DNA origami for 3D culture, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are a DNA origami for 3D culture, a preparation method therefor, and a use thereof. The DNA origami comprises a scaffold strand and staple strands, the scaffold strand and the staple strands being hybridized according to the principle of complementary base pairing. The staple strands comprise cell staple strands, connecting staple strands, and fixing staple strands. The DNA origami has a cross-shaped structure. The present invention designs a specific DNA origami, utilizes self-assembled DNA origami to decorate living cells, and mediates cell aggregation and self-assembly to generate designed spheres with programmable three-dimensional structures, which can rapidly produce feasible and functionally normal spheres with reproducible size uniformity, and can achieve precise spatial arrangement, simulate natural microanatomical structures, and achieve combining the complexity of tissue-like structures with drug screening throughput.

Description

DNA origami for 3D culture and preparation method and application thereof

[0001] Related applications

[0002] The present application claims priority to Chinese Patent Application No. 2024108659311, filed on July 1, 2024, entitled "DNA origami for 3D culture and preparation method and application thereof". TECHNICAL FIELD

[0003] The present application belongs to the technical field of biological medicine, and relates to a DNA origami for 3D culture and a preparation method and application thereof. BACKGROUND

[0004] Standard cell culture and animal models exhibit limited translational fidelity in predicting human drug responses, highlighting the need for more advanced in vitro model systems that more accurately mimic the multicellular complexity and structural complexity of human tissues.

[0005] Current 3D culture platforms aim to reconstitute tissue-like structures under physiological conditions. One common approach is to use carefully designed biomimetic scaffolds to direct cell organization through the presentation of local biochemical and biophysical signals. For example, hydrogels with defined mechanical properties and adhesion ligand display can guide human stem cells or cancer cells to self-assemble into organoid-like structures. However, recapitulating the fine compositional heterogeneity of native tissues composed of multiple interacting cell types in a physiologically accurate spatial context remains a prominent challenge. While emerging microphysiological system technologies aim to improve physiological relevance, excessive structural complexity often hinders integration with automated, cost-effective, high-throughput drug screening pipelines that are central to preclinical drug discovery.

[0006] Traditional cell spheroid models generated by simple cell aggregation provide a viable high-throughput screening platform. However, they fail to recapitulate the compositional heterogeneity of multiple cell types and the structural complexity of native tissues, severely limiting the accuracy of modeling complex multifaceted diseases. CN116396920A discloses a primary hepatocyte co-culture three-dimensional liver microsphere model, which prepares liver parenchymal cells, liver sinus endothelial cells, Kupffer cells and hepatic stellate cells into a mixed cell suspension according to the physiological proportion of liver cells in vivo; the mixed cell suspension is inoculated into an ultra-low adsorption well plate for culture to obtain the primary hepatocyte co-culture three-dimensional liver microsphere model.

[0007] In summary, it is of great significance to develop new biological biomimetic tissue engineering strategies to strike a balance between model complexity simulating disease states and repeatable large-scale pharmacological analysis. SUMMARY

[0008] In view of the deficiencies of the prior art and actual needs, the present application provides a DNA origami for 3D culture and a preparation method and application thereof, a nano nucleic acid material based on the DNA origami, which mediates cell aggregation self-assembly into a three-dimensional structure, and balances between model complexity simulating disease states and repeatable large-scale pharmacological analysis.

[0009] To achieve the above object, the present application adopts the following technical solutions.

[0010] In a first aspect, the present application provides a DNA origami for 3D culture, which comprises a scaffold chain and staple chains, and the scaffold chain and the staple chains are hybridized by base complementary pairing principle; the staple chains comprise a cell staple chain, a connecting staple chain and a fixing staple chain; the nucleic acid sequence of the cell staple chain comprises the sequence shown in SEQ ID NO. 1-8, and the 5' end of the cell staple chain is modified with cholesterol (chol); the nucleic acid sequence of the connecting staple chain comprises the sequence shown in SEQ ID NO. 9-16, or the sequence shown in SEQ ID NO. 17-24; the nucleic acid sequence of the fixing staple chain comprises the sequence shown in SEQ ID NO. 25-242; the DNA origami has a cross-shaped structure, and the scaffold chain is M13mp18 single-stranded DNA.

