CN219409758U

CN219409758U - Three-dimensional culture chip

Info

Publication number: CN219409758U
Application number: CN202222355812.0U
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Jiangsu Aiweide Biotechnology Co ltd
Current assignee: Jiangsu Aiweide Biotechnology Co ltd
Priority date: 2022-09-05
Filing date: 2022-09-05
Publication date: 2023-07-25
Anticipated expiration: 2032-09-05

Abstract

The utility model provides a three-dimensional culture chip, which comprises: a substrate, an intermediate layer and a cover plate which are stacked in sequence; the substrate comprises a first micro-channel and a first cavity matrix; the intermediate layer comprises a porous biological membrane embedded in the intermediate layer and a second cavity matrix which is correspondingly communicated with the first cavity matrix at intervals; the cover plate comprises a second micro-channel and a third cavity matrix corresponding to the second cavity matrix and communicated with the second micro-channel, and the first cavity matrix and the second cavity matrix exchange substances through the porous biological membrane; the first and second microchannels are for providing a fluidic environment for the first and second chamber matrices, respectively. The three-dimensional culture chip can be used for rapidly producing a large number of cell microspheres, and the culture process is automatic. The chip is divided into an upper runner layer and a lower runner layer, so that different organs can be cultured, and multicellular interaction and immune interaction research can be performed.

Description

Three-dimensional culture chip

Technical Field

The utility model belongs to the technical field of biological tissue engineering and biological medicine, and particularly relates to a three-dimensional culture chip.

Background

The organ chip is a novel biochip, and the micro artificial organ composed of living cells is integrated on the micro-fluidic chip through the tissue engineering technology and the micro-processing technology so as to realize the real-time culture and detection of the micro organ, and the micro physiological system is infinitely close to the human organ to be studied. Through the organ chip, the development of pathological changes and diseases of tissues and organs can be deeply known, the early screening and evaluation of effective medicaments are promoted, researchers are helped to explore the pathogenesis, and the clinical treatment effect is improved. Human organ chip is one of the forefront and most transformation value research fields in biomedical engineering research, and is evaluated as one of ten emerging technologies by the Darworth forum in 2016. Has wide application prospect in the fields of new medicine research and development, disease model, personalized medical treatment, aerospace medicine and the like.

The existing two-dimensional cell culture lacks three-dimensional tissue structure of organs, cell-cell and cell-matrix interaction, and micro-environment similar to cell culture of human body, and can not comprehensively analyze action mechanism and toxic influence on other organs.

The traditional three-dimensional cell culture adopts U-shaped low adsorption plates, hanging Drop and other modes to prepare cell aggregates, the required sample size is large, and the immune environment in a human body and the interaction with other organs cannot be simulated. The conventional methods are laborious and time-consuming, both for the preparation of cell aggregates and for the detection of drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the utility model provides a cell aggregate three-dimensional culture chip capable of simulating the human body absorption process, and provides a brand new model for drug screening and disease research. The utility model is suitable for mixed culture of various tissues and organs such as skin, heart, liver, kidney, intestinal tract, spleen, pancreas and the like. Specifically, the technical scheme of the utility model is as follows:

the utility model provides a three-dimensional culture chip, comprising: a substrate, an intermediate layer and a cover plate which are stacked in sequence; wherein,,

the substrate comprises a first micro-channel and a first cavity matrix communicated with the first micro-channel, wherein the bottom of the first cavity matrix is suitable for culturing one or more of cell aggregates, cell spheres and organoids;

the intermediate layer comprises a porous biological membrane embedded in the intermediate layer and a second chamber matrix which is correspondingly communicated with the first chamber matrix at intervals;

the cover plate comprises a second micro-channel and a third cavity matrix which is communicated with the second micro-channel and corresponds to the second cavity matrix; the third cavity matrix is communicated with the second cavity matrix;

the first chamber matrix and the second chamber matrix exchange substances through the porous biological membrane; the first and second microchannels are used to provide a fluidic environment for the first and second chamber matrices, respectively.

