WO2023160687A1 - 一种基于微孔板的均一的单类器官模型及其制备方法 - Google Patents

一种基于微孔板的均一的单类器官模型及其制备方法 Download PDF

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WO2023160687A1
WO2023160687A1 PCT/CN2023/078352 CN2023078352W WO2023160687A1 WO 2023160687 A1 WO2023160687 A1 WO 2023160687A1 CN 2023078352 W CN2023078352 W CN 2023078352W WO 2023160687 A1 WO2023160687 A1 WO 2023160687A1
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organoids
organoid
model based
microwell plate
microwell
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French (fr)
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陈璞
金梦龙
江善青
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武汉大学
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

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  • the invention relates to the technical field of cell and tissue engineering, in particular to a uniform single organoid model based on a microwell plate and a preparation method thereof.
  • Organoids are collections of organ-specific cells that develop from stem cells or organ progenitor cells and can self-organize through cell sorting and spatially restricted lineage differentiation in a manner similar to that in humans.
  • organoids are a three-dimensional (3D) in vitro cell culture system, which can replicate the complex spatial morphology of differentiated tissues, and can express the relationship between cells and between cells and their surrounding matrix. Interactions and spatial location patterns.
  • Organoids can have similar physiological responses to differentiated tissues in the human body, and have a high similarity to human-derived tissues. Compared with the traditional two-dimensional (2D) cell culture model, organoids have substantial improvements.
  • Organoids contain a variety of cell types, breaking through the simple physical contact between cells, forming a tighter inter-cell, cell-matrix interaction, forming a functional "micro-organ", which can be better used for simulation
  • the development process and physiological and pathological state of organs and tissues have broad application prospects and commercial value in basic research and clinical diagnosis and treatment.
  • the method of culturing organoids is mainly based on Matrigel-based Matrigel drop method.
  • the general process is to disperse stem cells, cells differentiated from stem cells, and adult tissues into small cell clusters or wrap single cells in Matrigel and culture them in a special medium for a period of time to obtain organoids.
  • the technology of culturing organoids by the glue drop method is relatively mature, and the success rate of constructing organoids is over 90%, which is suitable for the cultivation of most organoids.
  • Organoids obtained by the Matrigel drop method show great heterogeneity, such as large differences in the time taken for organoid formation, large differences in final morphology, and organoid functions.
  • the purpose of the present invention is to provide a uniform single organoid model based on a microwell plate and its preparation method.
  • the organoids cultured in the present invention have a high degree of singleness, and it is almost certain that there is only one organoid per well.
  • the present invention has high repeatability and operation Strong controllability and other advantages have good advantages for ordinary laboratories and mass production.
  • a method for preparing a uniform single organoid model based on a microwell plate comprising:
  • the blend is placed in a microwell plate with a hydrophobic or superhydrophobic bottom, centrifuged to allow cells to aggregate at the bottom of the microwell plate, and then organoid medium is added for routine culture to obtain a uniform microwell plate-based single organoid model.
  • the centrifugal force is 100g-400g, and the centrifugation time is 3min-5min.
  • microplate is one of 96-well plate, 384-well plate and 1536-well plate.
  • the bottom of the microwell plate is a U-shaped bottom or a V-shaped bottom.
  • the time of the conventional culture is 1-6 days.
  • the subculturable organoids include organoids derived from adult stem cells, normal tissues, and tumor tissues.
  • organoids include liver organoids, small intestine organoids, pancreas-related organoids, gastric organoids, lung organoids, breast organoids, lung cancer organoids, colorectal cancer organoids, gastric cancer organoids, liver cancer organoids, breast cancer organoids, pancreas One of cancer organoids, esophageal cancer organoids, prostate cancer organoids, cervical cancer organoids, etc.
  • a uniform single organoid model based on a microwell plate prepared by the method is provided.
  • each microwell in the microwell plate contains only one organoid and is located in the central area of the microwell.
  • the size of a single organoid in the uniform single organoid model based on a microwell plate and the preparation method thereof is 50 ⁇ m to 500 ⁇ m.
  • the area coefficient of variation of a single organoid in the uniform single organoid model based on a microwell plate and its preparation method is less than 35%.
  • the homogeneous single organoid model based on the microwell plate of the present invention has obvious advantages compared with the widely used classic glue drop method for culturing organoids.
  • the present invention utilizes the technology means of organoid fusion and cell self-organization, by blending the low-concentration Matrigel glue into the organoid culture medium, and further making the Matrigel glue and cells sink to the center of the microwell plate by centrifugation to achieve the Cells aggregate and the aggregated cells are embedded in high-concentration Matrigel gel, thereby creating a standardized organoid model and its fabrication
  • the preparation method the relevant parameters of the preparation method have been determined, the organoid formation position, shape, and size are highly uniform, and the coefficient of variation of the organoid area between each well is less than 35%.
  • the model and production method created by the present invention have incomparable advantages, and solve the problem of significant heterogeneity in organoid culture in the past ten years.
  • the specific advantages are:
  • the preparation method of a uniform single organoid model based on a microwell plate provided by the present invention can quickly construct organoids and cultivate products that meet the experimental requirements in a short time.
  • the organoids cultured in the present invention have a high degree of singleness, and it is almost certain that there is only one organoid per well.
  • the present invention has the advantages of high repeatability and strong operation controllability, and has better advantages for ordinary laboratories and mass production.
  • the present invention can construct homogeneous adult stem cell-derived organoids, tumor organoids, and tissue-derived organoids in large quantities.
  • the present invention provides a brand-new solution for the standardization of organoids, which can greatly promote the application of organoids in basic research and clinical translational research, and is expected to accelerate the development of organoid quality standardization and marketization.