[0011] The present application designs a DNA origami with a specific structure (referred to as NAC-Linker in the present application), which folds and assembles the scaffold chain into a cross-shaped structure by the staple chains, in addition, the cell staple chain and the connecting staple chain have special extension sequences, the cell staple chain in the staple chain has an extension sequence and the sequence end is modified with cholesterol, which can extend from one side of the DNA origami and combine with cells, and the connecting staple chain in the staple chain is divided into two types, and the two types of chains are complementary through the extension sequences thereon, and the DNA origami has one of the two types, which extends from the other side, and the overall structure is shown in FIG. 1, so as to correspond to the two types of DNA origami, the two types of DNA origami are connected through the complementary connecting staple chains, and thus, if the two types of DNA origami are modified on the surface of cells respectively, the cells can be aggregated and self-assembled into a three-dimensional structure through the connection between the DNA origamis.

[0012] The M13mp18 single-stranded DNA refers to single-stranded circular viral DNA from M13mp18. The M13mp18 single-stranded DNA is a product that has been commercialized, and its sequence has been disclosed. The M13mp18 single-stranded DNA commonly used in the art for DNA origami technology is suitable for the present application, and the length can be 6000-8000 bp. In an embodiment of the present application, the length of the M13mp18 single-stranded DNA is 7249 bp.

[0013] Preferably, the cross-sectional height of the DNA origami is 1-3 nm.

[0014] In a second aspect, the present application provides a method for preparing the DNA origami of the first aspect, the method comprising:

[0015] Mixing and incubating the scaffold strand and staple strands to obtain the DNA origami.

[0016] Preferably, the method for preparing the DNA origami comprises:

[0017] Mixing the scaffold strand, the cell staple strand, the linking staple strand, and the fixing staple strand with a buffer and incubating.

[0018] Preferably, the buffer contains Tris, EDTA, and magnesium acetate.

[0019] Preferably, the concentration of the scaffold strand is 10-30 nM, including but not limited to 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28, or 29 nM, and the like.

[0020] Preferably, the concentration ratio of the scaffold strand, the cell staple strand, the linking staple strand, and the fixing staple strand is 1:(3-6):(3-6):(3-6), including but not limited to 1:4:5:4, 1:5:5:5, 1:5:4:4, 1:5:4:4, or 1:4:5:5, and the like.

[0021] Preferably, the procedure of the incubation comprises:

[0022] Incubating at 93-96℃ for 3-6 min, and then decreasing by 0.1-0.2℃ every 10 seconds to 18-21℃.

[0023] In a third aspect, the present application provides the use of the DNA origami of the first aspect in constructing an organoid model.

[0024] In a fourth aspect, the present application provides a method for constructing an organoid model, the method comprising:

[0025] Incubating the DNA origami of the first aspect with cells to modify the cells, mixing the modified cells, and culturing to obtain the organoid model.

[0026] The present application proposes a novel method of using self-assembling DNA origami to decorate living cells, referred to as NAC-Linker, to generate designed spheroids with programmable three-dimensional structures, capable of rapidly producing viable and functionally normal spheroids with reproducible size uniformity. It is crucial that, due to the programmability and highly specific sequence-based interactions of DNA origami, precise spatial arrangements can be achieved, which allow the introduction of tissue-like complexity, such as controlled positioning of multiple cell types, to mimic natural microanatomy. The present application specifically demonstrates the generation of heterotypic spheroids containing parenchymal hepatocytes and non-parenchymal stromal cells, with detailed organ function, in addition, replicating the complex pathological features of chronic diseases, successfully reproducing the characteristics of non-alcoholic fatty liver disease with different stages of development from steatosis to inflammation and fibrosis, in order to improve physiological relevance, we integrated immune cells into the spheroids to establish a tumor microenvironment with immune interactions. The method of the present application is expected to reproducibly generate spheroid models with clear structures with precise spatial control; this advanced in vitro system combining tissue-like complexity with drug screening throughput has great potential in transforming preclinical evaluation and personalized medicine.