Optionally, the device further comprises a porous biological membrane limiting block, wherein the porous biological membrane limiting block is embedded on the middle layer, and the second cavity matrix is arranged on the limiting block.

Optionally, the middle layer is further provided with a porous biological membrane fixing groove, and the porous biological membrane fixing groove is used for positioning the porous biological membrane.

Optionally, the cover plate is further used for generating a flow path structure with different concentrations, wherein the second micro-channel at least comprises a first concentration inlet and a second concentration inlet.

Optionally, the second micro flow channel has a multi-stage multi-percent mixing structure to form flow paths with different concentrations, and the flow paths with different concentrations are respectively communicated with the third cavity matrix.

Optionally, the device further comprises a first sample adding hole and a second sample adding hole which are arranged on the cover plate, wherein the first sample adding hole is configured to be communicated with the first cavity matrix, and the second sample adding hole is configured to be communicated with the second cavity matrix.

Optionally, the number of the first wells and the second wells is the same as the number of rows or columns of the first chamber matrix or the second chamber, respectively.

Optionally, the device further comprises a first vent hole and a second vent hole, wherein the first vent hole and the second vent hole are arranged on the cover plate, the first vent hole is configured to be communicated with the first cavity matrix, and the second vent hole is configured to be communicated with the second cavity matrix.

Optionally, the device further comprises a fluid inlet and a fluid outlet which are arranged on the cover plate, wherein the fluid inlet is communicated with the first micro-channel of the substrate through the middle layer; the fluid outlets are connected to the first matrix of chambers through the intermediate layer.

Alternatively, the first chamber adopts a wide-mouth structure with a spherical bottom and an elliptic upper surface.

The utility model provides a three-dimensional culture chip which can rapidly produce a large amount of cell microspheres, has small required sample size and is beneficial to the treatment of precious samples. The culture process is automatic, and manual operation is greatly reduced. In addition, the chip is divided into an upper runner layer and a lower runner layer, so that different organs can be cultured, detection of an object to be detected can be performed based on the generated bionic organ, and research on multicellular interaction and immune interaction can be performed.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements, and in which the figures of the drawings are not to be taken in a limiting sense, unless otherwise indicated.

FIG. 1 is a schematic diagram of a three-dimensional culture chip according to an embodiment of the utility model;

FIG. 2 is a schematic diagram showing a structure of a substrate of a three-dimensional culture chip according to an embodiment of the utility model;

FIG. 3 is a schematic diagram showing the structure of an intermediate layer of a three-dimensional culture chip according to an embodiment of the utility model;

FIG. 4 is a schematic diagram showing the structure of a cover plate of a three-dimensional culture chip according to an embodiment of the utility model;

FIG. 5 is a schematic view in section B-B of FIG. 2;

fig. 6 is an enlarged schematic view of a top view of the first chamber at C in fig. 2.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present utility model more apparent, the following detailed description of the embodiments of the present utility model will be given with reference to the accompanying drawings. However, those of ordinary skill in the art will understand that in various embodiments of the present utility model, numerous technical details have been set forth in order to provide a better understanding of the present application. However, the technical solutions claimed in the present application can be implemented without these technical details and with various changes and modifications based on the following embodiments. The following embodiments are divided for convenience of description, and should not be construed as limiting the specific implementation of the present utility model, and the embodiments can be mutually combined and referred to without contradiction.

Example 1

The utility model provides a three-dimensional culture chip capable of simulating the human body absorption process, in particular to a three-dimensional culture chip, which comprises: a substrate, an intermediate layer and a cover plate which are stacked in sequence; wherein,,

the cover plate comprises a second micro-channel and a third cavity matrix corresponding to the second cavity matrix and communicated with the second micro-channel; the third chamber matrix is communicated with the second chamber matrix.

The first chamber matrix and the second chamber matrix exchange substances through the porous biological membrane; the first and second microchannels are for providing a fluidic environment for the first and second chamber matrices, respectively.