  • the organoids produced by the present invention have the advantages of less required cells and short culture time, and can be used to construct organoids from a small amount of tumor tissue such as clinical biopsy or endoscopic biopsy, and further used in drug sensitivity tests and personality tests of clinical tumor patients.
  • the development of personalized treatment plans promotes the precise treatment of tumors.
  • Figure 1 is a schematic diagram of a homogeneous single organoid culture process and a diagram of expected culture effects
  • Figure 2 is the effect diagram of the effect of different cell numbers on the growth of organoids
  • Figure 2A is the bright field images at the beginning of organoid culture and the sixth day, and the scale bars are 500 ⁇ m and 100 ⁇ m respectively
  • Figure 2B is the diameter of organoid culture on the sixth day Quantitative statistics
  • Figure 2C is the quantitative statistics of the coefficient of variation of the diameter of organoid culture on the sixth day;
  • Figure 3 is the effect diagram of the effect of different Matrigel concentrations on the growth of organoids;
  • Figure 3A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 3B is the diameter of organoid culture on the sixth day Quantitative statistics;
  • Figure 3C is the quantitative statistics of the coefficient of variation of the diameter of organoid culture on the sixth day;
  • Figure 4 is the effect diagram of the effect of different culture medium addition time points on the growth of organoids;
  • Figure 4A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 4B is the sixth day of organoid culture Quantitative statistics of the diameter of the first day;
  • Figure 4C is the quantitative statistics of the coefficient of variation of the diameter of the sixth day of organoid culture;
  • Figure 5 is a graph showing the effect of different centrifugal forces on the growth of organoids;
  • Figure 5A is the bright field images at the beginning of organoid culture and the sixth day, and the scale bars are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 5B is the diameter quantification of organoid culture on the sixth day Statistics;
  • Figure 5C is the quantitative statistics of the coefficient of variation of the diameter of the organoid culture on the sixth day;
  • Figure 6 is a graph showing the effect of different centrifugation times on the growth of organoids;
  • Figure 6A is the bright field images at the beginning of organoid culture and the sixth day, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 6B is the diameter of organoid culture on the sixth day Quantitative statistics;
  • Figure 6C is the quantitative statistics of the coefficient of variation of the diameter of organoid culture on the sixth day;
  • Figure 7 is the effect diagram of the effect of different orifice plate specifications on the growth of organoids;
  • Figure 7A is the bright field images at the beginning of organoid culture and the sixth day, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 7B is the organoid culture on the sixth day Quantitative statistics of diameter;
  • Figure 7C is the quantitative statistics of the coefficient of variation of diameter on the sixth day of organoid culture;
  • Figure 8 is the effect diagram of the effect of different plate characteristics on the growth of organoids;
  • Figure 8A is the bright field images at the beginning of organoid culture and the sixth day, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 8B is the organoid culture on the sixth day Quantitative statistics of diameter;
  • Figure 8C is the quantitative statistics of the coefficient of variation of diameter on the sixth day of organoid culture;
  • Figure 9 shows the expression of characteristic markers of mouse liver organoids cultured to day 6.
  • Figure 10 is the effect diagram of the effect of different Matrigel concentrations on the growth of organoids;
  • Figure 10A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 10B is the results in each well under different Matrigel concentrations Statistics on the number of organoids;
  • Figure 10C is the quantitative statistics of the diameter of organoids on the sixth day of culture at different Matrigel concentrations;
  • Figure 10D is the quantitative statistics of the diameter variation coefficient of organoids on the sixth day of culture at different Matrigel concentrations;
  • Figure 10E is the quantitative statistics of the diameters of organoids at different Matrigel concentrations on the sixth day Quantitative statistics of the area of the organoid culture on the sixth day;
  • Figure 10F is the quantitative statistics of the coefficient of variation of the area of the organoid culture on the sixth day of different Matrigel concentrations.
  • Figure 11 is a graph showing the effect of different centrifugal forces on the growth of organoids;
  • Figure 11A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 11B is the organoids in each well under different centrifugal forces Quantitative statistics;
  • Figure 11C is the quantitative statistics of the diameter of organoids cultured on the sixth day under different centrifugal forces;
  • Figure 11D is the quantitative statistics of the diameter variation coefficient of organoids cultured on the sixth day under different centrifugal forces;
  • Figure 11E is the sixth day of organoids cultured under different centrifugal forces Quantitative statistics of the area of the day;
  • Figure 11F is the quantitative statistics of the coefficient of variation of the area of the sixth day of organoid culture under different centrifugal forces.
  • Figure 12 is a graph showing the effect of different centrifugation times on the growth of organoids;
  • Figure 12A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 12B is the results in each well under different centrifugation times Statistics on the number of organoids;
  • Figure 12C is the quantitative statistics of the diameter of organoids cultured on the sixth day with different centrifugation times;
  • Figure 12D is the quantitative statistics of the coefficient of variation of diameters on the sixth day of organoids cultured at different centrifugation times;
  • Figure 12E is the quantitative statistics of organoids cultured at different centrifugation times Quantitative statistics of the area on the sixth day;
  • Figure 12F is the quantitative statistics of the area coefficient of variation on the sixth day of organoid culture at different centrifugation times.
  • Figure 13 is the effect diagram of the effect of different starting cell numbers on the growth of organoids;
  • Figure 13A is the bright field images at the beginning and the sixth day of organoid culture, and the scales are 500 ⁇ m and 100 ⁇ m respectively;
  • Figure 13B is different starting cell numbers per pore class
  • Figure 13C is the quantitative statistics of the diameter of organoids with different initial cell numbers on the sixth day of culture;
  • Figure 13D is the quantitative statistics of the diameter coefficient of variation of organoids with different initial cell numbers on the sixth day of culture;
  • Figure 13E is the quantitative statistics of different initial cell numbers Quantitative statistics of area on the sixth day of organoid culture under the cell number;
  • Figure 13F is the quantitative statistics of area coefficient of variation on the sixth day of organoid culture with different initial cell numbers.
