Organ-like culture patch and culture chip

Abstract

The utility model 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 utility model can realize co-culture of various cells, is good in compatibility with various experimental technologies, and has extremely high universality.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, and more specifically, relates to an organoid culture patch and culture chip. Background Technology

[0002] Microfluidics is a technology that controls tiny volumes of fluid to achieve various microscale physical, chemical, and biological processes. Microfluidics integrates many laboratory reactions onto microfluidic chips, hence the term "lab on a chip" in many contexts. The volume of fluid handled can also be larger or smaller in certain situations. Microfluidic chips typically have one or more fluid channels. Under the influence of various mechanisms such as external pressure, density, gravity, surface tension, capillary action, and mechanical action, fluids can be transported within the channels of the microfluidic chip.

[0003] Organoids are tissue analogs formed by cell self-assembly, possessing specific spatial structures and functions, capable of mimicking key organ functions, structures, and biological complexities. Organoid-on-a-chip integrates the advantages of both organoids and organ-on-a-chip, incorporating multiple functional structural units to provide organ physiological microsystems. They are primarily used in research and applications such as organ development, disease modeling, drug detection, regenerative medicine, and tumor immunotherapy.

[0004] Due to their self-organizing and heterogeneous nature, existing organoids are often developed through the self-organization of iPSCs or ESCs into spherical organoids. This self-organization process utilizes columnar or microporous microarrays as carriers. Subsequently, organoid differentiation is achieved through perfusion or static medium exchange culture until maturity. Mature organoids can then be used for downstream experimental validation, such as evaluating anticancer effects by adding specific drug combinations to the culture medium of tumor organoids, or assessing drug toxicity by adding drugs to liver organoid microarrays.

[0005] Existing chips can culture multiple organoids in a set of chambers in an array, but existing structures often can only culture a single type of organoid, which seriously affects the efficiency and consistency of organoid experiments. Utility Model Content

[0006] To address the above-mentioned deficiencies or improvement needs of existing technologies, this utility model provides an organoid culture patch and 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. The culture area is arranged in an array or in a connected manner with multiple culture units. This culture patch can realize the co-culture of multiple cells, has good compatibility with various experimental techniques, and has extremely high versatility.

[0007] To achieve the purpose of this utility model, according to the first aspect of this utility model, an organoid culture patch is provided, comprising: a polyimide film;

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

[0009] Preferably, the raised areas formed by the graphene film are obtained by patterning and laser engraving on the polyimide film.

[0010] Preferably, the surface of the raised area has a hydrophobic modification layer.

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

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

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

[0014] Preferably, when the culture region is composed of multiple culture units arranged in a connected manner, the number of culture units is 3 to 5.

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

[0016] According to a second aspect of the present invention, an organoid culture chip is provided, using the organoid culture patch described in 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] In summary, compared with the prior art, the above-described technical solution conceived by this utility model has the following main technical advantages:

[0019] 1. The organoid culture patch of this invention is a polyimide film, the upper surface of which consists of a raised region formed by a graphene film and a culture region not covered by the graphene film. The culture region comprises multiple culture units arranged in an array or in a connected manner. The raised region on the upper surface of this culture patch, formed by the graphene film, and the designability of the shape of this raised region, enable this invention to achieve co-culture of multiple cell types, high throughput, and controlled culture.

[0020] 2. When the culture area is composed of multiple culture units arranged in a connected manner, the culture area is a closed symmetrical shape, which can form a multi-path culture patch. The biological culture medium will restrict the flow in the path. Through the strong hydrophobicity of the biochip, multiple cells or organoids can be controlled to exist stably in different paths, thereby realizing the co-culture of two types of cells or organoids.

[0021] 3. When the culture area consists of multiple interconnected culture units, each point contains different types of cells or organoids. The connection points have extremely small flow channels. Under the constraint of strong hydrophobicity, different types of cells or organoid culture media are connected through specific cell types in the flow channels, achieving vascularized culture of cells or organoids or interaction of multiple organoids. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of an organoid culture patch as an example of this utility model;

[0023] Figure 2 This is a schematic diagram of the structure of an organoid culture chamber chip, which is an example of this utility model.

[0024] Figure 3 This is a schematic diagram showing the distribution of tumor organoids on a patch during a co-culture experiment of colorectal cancer and liver cancer organoids, which is an example of this utility model.