A large amount of three-dimensional cell tissue, such as one or more of cell aggregates, organoids, biological tissue, can be rapidly produced by a three-dimensional cell tissue culture matrix; specifically, the first chamber matrix and the second chamber matrix can exchange substances through the porous biological membrane, and the first micro flow channel and the second micro flow channel can be respectively used for providing fluid environments for the first chamber matrix and the second chamber matrix, so that the automation degree and the flux of the culture process are improved, different organs can be cultured, and multicellular interaction and immune interaction research can be carried out.

In some embodiments, the chip may further include a porous bio-membrane stopper embedded on the intermediate layer and a second matrix of chambers disposed on the stopper.

In some embodiments, the cover plate may also be used to create a flow path structure of different concentrations, wherein the fluid inlet may include at least a first concentration inlet and a second concentration inlet.

In some embodiments, the second fluidic channel has a multi-stage multi-percent mixing structure, flow paths having different concentrations may be formed, and the flow paths having different concentrations may be respectively in communication with the third matrix of chambers.

In some embodiments, the three-dimensional culture chip may further include a fluid inlet and a fluid outlet disposed on the cover plate, the fluid inlet communicating with the first fluidic channel of the substrate through the intermediate layer; the fluid outlet is connected to the first matrix of chambers through the intermediate layer.

Referring to fig. 1 to 6, the three-dimensional culture chip of the utility model mainly comprises three parts, namely a substrate A1, an intermediate layer A2 and a cover plate A3 which are stacked in sequence.

The substrate A1 provides a fluid culture environment for cell aggregates through the first micro flow channel A1-1; a first chamber matrix A1-2 is formed on the cell culture layer A1, and the first micro-channel A1-1 is communicated with the first chamber matrix A1-2 to provide cell culture nutrient substances or medicine stimulation in medicine test for the first chamber matrix A1-2.

The middle layer A2 is sandwiched between the substrate A1 and the cover plate A3, and includes a porous bio-membrane A4 embedded therein and a second cavity matrix (not shown) with the porous bio-membrane A4 being correspondingly communicated with the first cavity matrix A1-2.

The cover plate A3 comprises a second micro-channel A3-1 and a third cavity matrix A3-2 which is communicated with the second micro-channel A3-1 and corresponds to the second cavity matrix.

The first chamber matrix A1-2 and the second chamber matrix can exchange substances through the porous biological membrane A4, and the first micro-flow channel A1-1 and the second micro-flow channel A3-1 are respectively used for providing fluid environment for the first chamber matrix A1-2 and the second chamber matrix, for example, can provide cell culture nutrient substances or medicine stimulation in medicine test.

In this embodiment, the first cavity matrix A1-2, the second cavity matrix and the third cavity matrix A3-2 are mutually corresponding, and when the three layers are stacked together to form a chip, the first cavity matrix A1-2, the second cavity matrix and the third cavity matrix A3-2 jointly form a cell aggregate culture cavity matrix.

In this embodiment, the first micro flow channel A1-1 and the second micro flow channel A3-1 in the upper layer and the lower layer can respectively provide different concentrations of drug stimulation in a required nutrient substance or drug test for tissue or organ culture, and simultaneously provide fluid shear force in a bionic environment for tissue or organ culture.

The chip culture cavity is divided into an upper part and a lower part by a porous biological membrane, and the upper part is composed of a second cavity matrix and a third cavity matrix A3-2 which are overlapped and used for providing medicines with certain concentration; the lower chamber consists of a first chamber matrix A1-2 and is used for culturing one or more of cell aggregates, cell balls and organoids, and the upper chamber and the lower chamber exchange substances through a porous biological membrane A4; at the same time, the porous biological membrane can provide enough space and a certain physical support for the growth of the culture tissue or organ on the membrane.

Optionally, in this embodiment, a porous biological membrane limiting block A5 is provided on the intermediate layer A2, and the porous biological membrane limiting block A5 may partition the porous biological membrane limiting block A5 to form a second cavity matrix. Specifically, the middle layer A2 may be further provided with a porous biological membrane fixing groove A2-4, for example, the number of the porous biological membrane fixing grooves A2-4 may be 8, corresponding to 8 concentration gradients, for positioning 8 porous biological membranes A4.