  • a uniform single organoid model based on a microwell plate and a preparation method thereof are provided, the method comprising:
  • Step S1 digesting the subculturable organoids into single cells
  • the subculturable organoids include organoids derived from adult stem cells, normal tissues, and tumor tissues. Examples include liver organoids, small intestine organoids, pancreas-related organoids, gastric organoids, lung organoids, breast organoids, lung cancer organoids, colorectal cancer organoids, gastric cancer organoids, liver cancer organoids, breast cancer organoids, pancreas One of cancer organoids, esophageal cancer organoids, prostate cancer organoids, cervical cancer organoids, etc.
  • Step S2 blending the single cell with the organoid medium and 5%-10% Matrigel to obtain a blend
  • the Matrigel includes, but is not limited to, Matrigel, Cultrex, and other commercial and non-commercial Matrigels suitable for organoid culture.
  • the organoids cannot grow; if it is greater than 10%, there is an adverse effect that the organoids are easily differentiated;
  • the centrifugal force is 100g-400g, and the centrifugation time is 3min-5min.
  • centrifugal force is less than 100g, there is an adverse effect that cells cannot aggregate to form a single organoid, and if it is greater than 400g, there is an adverse effect that the cells are damaged and the organoid grows slowly and the volume is small;
  • Step S3 placing the blend in a microwell plate with a hydrophobic or superhydrophobic bottom, adding organoid medium for conventional culture, and centrifuging to make the cells aggregate at the bottom of the microwell plate to obtain a microwell-based Plate homogeneous single organoids Model.
  • the bottom of the microwell plate is hydrophobic or superhydrophobic, and the commercially available bottom is a low-adhesion or ultra-low-adhesion microplate.
  • the microporous plate is one of a 96-well plate, a 384-well plate and a 1536-well plate.
  • the bottom of the microwell plate is a U-shaped bottom or a V-shaped bottom. If the bottom of the microwell plate is flat, there will be adverse effects of organoids floating and scattered growth;
  • the time for the conventional culture is 1-6 days.
  • the initial inoculated cell quantity is 100-5000/well. If it is less than 100/well, there is an adverse effect of too small organoid volume, and if it is more than 5000/well, there is an adverse effect of too large organoid volume.
  • a uniform single organoid model based on a microwell plate prepared by the method is provided.
  • Each microwell in the microplate contains only one organoid and is in the central region.
  • the size of a single organoid in the uniform single organoid model based on a microwell plate is 50 ⁇ m to 500 ⁇ m.
  • the organoid size required for the experiment can be controlled by the initial cell number and culture time.
  • the coefficient of variation of the area of a single organoid in the uniform single organoid model based on the microwell plate is less than 35%.
  • the present invention has invented a new form of organoid culture and construction using micro-well plates (96, 384, 1536 wells) with high throughput and highly controllable uniformity.
  • the method and application are characterized in that there is an organoid in the center of the bottom of each microwell, and the size of the organoid is 50 ⁇ m to 500 ⁇ m, which is suitable for high-throughput imaging and quantitative analysis.
  • Organoids with controllable size are formed within 1-6 days.
  • This method overcomes the difficulty in allowing cells to aggregate into single cell aggregates in high-viscosity Matrigel, breaks the need for cultivating 10% or more of the Matrigel concentration necessary in the original organoid Matrigel drop method, and finds a new method Optimal cell aggregation protocols and culture substrates and materials.
  • the present invention is applicable to the construction of high-throughput uniform and controllable organoids of different cell sources or different tissue types. It is especially suitable for the construction of one organoid per unit of tumor tissue origin, normal tissue origin and adult origin.
  • the present invention provides a brand-new solution for the standardization of organoids, and finds the surface characteristics of microwell plates suitable for the formation of individual organoids. It can greatly promote the commercial application of organoids in basic research and clinical translational research, and is expected to accelerate the development of organoid quality standardization and marketization.
  • Example 1 A uniform single organoid model based on a microwell plate and its preparation method
  • the initial seeding cell number is 2000/well, and other operations are the same as in embodiment 1.
  • Add culture medium group after centrifugation prepare a mixture containing 5% Matrigel and complete HepatiCult TM organoid growth medium (mouse), mix with a certain number of cells, add to the well plate, set 4 duplicate wells for each group, each group The theoretical cell number was set at 500. First add 50 ⁇ L of the mixed solution to each well, centrifuge at 100 g for 3 minutes at 4°C to allow the cells to enter the well, let stand at 37°C for 30 minutes, then add 150 ⁇ L of complete HepatiCult TM organoid growth medium (mouse), and routinely nourish. Take pictures in bright field every two days to observe and count the number and area of organoids in each well.
  • the centrifugal force is 200g, and other steps are the same as in embodiment 1.
  • the centrifugation time is 5 min, and other steps are the same as in embodiment 1.
  • the Matrigel gel concentration is 10%, and other steps are the same as in embodiment 1.
  • the initial seeding cell number is 100/well, and other steps are the same as in embodiment 1.
  • the initial seeding cell number is 5000/well, and other steps are the same as in embodiment 1.
  • the centrifugal force is 400g, and other steps are the same as in embodiment 1.