[0027] Preferably, the cells comprise hepatocytes.

[0028] In a fifth aspect, the present application provides an organoid model, which is constructed by the method for constructing an organoid model according to the fourth aspect.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The present application designs specific DNA origami, uses self-assembling DNA origami to decorate living cells, mediates cell aggregation self-assembly, generates designed spheroids with programmable three-dimensional structures, can rapidly produce viable and functionally normal spheroids with reproducible size uniformity, and can achieve precise spatial arrangement, simulate natural microanatomy, and realize the combination of tissue-like complexity and drug screening throughput. BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 is a schematic diagram of a DNA origami structure;

[0032] FIG. 2 is an atomic force microscope (AFM) image and cross-sectional height analysis result diagram of the DNA origami, with a scale of 50 nm;

[0033] FIG. 3 is a DNA agarose gel electrophoresis characterization result diagram of the DNA origami, from left to right, DNA Marker, M13mp18 DNA and DNA origami (NAC-Linker);

[0034] Figure 4 is a schematic diagram of the organoid construction process;

[0035] Figure 5A is a graph showing the results of flow cytometry analysis of cells incubated with 20 nM AF488-labeled NAC-Linker at room temperature for different time periods;

[0036] Figure 5B is a graph showing the results of flow cytometry analysis of cells incubated with different concentrations of AF488-labeled NAC-Linker for 30 min at room temperature;

[0037] Figure 6 is a representative confocal microscopy image of cells modified with or without AF488-labeled NAC-Linker, scale bar 10 pm;

[0038] Figure 7 is a graph showing the biocompatibility of NAC-Linker, where panel a shows the results of a proliferation test of cells after incubation with different concentrations of NAC-Linker for 96 h, the data is the mean ± standard deviation of 3 independent experiments, panel b shows the viability of cells after incubation with different concentrations of NAC-Linker, the data is the mean ± standard deviation of 5 independent experiments, and panel c shows the results of flow cytometry analysis of cells stained with propidium iodide (PI) after incubation with different concentrations of NAC-Linker for 96 h, the data is from 3 independent experiments;

[0039] Figure 8 is a representative fluorescence image of the cell culture process for organoid construction, scale bar 200 pm;

[0040] Figure 9 is a graph showing the results of a 12 h cell culture using paired NAC-Linker (A and B), NAC-Linker A alone, NAC-Linker B alone, and no NAC-Linker modification, scale bar 200 pm;

[0041] Figure 10 is a representative image of organoids with different initial cell numbers cultured for 1 day or 7 days, scale bar 200 pm;

[0042] Figure 11 is a phase contrast image of organoids generated from different initial cell numbers of the same batch cultured for 7 days, scale bar 500 pm;

[0043] Figure 12 is a size distribution plot of organoids generated from different initial cell numbers of the same batch cultured for 7 days;

[0044] Figure 13A is a heatmap showing the differences in expression of DMEs and transporters between freshly isolated mouse liver tissue (fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4), and 3D NAC-livers constructed from primary mouse hepatocytes at day 1 and day 4 (3D day 1, 3D day 4), genes are ordered from high to low according to their tissue expression levels, and genes with very low expression are excluded;

[0045] Figure 13B is a principal component analysis (PCA) showing changes in DMEs and transporters in freshly isolated mouse liver tissue (Fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4) and 3D NAC-livers at day 1 and day 4 (3D day 1, 3D day 4), grey arrows indicate temporal changes in transcriptomic markers of 2D primary mouse hepatocytes, red arrows indicate changes in NAC-livers;

[0046] Figure 14 is a graph of the results of transcriptomic profiling of liver functions, wherein a) is a heatmap showing expression of liver function genes in freshly isolated mouse liver tissue (Fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4) and 3D NAC-livers constructed from primary mouse hepatocytes at day 1 and day 4 (3D day 1, 3D day 4); b) is a principal component analysis of liver function expression, grey arrows indicate temporal changes in transcriptomic markers of 2D primary mouse hepatocytes, red arrows indicate changes in NAC-livers, DME, drug metabolizing enzymes. DETAILED DESCRIPTION

[0047] To further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with the examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0048] If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art, or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0049] Example 1

[0050] In this example, DNA origami (referred to as NAC-Linker) for 3D culture was prepared, and whole assembly based on DNA origami and in vitro characterization were carried out.

