Organ-like culture patch and culture chip

Abstract

The invention belongs to the field of biomedical engineering, and discloses an organoid culture patch and a culture chip. The organ-like culture patch comprises: a polyimide film; the upper surface of the polyimide film comprises a convex area formed by the graphene film and a culture area which is not covered by the graphene film; wherein the culture area is formed by arranging a plurality of culture units in an array manner or in a communicated manner. The biological patch disclosed by the invention can realize co-culture of various cells, is good in compatibility with various experimental technologies, and has extremely high universality.

Description

Technical Field

[0001] The present invention belongs to the technical field related to biomedical engineering, and more specifically, relates to an organoid culture patch and a culture chip. Background Art

[0002] Microfluidics is a technology that controls fluids of tiny volumes to achieve various microscale physical, chemical, and biological processes. Microfluidics can integrate many reactions carried out in laboratories onto a microfluidic chip, so it is called a laboratory on a chip in many scenarios. In some specific cases, the volume of the fluid to be processed can be larger or smaller. Usually, a microfluidic chip has one or more fluid channels. Under the action of different acting mechanisms such as external pressure, density, gravity, surface tension, capillary action, and mechanical action, the fluid can be transported within the flow channels of the microfluidic chip.

[0003] Organoids are tissue analogs with a certain spatial structure and function formed by self-assembly of cells, and can simulate the key functions, structures, and biological complexities of organs. Organoid chips integrate the advantageous features of organoids and organ chips, integrate multiple functional structural units, and can provide an organ physiological microsystem, which is mainly used for research and applications such as organ development, disease modeling, drug detection, regenerative medicine, and tumor immunotherapy.

[0004] Due to the self-organization and heterogeneity of organoids, existing organoids often self-organize into spherical organoids from iPSCs cells or ESCs cells. The self-organization process uses a columnar chip or a microporous chip as a carrier, and then the differentiation of organoids until maturity is achieved through perfusion culture or static medium replacement culture. The mature organoids can be used for subsequent experimental verification, such as evaluating the anti-cancer effect by adding a specific concentration of drug combination to the culture medium of tumor organoids, or evaluating the drug toxicity by adding drugs to a liver organoid chip.

[0005] Existing chips can culture multiple organoids in a group of chambers in an array manner, but the existing structures can often only culture single-type organoids, seriously affecting the efficiency of organoid experiments and the consistency of results. Summary of the Invention

[0006] Aiming at the above defects or improvement requirements of the prior art, the present invention provides an organoid culture patch and a culture chip. The culture patch includes a polyimide film, the upper surface of which includes a raised area formed by a graphene film and a culture area not covered by the graphene film; and the culture area is arranged in an array or in a connected manner in multiple culture units. The culture patch can achieve co-culture of multiple cells, has good compatibility with various experimental techniques, and has extremely high versatility.

[0007] To achieve the object of the present invention, according to the first aspect of the present invention, there is provided an organoid culture patch, comprising: a polyimide film;

[0008] The upper surface of the polyimide film includes: a raised area formed by a graphene film, and a culture area not covered by the graphene film; wherein, the culture area is composed of a plurality of culture units arranged in an array or a connected arrangement.

[0009] Preferably, the raised area formed by the graphene film is obtained by patterning laser engraving on the polyimide film.

[0010] Preferably, the surface of the raised area is provided with a hydrophobic modification layer.

[0011] Preferably, the diameter or the diameter of the minimum circumscribed circle of the culture unit is 0.5-4 mm.

[0012] Preferably, when the culture area is composed of a plurality of culture units arranged in an array, each of the culture units is in a closed shape, and the shape is circular or polygonal.

[0013] Preferably, when the culture area is composed of a plurality of culture units arranged in an array, the sizes of the culture units are distributed in a gradient.

[0014] Preferably, when the culture area is composed of a plurality of culture units arranged in a connected arrangement, the number of the culture units is 3-5.

[0015] Preferably, when the culture area is composed of a plurality of culture units arranged in a connected arrangement, microchannels are provided between the culture units, and the width of the microchannels is 0.5-1 mm.