[0025] Figure 4 The following is an example of the co-culture experiment of colorectal cancer and liver cancer organoids according to this utility model, wherein a is a schematic diagram, b is a bright field diagram, c is a cell nuclear staining diagram, and d is a dead / live staining diagram.

[0026] In the figure, 1 represents a graphene film; 2 represents a cell culture unit. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this utility model clearer, the present utility model 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 merely illustrative of the present utility model and are not intended to limit the present utility model. Furthermore, the technical features involved in the various embodiments of the present utility model described below can be combined with each other as long as they do not conflict with each other.

[0028] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] like Figure 1 As shown, six different organoid culture patches are displayed: a patch with multiple identical circular culture units arranged in an array, a patch with multiple identical rectangular culture units arranged in an array, a patch with multiple identical hexagonal culture units arranged in an array, a patch with multiple identical triangular culture units arranged in an array, and two cross-shaped patches with four culture units arranged in a connected manner.

[0030] This invention includes, but is not limited to, patches of the shapes described above. By designing patches containing culture regions of different shapes and assembling them into culture chambers, it is applicable to the co-culture of various cells under different experimental conditions.

[0031] In this invention, an organoid culture patch is provided, comprising 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.

[0032] Among them, graphene films have a visible structure, providing spatial control for both high-viscosity and low-viscosity matrices.

[0033] Meanwhile, the bottom of the graphene film cultivation area is still a polyimide film, composed of multiple cultivation units arranged in an array or in a connected manner. For example... Figure 1 As shown, the graphene film 1 surrounds a culture region consisting of multiple culture units 2 arranged in an array or in a connected manner, wherein the culture units 2 are used to carry cultured cells or organoids, etc.

[0034] Specifically, the preparation method of the graphene film includes: using a CO2 laser cutting machine system (UNIVERSAL-ULR30 laser cutting machine platform) to laser-etch Kapton polyimide (PI) to obtain it.

[0035] Optionally, the raised regions, i.e., the graphene film, have a layered surface structure, which facilitates hydrophobic modification. The surface can be hydrophobically modified using hydrophobic reagents through a stepwise drop-addition process, resulting in a superhydrophobic surface for the graphene film.

[0036] Optionally, the relative diameter of the culture unit is 0.5 to 4 mm, which is suitable for the culture of various organoids, including but not limited to stomach, intestine, liver, etc.

[0037] Optionally, when the culture area is composed of an array of culture units, each culture unit is a closed shape, and the shape may be circular or polygonal. For example... Figure 1The array-shaped circular biopatches shown, due to their strong hydrophobic properties and edge effect, can control the formation of uniformly sized circles on the circular culture units of the bioculture substrate, enabling organoids / cells to grow three-dimensionally and uniformly. Similarly, triangular, square, hexagonal, and other polygonal biopatches are highly versatile because the different shapes of the culture units can adapt to the growth environments of different cell species.

[0038] Optionally, when the culture region is composed of multiple interconnected culture units, the culture region is closed, enabling the formation of multi-pathway culture patches. This allows for the stable existence of multiple cells or organoids in different pathways, thereby achieving the co-culture of two types of cells or organoids. The number of culture units is four, for example, to prepare... Figure 1 The cross-shaped patch shown.

[0039] Optionally, when the culture area is composed of multiple culture units arranged in an array, the spatial size of the culture units is gradient-distributed, which can be used for drug screening of organoid culture units of different sizes to obtain different results.

[0040] Optionally, microchannels are provided between culture units, with a width of 0.5 to 1 mm, which can be used for different culture units or to achieve vascularized culture.

[0041] This invention also provides an organoid culture chip, such as using the above-mentioned organoid culture patch as a culture chamber for co-culturing different organoids; or integrating the above-mentioned organoid culture patch as a culture chamber with a liquid peristaltic pump to form a microfluidic chip for high-throughput controllable culture.

[0042] Optionally, such as Figure 2 As shown, the culture chambers in organoid culture chips can be directly fabricated on the bottom of the chip using the above-described preparation method, or can be detachably assembled on the bottom of the organoid culture chip, enabling modular applications and personalized applications for different culture sizes and different culture types.