  • Mouse hepatic progenitor organoids were inoculated in the well plate to prepare a mixture containing 2% Matrigel and complete HepatiCult TM organoid growth medium (mouse), mixed with a certain number of cells, and added to the well plate, with 4 for each group The theoretical number of cells in each group was set at 500 cells/well. First add 50 ⁇ L of the mixed solution to each well, then add 150 ⁇ L of complete HepatiCult TM organoid growth medium (mouse), centrifuge at 100g, 4°C for 3 minutes to allow the cells to enter the well, let stand at 37°C for 30 minutes, and take pictures Routine cultivation. Take pictures in bright field every two days to observe and count the number and area of organoids in each well.
  • centrifugal force is 200g, and other steps are all with embodiment 10.
  • the centrifugal force is 400g, and other steps are the same as in embodiment 10.
  • the centrifugal force is 600g, and other steps are the same as in embodiment 10.
  • the centrifugation time is 5 minutes, and other steps are the same as in embodiment 10.
  • the centrifugation time is 10 min, and other steps are the same as in embodiment 10.
  • the centrifugation time is 1 min, and other steps are the same as in embodiment 10.
  • the initial number of cells in each well is 100, and other steps are the same as in embodiment 10.
  • This comparative example 1 uses a 96-well plate with a flat bottom and good adhesion, and other steps are the same as in Example 1.
  • the Matrigel gel concentration was 50%, and other steps were the same as in Example 1.
  • Comparative Example 1 a flat-bottomed and well-adhesive microwell plate was used, and the number of growths was 39, and a single homogeneous organoid could not be obtained;
  • the centrifugation time was 1 min, which was less than the range of 3 to 5 min in the embodiment of the present invention, and the number of growths was 64, and a single homogeneous organoid could not be obtained;
  • Comparative Example 6 a 384 microwell plate with a flat bottom and good adhesion was used, and the number of growths was 36, and a single homogeneous organoid could not be obtained;
  • the concentration of Matrigel gel was 50%, which was greater than the range of 5% to 10% in the embodiment of the present invention, and the outgrowth rate was only 25%, and there was a high probability that organoids could not be obtained.
  • the organoids obtained in Examples 1 to 9 of the present invention have a high degree of singleness, and it can be determined that there is only one organoid per well.
  • the organoids obtained in Example 10-Example 17 of the present invention have a high degree of singleness, and there is only one organoid in more than 90% of the wells.
  • the organoids obtained in Comparative Example 1 to Comparative Example 9 of the present invention hardly appeared in a single form, and even failed to grow normally, and their uniformity was poor.