In order to form different concentration gradients, at least two inlets of different concentrations may be included in the cover plate A3, for example in this embodiment a first concentration inlet A3-3 and a second concentration inlet A3-4, wherein the first concentration is for example greater than the second concentration.

In some embodiments, the second micro-fluidic channel may adopt a christmas tree structure, and form a flow path structure with different concentrations through a multi-stage multi-ratio mixing structure, for example, in this embodiment, 8 concentration gradients are generated, and the flow paths with different concentrations are respectively communicated with the third chamber matrix, so as to provide different fluid environments for culturing cell aggregates, cell balls, organoids, and the like.

Through the multistage multiple percentages of the micro-channels of the Christmas tree structure, the embodiment adopts 6-level 2 percentages, and finally 8 flow paths with different concentrations are formed, and the flow paths with different concentrations are communicated with the third cavity matrix A3-2, for example, medicines with different concentrations are provided for each row of cavities.

The upper and lower chambers are provided with special sample adding channels for surface treatment and cell perfusion of the porous biomembrane of the chambers. For example, a fluid inlet A3-5 is provided on the upper cover plate A3, and the fluid inlet is used for conveying the perfused culture medium to the first chamber matrix A1-2 through the first micro flow channel A1-1 via the hole A2-1 on the middle layer A2 and the hole A1-4 on the base plate A1.

The cover plate A3 is provided with a first sample adding hole A3-6 and a first vent hole A3-7 corresponding to the first cavity matrix A1-2, the first sample adding hole and the first vent hole respectively comprise a plurality of sample adding holes, the specific number of the sample adding holes and the first vent hole can be the same as the number of rows or columns of the first cavity matrix A1-2, for example, the first sample adding hole A3-6 in the embodiment can comprise eight first sample adding holes arranged in columns, the first vent hole A3-7 also comprises eight first vent holes arranged in columns, the number of the sample adding holes A3-6 is the same as the number of rows of the first cavity matrix A1-2, namely, the number of concentration gradients, and the first sample adding holes A3-6 are respectively communicated with a plurality of holes A2-2 on the middle layer A2 and are connected with a cavity at the lower layer of the porous biological membrane A4. Through the first well A3-6, a cell solution can be added to the first matrix of chambers A1-2. Accordingly, the first exhaust holes A3-7 are communicated with a plurality of holes A2-3 on the middle layer and are connected with a chamber on the lower layer of the porous biological membrane A4. The first loading holes A3-6 are symmetrically arranged with the first exhaust holes A3-7, and the first loading holes A3-6 and the first exhaust holes A3-7 can be interchanged according to the requirement.

The cover plate A3 is provided with a second sample adding hole A3-8 and a second vent hole A3-9 corresponding to the second chamber matrix, that is, sample adding holes corresponding to the upper chamber of the culture chamber, the second sample adding holes A3-8 and the second vent holes A3-9 respectively comprise a plurality of sample adding holes, and the specific number of the sample adding holes can be the same as the number of rows or columns of the second chamber matrix, for example, the second sample adding holes A3-8 comprise eight second sample adding holes arranged in columns, and the second vent holes A3-9 also comprise eight second vent holes arranged in columns, both of which are the same as the number of rows of the second chamber matrix, that is, the number of concentration gradients. Endothelial cell solution may be added to the second matrix of chambers through the second loading wells A3-8. The second sample addition holes A3-8 are symmetrically arranged with the second vent holes A3-9, and the second sample addition holes A3-8 and the second vent holes A3-9 can be interchanged according to the requirement.

Optionally, the device further comprises a waste liquid recovery flow path arranged on the base plate A1 and the cover plate A3. Specifically, in this embodiment, the waste liquid in the lower culture chamber, i.e., the first chamber matrix A1-2, is mixed by combining 8 branches into one by 3 stages to form 1 total loop, and flows to the hole A1-3, where the hole A1-3 is communicated with the holes A2-5 and A3-10, and the waste liquid is recovered to the waste liquid bottle through the first waste liquid outlet A3-11 formed in the cover plate A3.