[0016] According to the second aspect of the present invention, there is provided an organoid culture chip, using the organoid culture patch according to the first aspect of the present invention as a culture chamber.

[0017] Preferably, the organoid culture patch is detachably assembled at the bottom of the organoid culture device.

[0018] Generally speaking, compared with the prior art by the above technical solution conceived by the present invention, the following technical advantages are mainly possessed:

[0019] 1. The organoid culture patch of the present invention is a polyimide film, whose upper surface has a raised area formed by a graphene film and a culture area not covered by the graphene film, and the culture area is arranged in an array or connected manner with multiple culture units. Based on the strong hydrophobicity and controllability of graphene, this culture patch can control the biological culture medium to form corresponding shapes on culture units of different shapes, realizing high-throughput and controllable culture of organoids; and based on the designability of the shape of the raised area, the present invention can achieve co-culture of multiple types of cells.

[0020] 2. The graphene film in the organoid culture patch of the present invention is obtained by patterning laser engraving on the polyimide film, so that the graphene film formed by laser engraving has a hierarchical surface structure, providing excellent spatial control for high-viscosity and low-viscosity matrices; at the same time, based on the precise control of the light source, the graphene formed by laser engraving has micron-level precision in terms of the size and shape of the graphene film.

[0021] 3. When the culture area of the present invention is composed of multiple culture units arranged in an array, each culture unit is in a closed shape, and through shape design, it can control the biological culture medium to form corresponding shapes on culture units of different shapes, adapting to the growth environments of different types of cells, with high versatility.

[0022] 4. When the culture area is composed of multiple culture units arranged in a connected manner, the culture area is in a closed symmetric shape, capable of forming a multi-path culture patch. The biological culture medium will be restricted in flow in the path, and through the strong hydrophobicity of the biochip, it can control multiple types of cells or organoids to stably exist in different paths, thereby realizing co-culture of two types of cells or organoids.

[0023] 5. When the culture area is composed of multiple culture units arranged in a connected manner, each point is a different type of cell or organoid, and there are extremely tiny channels at the joints. Under the restriction of strong hydrophobicity, the culture media of different types of cells or organoids are connected through specific cell types in the channels, achieving vascularized culture of cells or organoids or interactions between multiple types of organoids. Description of the Drawings

[0024] Figure 1 It is a schematic structural diagram of the organoid culture patch according to an example of the present invention;

[0025] Figure 2 It is a schematic structural diagram of the organoid culture chamber chip according to an example of the present invention.

[0026] Figure 3 It is a schematic diagram of the distribution of tumor organoids on the patch in the co-culture experiment of colorectal cancer and liver cancer organoids according to an example of the present invention.

[0027] Figure 4Testing of the co - culture of colorectal and liver cancer organoids as an example of the present invention, where a is a schematic diagram, b is a bright - field image, c is a nuclear staining image, and d is a live - dead staining image.

[0028] Reference numerals in the figure: 1 is a graphene film; 2 is a cell culture unit. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0031] As Figure 1 shown, six different organoid - culture patches are presented, namely patches with an array distribution of multiple identical circular culture units, patches with an array distribution of multiple identical rectangular culture units, patches with an array distribution of multiple identical hexagonal culture units, patches with an array distribution of multiple identical triangular culture units, and two cross - shaped patches with a connected arrangement of four culture units.

[0032] The present invention includes but is not limited to patches of the above - mentioned shapes. By designing patches with different - shaped culture regions and assembling them into culture chambers, it can be applicable to the co - culture of various cells under different experimental conditions.

[0033] In the present invention, an organoid - culture patch is provided, which includes a polyimide film, and its upper surface includes: a raised region formed by a graphene film, and a culture region not covered by the graphene film.

[0034] Among them, the graphene film is a visible structure to the naked eye, providing spatial control for high - viscosity and low - viscosity matrices.