[0051] 1. Dilute DNA origami short chains and mix chains

[0052] (1) Prepare 10×TAE solution, take 5 mL 10×TAE solution and dilute into 45 mL pure water to form 1×TAE solution;

[0053] 10×TAE formula: Tris 48.456 g, EDTA 7.44 g, magnesium acetate 26.81 g.

[0054] (2) Centrifuge the DNA short chain dry powder tube at 8000 rpm for 5 min to make the dry powder aggregate for easy dissolution.

[0055] (3) According to the instructions, add an appropriate amount of 1xTAE solution to the staple chain dry powder tube, and shake and centrifuge to store in a 4°C refrigerator. For long-term storage, place in a -20°C refrigerator.

[0056] (4) Classify and mix the staple chains, a total of 4 1.5 mL centrifuge tubes, including: cell staple chains (SEQ ID NO. 1-SEQ ID NO. 8); connecting staple chain A (SEQ ID NO. 9-SEQ ID NO. 16), connecting staple chain B (SEQ ID NO. 17-SEQ ID NO. 24), which are complementary chains; and the remaining staple chains (fixed staple chains, SEQ ID NO. 25-SEQ ID NO. 242). The first three types of chains each contain 8 DNA short chains, and the remaining staple chains include 218 DNA short chains.

[0057] System determination: when the number of staple chains is less than 100, the system is 800 μL, and the concentration of each chain is 1 μM;

[0058] When the number of staple chains is 100-200, the system is 1600 μL, and the concentration of each chain is 500 nM;

[0059] When the number of staple chains is more than 200, the system is 1600 μL, and the concentration of each chain is 400 nM.

[0060] Staple chain volume calculation example: in a total system of 800 μL, the concentration of each staple chain is 1 μM, and the required staple chain solution volume is calculated as follows: 800 μL x 1 μM ÷ 100 μM = 8 μL, i.e. the required volume of each short chain is 8 μL.

[0061] Functional regions (cell staple chains, connecting staple chain A, connecting staple chain B, 800 μL system, diluted to 1 μM):

[0062] Cell staple chains: 800 μL = 8 μL x 12 + 704 μL (1xTAE)

[0063] Connecting staple chain A or connecting staple chain B: 800 μL = 8 μL x 12 + 704 μL (1xTAE)

[0064] Non-functional regions (remaining staple chains, 1600 μL system, diluted to 500 nM):

[0065] Remaining staple chains: 1600 μL = 8 μL x 184 + 128 μL (1xTAE);

[0066] After mixing the tubes, label them and store them at 4°C.

[0067] (5)PCR instrument settings, the main program is 95℃, 5min, then every 10 seconds 0.1℃, and finally to 20℃.

[0068] Take PCR tube, 100 μL system per tube, M13mp18 single-stranded DNA (7249bp, BaYou, product number p-107) concentration is 20nM, the ratio of M13mp18 single-stranded DNA to other strands is 1:5, respectively prepare two kinds of DNA origami (NAC-LinkerA and NAC-LinkerB).

[0069] ①NAC-Linker A origami system:

[0070] ②NAC-Linker B origami system:

[0071] After mixing, shake the PCR tube and separate it, then put it into the PCR instrument to start the program (put the PCR tube in the middle as much as possible).

[0072] (6) After the program is completed, the origami needs to be purified, and the origami is mixed into a 1.5 mL centrifuge tube, a 100KD ultrafiltration tube is taken, and the ultra-pure water is washed and dried. Put it into the matching receiving tube, add 200 μL of NAC-Linker A or NAC-Linker B triangular origami to each ultrafiltration tube, then add 1×TAE to the boundary line above the ultrafiltration tube, 4℃, 5000×g centrifugation 3min, take out, discard the liquid in the receiving tube. Add 1×TAE to the boundary line again, 4℃, 5000×g centrifugation 3min, repeat the operation 3 times, after the last centrifugation, take out the ultrafiltration tube, take out the new receiving tube, cut off the bottle cap, place the ultrafiltration tube upside down in the receiving tube, 4℃, 2000×g centrifugation 5min, collect the liquid at the bottom of the receiving tube and store at 4℃. The used ultrafiltration tube is stored in a 50mL centrifuge tube containing ultrapure water.