[0043] For example, the organoid culture chip includes a cell culture chamber, wherein the cell culture chamber comprises, from bottom to top, a detachably assembled base plate, the aforementioned organoid culture patch, and a lid for sealing. It is understood that the device is not completely sealed after assembly to allow the biological cells to respire during culture.

[0044] This invention illustrates the preparation of an organoid culture patch, comprising: laser engraving of a 0.005-inch thick Kapton polyimide (PI) sheet using a CO2 laser cutting system (UNIVERSAL-ULR30 laser cutting platform). The laser engraving parameters are as follows: laser wavelength of 10.6 micrometers, pulse duration of 120 milliseconds; beam size of 4 millimeters; laser power systematically increased from 12 watts to 22 watts; laser system scanning speed maintained at 1270 millimeters per second; and all laser experiments performed at room temperature.

[0045] The organoid culture patch prepared by the above method in this invention is used as follows: The 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 then laid on a culture chip substrate, assembled, and covered. It is then placed in a carbon dioxide incubator for culture, with controlled culture conditions, and nerve growth factor and glial cell-derived neurotrophic factor are added for further culture.

[0046] In an embodiment of this utility model, a controllable culture experiment of an array-type organoid based on the device of this utility model specifically includes:

[0047] This invention utilizes an organoid culture patch containing an array of circular culture units arranged in a 2mm diameter. This ensures that colorectal cancer organoids are digested into cell clusters with an initial size of less than 30μm, which are then seeded onto the organoid culture patch with the array of circular culture units. This achieves uniform organoid size, enabling drug screening experiments at different organoid sizes to obtain the drug response effects of the same organoid at different sizes.

[0048] In an embodiment of this invention, an experiment on co-culturing colorectal cancer and liver cancer organoids based on the device of this invention specifically includes:

[0049] Organoid culture patches with a cross-shaped structure of connected raised areas were used. Liver cancer organoids were dropped onto the first line of the cross and colorectal cancer organoids were dropped onto the second line. After solidification in a cell culture incubator, co-culture medium containing Y-27632 was added. This medium consisted of colorectal cancer (CRC) organoid culture medium and liver cancer (LC) organoid culture medium.

[0050] Cultivation effect: such as Figure 3 and Figure 4 As shown, after culturing the two types of tumor organoids on organoid culture patches for 7 days, the activity of the co-cultured organoids was confirmed to be above 90% by activity assay.

[0051] In summary, the organoid culture patch of this invention has extremely high versatility and can be assembled into organoid culture chips with different chambers for the co-culture of multiple cell types. Unlike existing chips that can only culture single organoids, this chip provides an alternative solution for the co-culture of multiple cells and tissues in vitro, with key advantages including high throughput, good compatibility with various experimental techniques, simple operation, and low cost. On this chip, endothelial cells can be induced to self-assemble in vitro to form a perfusionable vascular network, allowing real-time online observation of the dynamic biological processes of vascular network formation and the interaction between tumor organoids and the vascular network. Furthermore, tumor organoids can be removed at any time for immunohistochemical staining, protein imprinting, transcriptome sequencing, and other detections. Due to the low reagent consumption and multifunctional unit stacking on the chip, it can meet the needs of high-throughput drug screening.

[0052] Obviously, those skilled in the art can make various modifications and variations to this utility model without departing from its spirit and scope. Therefore, if these modifications and variations fall within the protection scope of this utility model and its equivalents, this utility model also intends to include these modifications and variations. The above-described embodiments are merely preferred embodiments given to fully illustrate this utility model, and their protection scope is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on this utility model are all within the protection scope of this utility model.

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 cultivation area not covered by the graphene film; wherein the cultivation area is composed of multiple cultivation units arranged in an array or in a connected manner.

2. The organoid culture patch according to claim 1, characterized in that, The surface of the raised area has a hydrophobic modification layer.

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

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

5. The organoid culture patch according to claim 1, characterized in that, When the culture region is composed of an array of culture units, the size of the culture units is distributed in a gradient.

6. The organoid culture patch according to claim 1, characterized in that, When the culture region is composed of multiple culture units arranged in a connected manner, the number of culture units is 3 to 5.

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

8. An organoid culture chip, characterized in that, The organoid culture patch according to any one of claims 1-7 is used as the culture chamber.

9. The organoid culture chip according to claim 8, characterized in that, The organoid culture patch is detachably assembled at the bottom of the organoid culture device.

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