[0035] The bottom of the culture region is still a polyimide film, which is composed of multiple culture units arranged in an array or in a connected manner. As Figure 1 shown, the graphene film 1 encloses a culture region composed of multiple culture units 2 arranged in an array or in a connected manner, where the culture unit 2 is used to carry cells or organoids to be cultured, etc.

[0036] Specifically, the method for preparing the graphene film includes: using a CO2 laser cutting machine system (UNIVERSAL-ULR30 laser cutting machine platform) to fabricate Kapton polyimide (PI) by laser engraving.

[0037] Optionally, the raised area, that is, the graphene film has a hierarchical surface structure, which helps to further perform hydrophobic modification. Its surface can be hydrophobically modified by gradually dropping hydrophobic reagents, enabling the graphene film surface to have a superhydrophobic effect.

[0038] Optionally, the relative diameter of the culture unit is 0.5 - 4 mm, which is suitable for culturing various types of organoids such as those in the stomach, intestine, liver, etc.

[0039] Optionally, when the culture area is composed of an array of multiple culture units, each culture unit is in a closed shape, and the shape includes a circle or a polygon. For example Figure 1 As shown in the arrayed circular bio-patch, due to its strong hydrophobic property and edge effect, it can control the biological culture medium to form a circle with a uniform diameter on the circular culture unit, enabling the three-dimensional and uniform growth of organoids / cells, etc.; similarly, bio-patches in polygons such as triangles, squares, and hexagons have high versatility because different-shaped culture units can adapt to different cell growth environments.

[0040] Optionally, when the culture area is composed of a connected arrangement of multiple culture units, the culture area is closed, and it can form a multi-path culture patch to control the stable existence of multiple cells or organoids in different paths, thereby realizing the co-culture of two types of cells or organoids. The number of culture units is 4, for example, preparing a cross-shaped patch as Figure 1 shown.

[0041] Optionally, when the culture area is composed of an array of multiple culture units, the spatial dimensions of the culture units are distributed in a gradient, which can be used for drug screening of different-sized organoid culture units to obtain different results.

[0042] Optionally, microchannels are provided between the culture units, and the width dimension of the microchannels is 0.5 - 1 mm, which can be used for different culture units or to achieve vascularized culture.

[0043] The present invention also provides an organoid culture chip. For example, using the above-mentioned organoid culture patch as a culture chamber for co-culturing different organoids; or using the above-mentioned organoid culture patch as a culture chamber and integrating a liquid peristaltic pump to form a microfluidic chip for high-throughput controllable culture.

[0044] Optionally, as Figure 2As shown, the culture chamber in the organoid culture chip can be directly prepared at the bottom of the chip by the above preparation method, or can be detachably assembled at the bottom of the organoid culture chip to achieve modular application and personalized applications with different culture sizes and different culture types.

[0045] Exemplarily, the organoid culture chip includes: a cell culture chamber, wherein the cell culture chamber includes a bottom plate, the above-mentioned organoid culture patch, and a lid for sealing, which are detachably assembled and connected from the bottom to the top. It can be understood that in order to enable biological cells to carry out respiration during the culture process, the device is not completely sealed after assembly.

[0046] In the present invention, an example of the preparation of an organoid culture patch includes: using a CO2 laser cutting machine system (UNIVERSAL-ULR30 laser cutting machine platform) to perform laser engraving on Kapton polyimide (PI) with a thickness of 0.005 inches. The laser engraving parameters are: the wavelength of the laser is 10.6 microns, the pulse duration is 120 milliseconds; the beam size is set to 4 mm, and the laser power is systematically increased from 12 W to 22 W; the scanning speed of the laser system is maintained at 1270 mm / s; and all laser experiments are carried out under normal temperature conditions.

[0047] In the present invention, the organoid culture patch prepared based on the above method is used as follows: the above-mentioned organoid culture patch is used as a culture chamber, and a cell suspension is coated on the surface of the cell culture unit membrane. The organoid culture patch is laid on the bottom plate of the culture chip, assembled, and covered with a lid, and then placed in a carbon dioxide incubator for culture. The culture conditions are controlled, and nerve growth factor and glial cell-derived neurotrophic factor are added for culture.