In some embodiments, the chip may further include a fluid inlet (A3-5) and a fluid outlet (A3-11) disposed on the cover plate A3, the fluid inlet (A3-5) communicating with the first micro flow channel A1-1 of the substrate A1 through the intermediate layer A2, and the fluid outlet (A3-11) connecting with the first chamber matrix A1-2 through the intermediate layer A2.

The waste liquid in the upper culture chamber, i.e. the second chamber matrix and the third chamber matrix, is mixed by 8 branches through 3 stages to form 1 total loop, flows to the second waste liquid outlet A3-11 on the cover plate A3, and is recovered to the waste liquid bottle.

Optionally, the whole chip is made of high-transparency materials, so that the cell aggregate can be conveniently detected and observed by using an optical instrument. Wherein A1, A2, A3 and A5 can be processed by high transparent polymer materials such as PMMA, PC, PS, COC, die-casting or injection molding, and can also be processed by materials such as glass. A4-porous biological membrane can be a porous structure membrane made of PET, PC, PTFE and other materials.

Alternatively, the first and second micro flow channels A1-1, A3-1 in the present embodiment have a width of about 0.25mm and a depth of about 0.25mm, for example.

Alternatively, the upper surface of the substrate A1 and the lower surface of the intermediate layer A2 may be encapsulated by means of double-sided adhesive or bio-adhesive, thermal compression bonding, ultrasonic bonding, laser bonding, or the like.

Alternatively, the upper surface of the substrate A1 and the lower surface of the porous bio-membrane A4 may be encapsulated by double-sided adhesive or bio-adhesive, thermal compression bonding, ultrasonic bonding, laser bonding, or the like.

Alternatively, the upper surface of the intermediate layer A2 and the lower surface of the cover plate A3 may be encapsulated by means of double-sided adhesive or bio-adhesive, thermal compression bonding, ultrasonic bonding, laser bonding, etc.

Alternatively, the porous biological membrane lower chamber, i.e. the first chamber, adopts a wide-mouth structure with a spherical bottom and an elliptical upper surface, the spherical bottom has a diameter of about phi 1.00mm, the upper elliptical structure is shown in fig. 6, and the major axis is about 2.00mm and the minor axis is about 1.25mm. The cavity at the upper layer of the porous biological membrane is of an elliptic structure corresponding to the lower layer. A1-5 in the sectional view B-B shown in FIG. 5 is a cell aggregate culture chamber 6 cells in one branch.

Example 2

For the three-dimensional culture chip proposed in embodiment 1, this embodiment performs a specific application example:

the method for constructing the bionic organ chip adopts the three-dimensional culture chip, and comprises the following steps:

at the bottom of the first matrix of chambers of the substrate, the cell aggregates are formed by culturing, which may include, in particular, one or more of cell spheres, organoids, biological tissues.

And culturing a cell structure layer of endothelial cells and/or epithelial cells on the porous biosphere at the bottom of the second chamber matrix of the intermediate layer, wherein the endothelial cells can comprise one or more of vascular endothelial cells, lung endothelial cells and heart endothelial cells.

In some embodiments, taking culturing skin, heart, liver, kidney, intestinal tract, spleen, pancreas and other tissue organs as an example, the steps of constructing the bionic organ chip may specifically include:

1) Cells differentiated by human-derived multifunctional stem cells (IPSCs) of various tissues and organs such as skin, heart, liver, kidney, intestinal tract, spleen, pancreas and the like are prepared. Specifically, cells in human urine or blood can be collected, reprogrammed to Induce Pluripotent Stem Cells (IPSCs), and induced to differentiate into cells of various desired organ types to obtain cells required for constructing the organ.