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Abstract

本发明公开了一种基于微孔板的均一的单类器官模型及其制备方法，所述方法包括：将可传代培养的类器官消化成单个细胞；将所述单个细胞与含有2％～10％的基质胶的类器官培养基共混，获得含有细胞-基质胶-培养基的共混物；将所述共混物置于底部为低黏附或超低黏附的微孔板中，然后离心以使细胞聚集于所述微孔板底部，再加入类器官培养基进行常规培养，获得基于微孔板的均一的单类器官模型。

Description

一种基于微孔板的均一的单类器官模型及其制备方法 技术领域
本发明涉及细胞和组织工程技术领域，特别涉及一种基于微孔板的均一的单类器官模型及其制备方法。
背景技术
类器官是器官特异性细胞的集合，这些细胞从干细胞或器官祖细胞发育而来，并能以与人体内相似的方式经细胞分序和空间限制性的系别分化而实现自我组建。简而言之，类器官是一种基于三维(Three-dimensional，3D)体外细胞培养系统，可复制出已分化组织的复杂空间形态，并能够表现出细胞与细胞之间，细胞与其周围基质之间的相互作用和空间位置形态。类器官能做到与人体内分化的组织具有相似的生理反应，与人体来源组织具有极高的相似性。与传统二维(Two-dimensional，2D)细胞培养模式相比，类器官具有实质性的改进。类器官包含多种细胞类型，突破了细胞间单纯的物理接触联系，形成了更加紧密的细胞间、细胞与基质间高度相互作用，形成具有功能的“微器官”，能更好地用于模拟器官组织的发生过程及生理病理状态，在基础研究以及临床诊疗方面具有广阔的应用前景和商业价值。
目前，类器官的培养方法以基于Matrigel的基质胶滴法为主。其大致流程是将干细胞、干细胞分化的细胞及成体组织分散成小细胞团或者是单个细胞包裹在Matrigel中用特殊的培养基培养一段时候后得到类器官。目前，胶滴法培养类器官技术相对成熟，构建类器官的成功率达90％以上，适合于大多数类器官的培养。利用基质胶滴法得到的类器官表现出极大的异质性，例如类器官形成所用的时间差别较大、在最终形态上也具备较大的差异、类器官所具备的功能也在某些方面上无法保持一致，这严重影响了类器官培养的质量可控性。此外，在基质胶中培养的过程中，单个胶滴中含有多个类器官、类器官之间的相对位置随机，并且分布在不同焦平面，难以定位观察、个体差异化明显。上述局限性导致了类器官实验中难以进行定量，限制了类器官在基础研究和临床前药物开发的应用。
因此，有必要开发一种均一的单类器官模型，以用于基础研究和临床前药物开发中。
发明内容
本发明目的是提供一种基于微孔板的均一的单类器官模型及其制备方法，本发明培养的类器官具有高度的单一性，几乎可以确定每孔仅有一个类器官。本发明重复性高且操作 可控性强等优势，对于普通实验室和批量生产均有较好的优势。
为了实现上述目的，本发明采用如下技术方案：
在本发明的第一方面，提供了一种基于微孔板的均一的单类器官模型的制备方法，所述方法包括：
将可传代培养的类器官消化成单个细胞；
将所述单个细胞与类器官培养基和体积比5％～10％的基质胶共混，获得共混物；
将所述共混物置于底部为疏水或超疏水的微孔板中，后离心以使细胞聚集于所述微孔板底部，再加入类器官培养基进行常规培养，获得基于微孔板的均一的单类器官模型。
进一步地，所述离心中，离心力为100g～400g，离心时间为3min～5min。
进一步地，所述微孔板为96孔板、384孔板和1536孔板中的一种。
进一步地，所述微孔板的底部为U形底或V形底。
进一步地，所述常规培养的时间为1～6d。
进一步地，所述可传代培养的类器官包括成体干细胞、正常组织、肿瘤组织来源的类器官。例如肝脏类器官、小肠类器官、胰腺相关类器官、胃类器官、肺类器官、乳腺类器官、肺癌类器官、结直肠癌类器官、胃癌类器官、肝癌类器官、乳腺癌类器官、胰腺癌类器官、食管癌类器官、前列腺癌类器官、宫颈癌类器官等中的一种。
在本发明的第二方面，提供了所述方法制备得到的基于微孔板的均一的单类器官模型。
进一步地，所述微孔板中每个微孔仅含有一个类器官且在微孔的中心区域。
进一步地，所述基于微孔板的均一的单类器官模型及其制备方法中单个类器官的大小为50μm～500μm。
进一步地，所述基于微孔板的均一的单类器官模型及其制备方法中单个类器官的面积变异系数小于35％。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
本发明的基于微孔板的均一的单类器官模型与已有的被广泛采用的经典的胶滴法培养类器官对比具有明显的优势，具体表现在：经典的胶滴法培养的类器官在最终形态、体积大小、形成位置上都具有显著的异质性；形成类器官并使其聚集的时间在1～2周不等；类器官培养都依赖于50％～100％浓度的基质胶如Matrigel胶，但由于所用高浓度基质胶黏度高，用离心等其他手段无法使细胞高度聚集。以上这些问题都是影响类器官标准化的障碍。而本发明利用类器官融合的和细胞自组织的技术手段，通过将低浓度的Matrigel胶融入到类器官培养液中，再进一步通过离心使Matrigel胶和细胞下沉到微孔板中心达到了使细胞聚集并且使聚集的细胞包埋在高浓度Matrigel胶中，从而创造了一种标准化的类器官模型及其制 备方法，制备方法的相关参数已被确定，类器官形成位置、形态、大小都具有高度的均一性，各个孔间的类器官面积变异系数小于35％。本发明创造的模型及制作方法对比传统的胶滴法具有无可比拟的优势，解决了十年来类器官培养具有显著异质性的难题。具有的优点具体有：
(1)本发明提供的一种基于微孔板的均一的单类器官模型的制备方法可以快速构建类器官，在短时间内培养出符合实验要求的产品。
(2)本发明培养的类器官具有高度的单一性，几乎可以确定每孔仅有一个类器官。
(3)本发明重复性高且操作可控性强等优势，对于普通实验室和批量生产均有较好的优势。
(4)本发明可以大批量构建均一性的成体干细胞来源的类器官、肿瘤类器官、组织来源类器官。
(5)本发明为类器官标准化提供了一个全新的解决方案，可极大地推动类器官在基础研究和临床转化研究中的应用，有望加速类器官质量标准化和市场化的发展。
(6)本发明产生的类器官具有所需细胞量少，培养时间短等优势，可用于临床穿刺活检或内镜活检等少量肿瘤组织构建类器官，进一步用于临床肿瘤患者药敏试验和个性化的治疗方案开发，促进肿瘤的精准治疗。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为均一性的单一类器官培养流程示意图及预期培养效果图；