[0048] In an embodiment of the present invention, an array-type organoid controllable culture experiment based on the device of the present invention specifically includes:

[0049] An organoid culture patch containing a circular culture unit array arrangement culture area of the present invention is adopted. The size of the circular culture unit is 2 mm. Ensure that colorectal cancer organoids are digested into cell clusters with an initial size of less than 30 μm and inoculated on the organoid culture patch of the array-type circular culture unit. The uniformity of the organoid size is achieved, and a drug screening experiment is carried out on the organoids under different sizes to obtain the drug response effects of the same type of organoids under different sizes.

[0050] In an embodiment of the present invention, an experiment on co-culturing colorectal cancer and liver cancer organoids based on the device of the present invention specifically includes:

[0051] An organoid culture patch with a cross-shaped structure where the raised areas are connected. Add liver cancer organoids to the first line of the cross and colorectal cancer organoids to the second line. After solidification in a cell culture incubator, add a co-culture medium containing Y-27632, which is prepared from a colorectal cancer (CRC) organoid medium and a liver cancer organoid (LC) medium.

[0052] Culture effect: As Figure 3 and Figure 4 shown, after culturing the two types of tumor organoids on the organoid culture patch for 7 days and performing viability assays, it was confirmed that the viability of the co-cultured organoids was above 90%.

[0053] In summary, the organoid culture patch shown in the present invention has extremely high versatility and can be assembled into an organoid culture chip with different chambers for co-culturing multiple types of cells. Different from the existing chips that can only culture single-type organoids, the chip of the present invention provides an alternative solution for the co-culture of multiple types of cells and tissues in vitro, with prominent advantages of high throughput, good compatibility with various experimental techniques, simple operation, and low cost. On the chip of the present invention, endothelial cells can be induced to self-assemble in vitro to form a perfusable vascular network, and the dynamic biological process of vascular network formation and the interaction between tumor organoids and the vascular network can be observed in real time online. And the tumor organoids can be taken out at any time for immunohistochemical staining, Western blotting, transcriptome sequencing and other detections. Due to the characteristics of less reagent consumption and superposition of multifunctional units on the chip, it can meet the needs of high-throughput drug primary screening.

[0054] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the protection of the present invention and its equivalent technologies, the present invention also intends to include these changes and modifications. The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the scope of its protection is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the scope of protection of the present invention.

Claims

1. An organoid culture patch, characterized in that: include: Polyimide film; The upper surface of the polyimide film includes: a raised area formed by the graphene film, and a culture area not covered by the graphene film; wherein the culture area is composed of a plurality of culture units arranged in an array or in a connected manner.

2. The organoid culture patch according to claim 1, characterized in that: The raised area formed by the graphene film is obtained by patterning laser engraving on a polyimide film.

3. The organoid culture patch according to claim 1, characterized in that: The surface of the raised area is provided with a hydrophobic modification layer.

4. The organoid culture patch according to claim 1, characterized in that: The diameter or the minimum circumscribed circle diameter of the culture unit is 0.5 to 4 mm.

5. The organoid culture patch according to claim 1, characterized in that: When the culture area is composed of a plurality of culture units arranged in an array, each of the culture units is in a closed shape, and the shape is circular or polygonal.

6. The organoid culture patch according to claim 1, characterized in that: When the culture area is composed of a plurality of culture units arranged in an array, the sizes of the culture units are distributed in a gradient.

7. The organoid culture patch according to claim 1, characterized in that: When the culture area is composed of a plurality of culture units arranged in a connected manner, the number of the culture units is 3 to 5.

8. The organoid culture patch according to claim 1, characterized in that: When the culture area is composed of a plurality of culture units arranged in a connected manner, microchannels are arranged between the culture units, and the width of the microchannels is 0.5 to 1 mm.

9. An organoid culture chip, characterized in that: The organoid culture patch according to any one of claims 1 to 8 is used as a culture chamber.

10. The organoid culture chip according to claim 9, characterized in that: The organoid culture patch is detachably assembled at the bottom of the organoid culture device.

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