventilation flow channel D10-1. As shown in FIGS. 1 and 3, the second sub-culture layer A4 is further provided with a second perfusion flow channel C11-1 and a second vent flow channel C8-2, and the second sub-culture layer A4 is further provided with a second introduction through hole C5 and a second discharge through hole C10 throughout the thickness thereof.
Specifically, as shown in fig. 2 and 3, the second perfusion port B11 is connected to the inlet C11 of the second perfusion channel C11-1, and the outlet of the second perfusion channel C11-1 is connected to the second culture chamber C6. The inlet of the second channel C8-2 is connected to the second culture chamber C6, and the outlet C8-1 of the second channel C8-2 is connected to the second vent B8.
As shown in fig. 2, 3 and 4, the first perfusion port B5 is connected to the second introduction through hole C5, the second introduction through hole C5 is connected to the inlet D5 of the first perfusion flow channel D5-1, the outlet of the first perfusion flow channel D5-1 is connected to the first culture chamber D6, the inlet of the first aeration flow channel D10-1 is connected to the first culture chamber D6, the outlet D10 of the first aeration flow channel D10-1 is connected to the second discharge through hole C10, and the second discharge through hole C10 is connected to the first aeration port B10.
The chip of the embodiment can be filled with the surface treatment reagent or the cell suspension into the culture chamber by virtue of the filling port, the filling flow channel and the ventilation flow channel, thereby further realizing the real process of in-vitro simulation of transportation and absorption of medicines, nutrition and the like in vivo through blood vessels, and replacing animal experiments as an important platform for medicine screening and medicine toxicity experiments.
Illustratively, as shown in FIG. 4, the first perfusion flow channel D5-1 and the first ventilation flow channel D10-1 are both disposed on a side of the first sub-culture layer A2 facing away from the sealing layer A1. The second perfusion flow channel C11-1 and the second vent flow channel C8-2 are both disposed on the side of the second sub-culture layer A4 facing the sealant A1.
It should be noted that, the size of the culture chamber is not limited, and for example, the culture chamber adopts a hollow rectangular groove structure, and the size can be selected to be 4 × 8 mm.
In addition, the sizes of the inlet, the inlet flow passage, the outlet flow passage and the like mentioned above are not limited. Illustratively, the diameter of the inlet can be selected to be 0.5 mm-2 mm, the width of the flow channel can be selected to be 0.1 mm-2 mm, and the depth of the flow channel can be selected to be 0.1 mm-0.5 mm. The diameter of the inlet of the chip is preferably 1mm, the width of the flow channel is 0.5mm, and the depth of the flow channel is 0.5 mm. Of course, besides, those skilled in the art may also design other sizes of the inlet and the flow channel according to actual needs, and the embodiment is not limited in this respect.
In another aspect of the present invention, a method for culturing organ tissue is provided, wherein the chip described above is adopted, and the specific structure of the chip can refer to the related descriptions above, which are not described herein again. In the case of culturing, the chip is required to be put into a culture system comprising medium 1 and medium 2, pump M1 and pump M2 as shown in FIG. 6. in FIG. 6, medium 1 is connected to the first culture chamber via pump M1, and medium 2 is connected to the second culture chamber via pump M2. The specific culture method is as follows:
using human-derived multifunctional stem cells to promote the differentiation into mature human-derived endothelial cells and human-derived myocardial cells respectively;
inoculating the chip into a culture system, and sterilizing the chip and the culture system by adopting ethylene oxide, ultraviolet rays or alcohol;
respectively injecting 70-90 ul of collagen solution into the first sub-culture chamber and the second sub-culture chamber in an aseptic culture environment, refrigerating and standing at 3-4 ℃ for 1.5-2 h, and performing cell adsorption treatment on the surface of the porous membrane in the culture chambers;
sucking out collagen in the culture, putting the collagen in a sterile oven, and drying for 1-1.5 h;
70 ul-80 ul with the concentration of 0.5x105~1x105Injecting a human endothelial cell solution per ml into the first sub-culture chamber, inverting the chip, placing the chip into a sterile incubator, and standing for 2-3 hours to ensure that endothelial cells are firmly attached to the lower surface of the porous membrane in the culture chamber;
taking out the chip from the incubator, and adding 70-80 ul of the chip with the concentration of 0.5x105~1x105Injecting the human cardiac cell solution per ml into a second culture chamber, placing the chip into an aseptic culture box and standing for 2-3 hours, so that the cardiac cells are firmly attached to the upper surface of the porous membrane in the culture chamber;
injecting 10 ml-15 ml of culture medium into each culture bottle, starting a culture system, and starting continuous perfusion culture of cells in the culture chamber;
putting the culture system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days until endothelial cells and heart cells show functional characteristics;
taking out the chip from the incubator, respectively loading sterile platinum electrodes on the chip in a sterile environment, and electrifying weak square wave current with the frequency of 0.5 Hz-1 Hz to start a perfusion culture system;
putting the system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days to stimulate the upper layer heart tissue in the culture chamber to have regular pulsation consistent with the electrifying frequency;
when the power is cut off and the current is switched on, the heart tissue can grow well and form spontaneous regular pulsation;
changing the culture medium corresponding to the second sub-culture layer to a concentration of 1 x10 in a sterile environment-6mol/L~1*10-6A mol/L culture medium of noradrenaline;
and starting a culture system, and automatically culturing the culture system and a 35-37 ℃ incubator of the chip house for 1-2 days to obtain the organ tissues.