图2为不同细胞数量对类器官生长的影响效果图；图2A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图2B为类器官培养第六天的直径量化统计；图2C为类器官培养第六天的直径变异系数量化统计；
图3为不同Matrigel浓度对类器官生长的影响效果图；图3A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图3B为类器官培养第六天的直径量化统计；图3C为类器官培养第六天的直径变异系数量化统计；
图4为不同培养液添加时点对类器官生长的影响效果图；图4A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图4B为类器官培养第六天的直径量化统计；图4C为类器官培养第六天的直径变异系数量化统计；
图5为不同离心力对类器官生长的影响效果图；图5A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图5B为类器官培养第六天的直径量化统计；图5C为类器官培养第六天的直径变异系数量化统计；
图6为不同离心时间对类器官生长的影响效果图；图6A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图6B为类器官培养第六天的直径量化统计；图6C为类器官培养第六天的直径变异系数量化统计；
图7为不同孔板规格对类器官生长的影响效果图；图7A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图7B为类器官培养第六天的直径量化统计；图7C为类器官培养第六天的直径变异系数量化统计；
图8为不同孔板特性对类器官生长的影响效果图；图8A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图8B为类器官培养第六天的直径量化统计；图8C为类器官培养第六天的直径变异系数量化统计；
图9为小鼠肝脏类器官培养至第6天特征性标记物表达情况。
图10为不同Matrigel浓度对类器官生长的影响效果图；图10A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图10B为不同Matrigel浓度下每个孔中类器官数量统计；图10C为不同Matrigel浓度下类器官培养第六天的直径量化统计；图10D为不同Matrigel浓度下类器官培养第六天的直径变异系数量化统计；图10E为不同Matrigel浓度下类器官培养第六天的面积量化统计；图10F为不同Matrigel浓度下类器官培养第六天的面积变异系数量化统计。
图11为不同离心力对类器官生长的影响效果图；图11A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图11B为不同离心力下每个孔中类器官数量统计；图11C为不同离心力下类器官培养第六天的直径量化统计；图11D为不同离心力下类器官培养第六天的直径变异系数量化统计；图11E为不同离心力下类器官培养第六天的面积量化统计；图11F为不同离心力下类器官培养第六天的面积变异系数量化统计。
图12为不同离心时间对类器官生长的影响效果图；图12A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图12B为不同离心时间下每个孔中类器官数量统计；图12C为不同离心时间类器官培养第六天的直径量化统计；图12D为不同离心时间类器官培养第六天的直径变异系数量化统计；图12E为不同离心时间类器官培养第六天的面积量化统计；图12F为不同离心时间类器官培养第六天的面积变异系数量化统计。
图13为不同起始细胞数量对类器官生长的影响效果图；图13A为类器官培养开始时及第六天的明场图，标尺分别为500μm、100μm；图13B为不同起始细胞数量每个孔中类器 官数量统计；图13C为不同起始细胞数量类器官培养第六天的直径量化统计；图13D为不同起始细胞数量类器官培养第六天的直径变异系数量化统计；图13E为不同起始细胞数量下类器官培养第六天的面积量化统计；图13F为不同起始细胞数量类器官培养第六天的面积变异系数量化统计。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
此外，在本申请的描述中，多个”、“若干个”的含义是两个或两个以上，除非另有明确具体的限定。
本申请的技术方案总体思路如下：
根据本发明的一种典型的实施方式，提供一种基于微孔板的均一的单类器官模型及其制备方法，所述方法包括：
步骤S1、将可传代培养的类器官消化成单个细胞；
所述可传代培养的类器官包括成体干细胞、正常组织、肿瘤组织来源的类器官。例如肝脏类器官、小肠类器官、胰腺相关类器官、胃类器官、肺类器官、乳腺类器官、肺癌类器官、结直肠癌类器官、胃癌类器官、肝癌类器官、乳腺癌类器官、胰腺癌类器官、食管癌类器官、前列腺癌类器官、宫颈癌类器官等中的一种。
步骤S2、将所述单个细胞与类器官培养基和5％～10％的基质胶共混，获得共混物；
所述基质胶胶包括但不限于Matrigel、Cultrex等适用于类器官培养的商品化及非商品化基质胶。
所述基质胶若未添加，则类器官无法生长；若大于10％有类器官极易分化的不利影响；
所述离心中，离心力为100g～400g，离心时间为3min～5min。
所述离心力若小于100g，有细胞无法聚集形成单一类器官的不利影响，若大于400g，有伤害细胞导致类器官生长缓慢，体积较小的不利影响；
步骤S3、将所述共混物置于底部为疏水或超疏水的微孔板中，再加入类器官培养基进行常规培养，后离心以使细胞聚集于所述微孔板底部，获得基于微孔板的均一的单类器官 模型。
使得所述微孔板的底部为疏水或超疏水具体可商品化的底部为低黏附或超低黏附的微孔板。
所述微孔板为96孔板、384孔板和1536孔板中的一种。
所述微孔板的底部为U形底或V形底。所述微孔板的底部若为平底有类器官漂浮、分散生长的不利影响；
所述常规培养的时间为1～6d。
所述初始接种细胞数量为100～5000个/孔。若小于100个/孔有类器官体积过小的不利影响，若大于5000个/孔有类器官体积过大不利影响。
根据本发明另一种典型实施方式，提供了所述方法制备得到的基于微孔板的均一的单类器官模型。
所述微孔板中每个微孔仅含有一个类器官且在中心区域。
所述基于微孔板的均一的单类器官模型中单个类器官的大小为50μm～500μm。得到实验所需的类器官尺寸可以通过初始接种的细胞数量及培养时间调控。
所述基于微孔板的均一的单类器官模型中单个类器官的面积变异系数小于35％。