2) Taking the assembled utility model chip and pipeline system, and sterilizing the chip and the pipeline system by using ethylene oxide, ultraviolet rays or alcohol;

3) In a sterile culture environment, the porous biological membrane is treated by adding extracellular matrix into each of the second sample adding holes A3-8 on the upper layer of the chip of FIG. 4, so that the cell growth is facilitated;

4) Sucking out extracellular matrix, placing into a sterile oven, and oven drying for 1 hr;

5) In a sterile culture environment, primary or IPSC differentiated cells corresponding to the organ to be cultured are added through each of the first wells A3-6 of the lower chip of FIG. 4. Injecting 20ul of cells with the concentration of 1x10 x 5 into a culture room under the chip, centrifuging the chip 1000 for 5 minutes, focusing the cells on the bottom of the chip and growing into balls;

6) Taking out the chip from the incubator, and respectively adding 20ul of endothelial cell solution with the concentration of 1x 10-5 into the on-chip culture chamber through the second sample adding holes A3-8 on the upper layer of the chip in FIG. 4, so that cells grow on the porous biological membrane in an adherence manner to construct a vascular endothelial structure;

7) Connecting the chip with a culture system, injecting 15ml of culture medium into each culture bottle, starting the culture system, and starting continuous automatic perfusion culture of cells in the culture chamber to obtain the bionic organ chip.

The application also provides an embodiment of a method for detecting a substance by using the bionic organ chip, which specifically includes the following steps:

adding an object to be detected with preset concentration into a second cavity matrix through a second micro-channel, wherein the object to be detected passes through a cell structure layer on the porous biomembrane and acts on a lower cell aggregate;

obtaining and analyzing the structural and functional changes of the cell aggregate under the action of the substances to be detected with different concentrations.

Example 3:

a method for substance detection using the biomimetic organ chip described above, the method comprising:

1) Taking out the cultured bionic organ chip, adding culture medium and high concentration medicine from ports A3-3 and A3-4 marked in figure 4, automatically generating concentration gradient after the medicine generates structure through concentration gradient, and acting on the lower cell aggregate after passing through the endothelial layer and the porous biological membrane.

2) The cell aggregates were analyzed for structural and functional changes at different concentrations.

3) Immune cells can be added into the upper layer flow of the chip to construct an immune environment.

The application additionally provides an embodiment of a bionic organ model, which comprises a cell aggregate matrix positioned at a bottom layer and an upper layer matrix for realizing substance interaction with the cell aggregate matrix through a porous biological membrane.

In some embodiments, the upper matrix has an endothelial structure matrix cultured on the porous biological membrane.

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the utility model and that various changes in form and details may be made therein without departing from the spirit and scope of the utility model.

Claims

1. A three-dimensional culture chip, comprising: a substrate, an intermediate layer and a cover plate which are stacked in sequence; wherein,,

2. The three-dimensional culture chip of claim 1, further comprising a porous bio-membrane stopper embedded on the intermediate layer, the second matrix of chambers being disposed on the stopper.

3. The three-dimensional culture chip of claim 2, wherein the intermediate layer is further provided with a porous bio-membrane fixing groove for positioning the porous bio-membrane.

4. The three-dimensional culture chip of claim 1, wherein the cover plate is further configured to create flow path structures of different concentrations, wherein the second fluidic channel comprises at least a first concentration inlet and a second concentration inlet.

5. The three-dimensional culture chip of claim 4, wherein the second micro flow channels have a multi-stage multi-percent mixing structure to form flow channels having different concentrations, the flow channels having different concentrations being respectively in communication with the third matrix of chambers.

6. The three-dimensional culture chip of claim 1, further comprising a first well and a second well disposed on the cover plate, the first well configured to communicate with the first matrix of chambers, the second well configured to communicate with the second matrix of chambers.

7. The three-dimensional culture chip of claim 6, wherein the number of the first wells and the second wells is the same as the number of rows or columns of the first chamber matrix or the second chamber, respectively.

8. The three-dimensional culture chip of claim 7, further comprising a first vent hole and a second vent hole disposed on the cover plate, the first vent hole configured to communicate with the first matrix of chambers, the second vent hole configured to communicate with the second matrix of chambers.

9. The three-dimensional culture chip of claim 1, further comprising a fluid inlet and a fluid outlet disposed on the cover plate, the fluid inlet communicating with the first fluidic channel of the substrate through the intermediate layer; the fluid outlets are connected to the first matrix of chambers through the intermediate layer.

10. The three-dimensional culture chip of claim 1, wherein the first chamber has a wide-mouth structure with a spherical bottom and an elliptical upper surface.