In the culture method of the embodiment, the upper layer of the chip culture chamber can also culture a plurality of organ tissues with various organ characterization functions, such as liver, kidney tissue, intestine, spleen, pancreas, skin and the like. Can realize the real process of simulating the transportation and absorption of medicines, nutrition and the like in vitro through blood vessels in vivo, and can replace animal experiments to be used as an important platform for medicine screening and medicine toxicity experiments.
It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and substance of the invention, and these modifications and improvements are also considered to be within the scope of the invention.

Claims (10)

1. An organ chip for three-dimensional culture of organ tissues is characterized by comprising a sealing layer, a culture layer and a connecting layer which are sequentially stacked, wherein the culture layer comprises a first sub-culture layer, a separation layer and a second sub-culture layer which are sequentially arranged on the sealing layer, the first sub-culture layer is provided with a first culture chamber, a first inlet flow channel, a first flow guide flow channel and a first outlet flow channel, the second sub-culture layer is provided with a second culture chamber, a second inlet flow channel, a second flow guide flow channel and a second outlet flow channel, a first bubble removing flow channel is arranged on the sealing layer, and an inlet, an outlet and a second bubble removing flow channel are arranged on the connecting layer; wherein,
the first inlet flow channel is respectively connected with the inlet and the first bubble removing flow channel, the first flow guide flow channel is respectively connected with the first bubble removing flow channel and the first culture chamber, and the first outlet flow channel is respectively connected with the first culture chamber and the outlet;
the second inlet flow channel is respectively connected with the introducing port and the second bubble removing flow channel, the second flow guide flow channel is respectively connected with the second bubble removing flow channel and the second culture chamber, and the second outlet flow channel is respectively connected with the second culture chamber and the discharge port.
2. The chip of claim 1, wherein the first inlet flow channel and the first outlet flow channel are both disposed on a side of the first sub-culture layer facing the sealing layer; and/or the presence of a gas in the gas,
the second inlet flow channel and the second outlet flow channel are both arranged on one side, away from the sealing layer, of the second sub-culture layer.
3. The chip according to claim 1, wherein the first sub-culture layer is provided with a first hollowed-out square culture groove penetrating through the first sub-culture layer in the thickness direction, and the first hollowed-out square culture groove forms the first culture chamber; and/or the presence of a gas in the gas,
the second sub-culture layer is provided with a second hollowed square culture groove penetrating through the second sub-culture layer in the thickness direction, and the second hollowed square culture groove forms the second culture chamber.
4. The chip according to any one of claims 1 to 3, wherein the introduction port comprises a first introduction port and a second introduction port, the discharge port comprises a first discharge port and a second discharge port, and the second sub-culture layer is further provided with a first introduction through-hole and a first discharge through-hole that penetrate through the thickness thereof; wherein,
the second inlet port is connected to the second inlet flow passage, and the second outlet port is connected to the second outlet flow passage;
the first introduction port is connected to the first inlet flow path through the first introduction through hole, and the first discharge port is connected to the first outlet flow path through the first discharge through hole.
5. The chip according to any one of claims 1 to 3, wherein the connection layer is further provided with an observation groove extending through the thickness thereof, the position of the observation groove corresponding to the position of the culture chamber;
the chip further comprises an observation window cover, and the observation window cover is arranged on the observation groove in a sealing mode.