本发明针对传统类器官培养形式和方法的局限性问题，发明了一种利用微孔板(96、384、1536孔)的高通量且均一性的高度可控的新型类器官培养形式和构建方法及应用，其特点是每个微孔底部正中央有一个类器官、该类器官的大小为50μm～500μm，适合于高通量成像和定量分析，其构建方法通过调节初始播种细胞数量能够在1～6天内形成大小可控的类器官。本方法克服了高粘度基质胶中难以让细胞聚集成为单个细胞聚集体的困难，打破了原有类器官基质胶滴法形式中所必需的10％或以上的Matrigel胶浓度的培养需求，找到了最佳的细胞聚集方案以及培养基质和材料。
本发明适用于不同细胞来源或者是不同组织类型的高通量的均一、可控的类器官的构建。尤其适合于肿瘤组织来源、正常组织来源和成体来源的每个单元一个类器官的构建。本发明为类器官标准化提供了一个全新的解决方案，找到了适合单个类器官形成的微孔板表面特性。可极大地推动类器官在基础研究和临床转化研究中的商业化应用，有望加速类器官质量标准化和市场化的发展。
下面将结合附图对本申请的一种基于微孔板的均一的单类器官模型和培养方法进行详细说明。
实施例1、一种基于微孔板的均一的单类器官模型及其制备方法
1.传代扩增：
1)吸除原培养液，用2mL/孔的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入100μL预冷的完全HepatiCultTM类器官生长培养基(小鼠)重悬类器官，混匀；
4)用预冷枪头加入100μL Matrigel，轻柔混匀。
5)用预冷枪头吸取50μL混合液加入24孔板中，传代比例为1:4。静置5min后放入37℃的培养箱中放置30min。
6)待Matrigel胶滴凝固后，每孔加入500μL完全HepatiCultTM类器官生长培养基(小鼠)，继续于37℃培养箱中常规培养。
7)培养至第三天时换液。
2.小鼠肝祖类器官消化
1)吸除原培养液，用2mL的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入1ml TrypLETM，37℃珠浴10min；
4)加入4ml Advanced DMEM/F-12终止消化，吹打混匀并计数；
3.小鼠肝祖类器官接种于孔板中
配制含有5％的Matrigel和完全HepatiCultTM类器官生长培养基(小鼠)混合液，与一定数量细胞混合，加入孔板中，每组设置4个复孔，每组的理论细胞数量设定为500个细胞/孔。每孔先加50μL的混合液，再加150μL的完全HepatiCultTM类器官生长培养基(小鼠)，100g、4℃度条件下离心3min以使细胞入孔，37度静置30min后,拍照后常规培养。每两天明场拍照观察并计数每孔类器官数量及面积。
实施例2
本发明实施例中，初始接种细胞数量为2000个/孔，其他操作同实施例1。
实施例3
本发明实施例中，
1.传代扩增(P12小鼠肝祖类器官)：
1)吸除原培养液，用2mL的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入100μL预冷的完全HepatiCultTM类器官生长培养基(小鼠)重悬类器官，混匀；
4)用预冷枪头加入100μL Matrigel，轻柔混匀。
5)用预冷枪头吸取50μL混合液加入24孔板中，传代比例为1:4。静置5min后放入37℃的培养箱中放置30min。
6)待Matrigel胶滴凝固后后每孔加入500μL完全HepatiCultTM类器官生长培养基(小鼠)，继续于37℃培养箱培养。
7)培养至第三天时换液。
2.小鼠肝祖类器官消化
1)吸除原培养液，用2mL的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入1mL TrypLETM，37℃珠浴10min；
4)加入4mL Advanced DMEM/F-12终止消化，吹打混匀并计数；
3.小鼠肝祖类器官接种于孔板中
离心后加培养基组：配制含有5％的Matrigel和完全HepatiCultTM类器官生长培养基(小鼠)混合液，与一定数量细胞混合，加入孔板中，每组设置4个复孔，每组的理论细胞数量设定为500个。每孔先加50μL的混合液，100g、4℃条件下离心3min以使细胞入孔，37℃静置30min后，再加150μL的完全HepatiCultTM类器官生长培养基(小鼠),拍照后常规培养。每两天明场拍照观察并计数每孔类器官数量及面积。
实施例4
本发明实施例中，离心力为200g，其他步骤均同实施例1。
实施例5
本发明实施例中，离心时间为5min，其他步骤均同实施例1。
实施例6
本发明实施例中，Matrigel胶浓度为10％，其他步骤均同实施例1。
实施例7
本发明实施例中，初始接种细胞数量为100个/孔，其他步骤均同实施例1。
实施例8
本发明实施例中，初始接种细胞数量为5000/孔，其他步骤均同实施例1。
实施例9
本发明实施例中，离心力为400g，其他步骤均同实施例1。
实施例10
1.传代扩增：
1)吸除原培养液，用2mL/孔的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入100μL预冷的完全HepatiCultTM类器官生长培养基(小鼠)重悬类器官，混匀；
4)用预冷枪头加入100μL Matrigel，轻柔混匀。
5)用预冷枪头吸取50μL混合液加入24孔板中，传代比例为1:4。静置5min后放入37℃的培养箱中放置30min。
6)待Matrigel胶滴凝固后，每孔加入500μL完全HepatiCultTM类器官生长培养基(小鼠)，继续于37℃培养箱中常规培养。
7)培养至第三天时换液。
2.小鼠肝祖类器官消化
1)吸除原培养液，用2mL的预冷Advanced DMEM/F-12溶解1个胶滴，吹打20次以使小鼠肝祖类器官碎片化，然后将混合液全部收集至15mL EP管中；
2)300g离心5min，尽量去除上清；
3)加入1ml TrypLETM，37℃珠浴10min；
4)加入4ml Advanced DMEM/F-12终止消化，吹打混匀并计数；
3.小鼠肝祖类器官接种于孔板中配制含有2％的Matrigel和完全HepatiCultTM类器官生长培养基(小鼠)混合液，与一定数量细胞混合，加入孔板中，每组设置4个复孔，每组的理论细胞数量设定为500个细胞/孔。每孔先加50μL的混合液，再加150μL的完全HepatiCultTM类器官生长培养基(小鼠)，100g、4℃度条件下离心3min以使细胞入孔，37度静置30min后,拍照后常规培养。每两天明场拍照观察并计数每孔类器官数量及面积。
实施例11
本发明实施例中，离心力为200g，其他步骤均同实施例10.
实施例12
本发明实施例中，离心力为400g，其他步骤均同实施例10。
实施例13
本发明实施例中，离心力为600g，其他步骤均同实施例10。
实施例14
本发明实施例中，离心时间为5min，其他步骤均同实施例10。
实施例15
本发明实施例中，离心时间为10min，其他步骤均同实施例10。
实施例16
本发明实施例中，离心时间为1min，其他步骤均同实施例10。
实施例17
本发明实施例中，每个孔中的起始细胞数量为100个，其他步骤均同实施例10。
对比例1
该对比例1使用平底、粘附性好的96孔板，其他步骤均同实施例1。
对比例2
该对比例中，离心时间为1min，其他步骤均同实施例1。
对比例3
该对比例中，不加入Matrigel胶，其他步骤均同实施例1。
对比例4
该对比例中，使用平底、超低粘附的96孔板，其他步骤均同实施例1。
对比例5
该对比例中，使用U形底、粘附性好的96孔板，其他步骤均同实施例1。
对比例6
该对比例中，使用平底、粘附性好的384孔板，其他步骤均同实施例1。
对比例7
该对比例中，使用平底、超低粘附的384孔板，其他步骤均同实施例1。
对比例8
该对比例中，使用平底、粘附性好的1536孔板，其他步骤均同实施例1。
对比例9
该对比例中，Matrigel胶浓度为50％，其他步骤均同实施例1。
实验例1
对上述实施例1-12和对比例1-8的每两天明场拍照观察并计数每孔类器官数量及面积进行统计，如表1所示，其中面积的标准差变异系数的计算方法为：变异系数C·V＝(标准偏差SD/平均值Mean)×100％；长出率是指第6天时有类器官长出的孔数与总的孔数的比值；长出个数为第6天统计；
表1


由表1的数据可知：
对比例1中，采用平底且黏附性好的微孔板，长出个数为39个，无法得到单一的均一性类器官；
对比例2中，离心时间为1min，小于本发明实施例3～5min的范围，长出个数为64个，无法得到单一的均一性类器官；
对比例3中，不加入Matrigel胶，类器官无法正常生长；
对比例4中，微孔板的底部为平底，长出个数为20个，无法得到单一的均一性类器官；
对比例5中，采用U形底且黏附性好的96微孔板，长出个数为52个，无法得到单一的均一性类器官；
对比例6中，采用平底且黏附性好的384微孔板，长出个数为36个，无法得到单一的均一性类器官；
对比例7中，微孔板的底部为平底，长出个数为41个，无法得到单一的均一性类器官；
对比例8中，采用平底且黏附性好的1536微孔板，长出个数为58个，无法得到单一的均一性类器官。
对比例9中，Matrigel胶浓度为50％，大于本发明实施例5％～10％的范围，长出率仅为25％，大概率无法得到类器官。
本发明实施例1-实施例9获得的类器官具有高度的单一性，可以确定每孔仅有一个类器官。本发明实施例10-实施例17获得的类器官具有高度的单一性，90％以上的孔中仅有一个类器官。本发明对比例1-对比例9获得的类器官几乎不以单一形式出现，甚至无法正常生长，均一性也较差。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。

Claims (21)

  1. 一种基于微孔板的均一的单类器官模型制备方法，其特征在于，所述方法包括：
    将可传代培养的类器官消化成单细胞；
    将所述单细胞与含有体积比5％～10％基质胶的类器官培养基共混，获得共混物；
    将所述共混物置于底部为低粘附或超低粘附的微孔板中，然后离心以使细胞和Matrigel胶聚集于所述微孔板底部，再加入类器官培养基进行常规培养或分化培养，获得一种基于微孔板的均一性的单类器官模型。
  2. 根据权利要求1所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述离心中，离心力为100g～400g，离心时间为3min～5min。
  3. 根据权利要求1所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述微孔板为96孔板、384孔板和1536孔板中的一种。
  4. 根据权利要求1所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述微孔板的底部为U形底或V形底。
  5. 根据权利要求1所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述得到单个类器官的常规培养的时间为1～6d。
  6. 根据权利要求1所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述可传代培养的类器官包括成体干细胞、正常组织、肿瘤组织来源的类器官。例如肝脏类器官、小肠类器官、胰腺相关类器官、胃类器官、肺类器官、乳腺类器官、肺癌类器官、结直肠癌类器官、胃癌类器官、肝癌类器官、乳腺癌类器官、胰腺癌类器官、食管癌类器官、前列腺癌类器官、宫颈癌类器官等中的一种。
  7. 一种采用权利要求1-6任一项所述方法制备得到的基于微孔板的均一的单类器官模型。
  8. 根据权利要求7所述的基于微孔板的均一的单类器官模型，其特征在于，所述微孔板中每个微孔仅含有一个类器官且在微孔的中心区域。
  9. 根据权利要求7所述的基于微孔板的均一的单类器官模型，其特征在于，所述基于微孔板的均一的单类器官模型中单个类器官的大小为50μm～500μm。
  10. 根据权利要求7所述的基于微孔板的均一的单类器官模型，其特征在于，所述基于微孔板的均一的单类器官模型中各孔间类器官面积变异系数小于35％。
  11. 一种基于微孔板的均一的单类器官模型制备方法，其特征在于，所述方法包括：
    将可传代培养的类器官消化成单细胞；
    将所述单细胞与含有体积比2％～5％基质胶的类器官培养基共混，获得共混物；
    将所述共混物置于底部为低粘附或超低粘附的微孔板中，然后离心以使细胞和Matrigel 胶聚集于所述微孔板底部，再加入类器官培养基进行常规培养或分化培养，获得一种基于微孔板的均一性的单类器官模型。
  12. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述离心中，离心力为100g～600g，离心时间为1min～10min。
  13. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述微孔板为96孔板、384孔板和1536孔板中的一种。
  14. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述微孔板的底部为U形底或V形底。
  15. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述得到单个类器官的常规培养的时间为1～6d。
  16. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述可传代培养的类器官包括成体干细胞、正常组织、肿瘤组织来源的类器官。例如肝脏类器官、小肠类器官、胰腺相关类器官、胃类器官、肺类器官、乳腺类器官、肺癌类器官、结直肠癌类器官、胃癌类器官、肝癌类器官、乳腺癌类器官、胰腺癌类器官、食管癌类器官、前列腺癌类器官、宫颈癌类器官等中的一种。
  17. 根据权利要求11所述基于微孔板的均一的单类器官模型制备方法，其特征在于，所述得到单个类器官所需的每个孔中的起始细胞数量为100～500个。
  18. 一种采用权利要求11-17任一项所述方法制备得到的基于微孔板的均一的单类器官模型。
  19. 根据权利要求18所述的基于微孔板的均一的单类器官模型，其特征在于，所述微孔板中每个微孔仅含有一个类器官且在微孔的中心区域。
  20. 根据权利要求18所述的基于微孔板的均一的单类器官模型，其特征在于，所述基于微孔板的均一的单类器官模型中单个类器官的大小为50μm～500μm。
  21. 根据权利要求18所述的基于微孔板的均一的单类器官模型，其特征在于，所述基于微孔板的均一的单类器官模型中各孔间类器官面积变异系数小于55％。
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