6. The chip of any one of claims 1 to 3, wherein the connection layer is further provided with a perfusion port and a vent port, the first sub-culture layer is further provided with a first perfusion channel and a first vent channel, and the second sub-culture layer is further provided with a second perfusion channel and a second vent channel; wherein,
the first perfusion flow channel is respectively connected with the perfusion port and the first culture chamber, and the first ventilation flow channel is respectively connected with the first culture chamber and the ventilation port;
the second perfusion channel is respectively connected with the perfusion opening and the second culture chamber, and the second ventilation channel is respectively connected with the second culture chamber and the ventilation opening.
7. The chip of claim 6, wherein the perfusion port comprises a first perfusion port and a second perfusion port, the vent port comprises a first vent port and a second vent port, and the second sub-culture layer is further provided with a second introduction through hole and a second discharge through hole penetrating through the thickness of the second sub-culture layer; wherein,
the second perfusion opening is connected with the second perfusion flow channel, and the second vent opening is connected with the second vent flow channel;
the first filling port is connected with the first filling flow channel through the second introducing through hole, and the first vent port is connected with the first ventilating flow channel through the second discharging through hole.
8. The chip of claim 7, wherein the first perfusion flow channel and the first ventilation flow channel are both disposed on a side of the first sub-culture layer facing away from the sealing layer; and/or the presence of a gas in the gas,
the second perfusion channel and the second ventilation channel are both arranged on one side, facing the sealing layer, of the second sub-culture layer.
9. The chip of any one of claims 1-3, wherein the separation layer is a porous film.
10. A method for culturing organ tissues, which comprises using the chip according to any one of claims 1 to 9, the method comprising:
using human-derived multifunctional stem cells to promote the differentiation into mature human-derived endothelial cells and human-derived myocardial cells respectively;
inoculating the chip into a culture system, and sterilizing the chip and the culture system by adopting ethylene oxide, ultraviolet rays or alcohol;
respectively injecting 70-90 ul of collagen solution into the first sub-culture chamber and the second sub-culture chamber in an aseptic culture environment, refrigerating and standing at 3-4 ℃ for 1.5-2 h, and performing cell adsorption treatment on the surface of the porous membrane in the culture chambers;
sucking out collagen in the culture, putting the collagen in a sterile oven, and drying for 1-1.5 h;
70 ul-80 ul with the concentration of 0.5x105~1x105Injecting a human endothelial cell solution per ml into the first sub-culture chamber, inverting the chip, placing the chip into a sterile incubator, and standing for 2-3 hours to ensure that endothelial cells are firmly attached to the lower surface of the porous membrane in the culture chamber;
taking out the chip from the incubator, and adding 70-80 ul of the chip with the concentration of 0.5x105~1x105Injecting the human cardiac cell solution per ml into a second culture chamber, placing the chip into an aseptic culture box and standing for 2-3 hours, so that the cardiac cells are firmly attached to the upper surface of the porous membrane in the culture chamber;
injecting 10 ml-15 ml of culture medium into each culture bottle, starting a culture system, and starting continuous perfusion culture of cells in the culture chamber;
putting the culture system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days until endothelial cells and heart cells show functional characteristics;
taking out the chip from the incubator, respectively loading sterile platinum electrodes on the chip in a sterile environment, and electrifying weak square wave current with the frequency of 0.5 Hz-1 Hz to start a perfusion culture system;
putting the system with the started perfusion culture and the chip into an incubator at 35-37 ℃, and automatically culturing for 3-5 days to stimulate the upper layer heart tissue in the culture chamber to have regular pulsation consistent with the electrifying frequency;
when the power is cut off and the current is switched on, the heart tissue can grow well and form spontaneous regular pulsation;
changing the culture medium corresponding to the second sub-culture layer to a concentration of 1 x10 in a sterile environment-6mol/L~1*10-6A mol/L culture medium of noradrenaline;
and starting a culture system, and automatically culturing the culture system and a 35-37 ℃ incubator of the chip house for 1-2 days to obtain the organ tissues.
CN202111460739.7A 2021-12-03 2021-12-03 Organ chip for three-dimensional culture of organ tissues and culture method of organ tissues Pending CN113862154A (en)

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CN103981096A (en) * 2014-05-27 2014-08-13 东南大学 Two-layer cell culture system organ chip and preparation method thereof
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CN103981096A (en) * 2014-05-27 2014-08-13 东南大学 Two-layer cell culture system organ chip and preparation method thereof

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