[0073] (7) Concentration measurement and calculation, take 1 μL of the purified origami and dilute it 10 times with 1×TAE, take the absorbance detection plate, wipe it with ethanol, add 2 μL of the diluted origami to each well, repeat twice, and add 1×TAE as a blank well to zero. Enzyme marker selects microplate-nucleic acid-other-layout-run, record the OD value at 260nm.

[0074] Take the average of the two repeated OD values recorded, and calculate the concentration (nmol) according to the formula: OD value × dilution factor × 12.6.

[0075] Recovery rate: concentration x volume (folded paper volume after purification) / (M13mp18 single-stranded DNA concentration (20 nmol / mL) x PCR system volume x number of PCR tubes).

[0076] Take NAC-Linker A folded paper as an example for analysis, the DNA folded paper atomic force microscope (AFM) image and cross-section height analysis results are shown in FIG. 2, the results show that the expected cross structure is successfully synthesized; the DNA folded paper agarose gel electrophoresis characterization results are shown in FIG. 3, from left to right are DNA Marker, M13mp18 DNA and DNA folded paper, the results show that the synthesis process has very high efficiency, and the obtained DNA folded paper band is clear.

[0077] Example 2

[0078] Based on NAC-Linker promoting cell aggregation into organoids, using DNA folded paper nanostructure to assemble up to millions of human primary cells into microspheres, this technology can program the interaction between cells, generating a unified organoid with complex function and scalable scale, the flowchart is shown in FIG. 4, after incubating cells with cholesterol-modified DNA folded paper (NAC-Linker A or NAC-Linker B) respectively, then mixed, inducing chemical remodeling of cell surface. Cells carrying complementary cell surface NAC-Linker combine to form temporary artificial adhesion, leading to rapid aggregation and eventually forming organoids.

[0079] Before starting the cell surface modification, cells were collected and washed with PBS. To optimize the incubation conditions, flow cytometry (FlowSight, Germany) was used to evaluate the cell surface fluorescence intensity after incubation with different concentrations of AF488-labeled NAC-Linker and different incubation times, as shown in FIG. 5A and FIG. 5B, flow cytometry analysis of cells after incubation with 20 nanomolar AF488-labeled NAC-Linker for different times at room temperature (20-30°C), flow cytometry analysis of cells after incubation with different concentrations of AF488-labeled NAC-Linker for 30 min at room temperature. According to the results obtained from this optimization process, 1x10^5 hepatocytes were resuspended in complete medium containing 20 nanomolar NAC-Linker A, and 1x10^5 immune cells were resuspended in complete medium containing 20 nanomolar NAC-Linker B, and incubated at 25°C for 30 min to achieve effective membrane modification. After the incubation period, unbound NAC-Linker was effectively removed by centrifugation at 1000 rpm for 3 min. To establish intercellular connections, cells modified with paired NAC-Linkers (NAC-Linker A and NAC-Linker B) (1:1) were mixed and incubated at 25°C for 30 min. Cells modified only with NAC-Linker A, cells modified only with NAC-Linker B, and unmodified cells were mixed as controls.

[0080] In addition, organoids were prepared using different numbers of initial cells for analysis.

[0081] Confocal microscopy results showed that NAC-Linker was stably anchored on the membrane (FIG. 6). NAC-Linker biocompatibility was detected, and the results are shown in FIG. 7, where a is a proliferation test of cells after incubation with different concentrations of NAC-Linker for 96 h, the result data are the average values ± standard deviations of 3 independent experiments, b is the survival rate of cells after incubation with different concentrations of NAC-Linker, the data are the average values ± standard deviations of 5 independent experiments, and c is flow cytometry analysis of cells stained with propidium iodide (PI) after incubation with different concentrations of NAC-Linker for 96 h, the data are derived from 3 independent experiments. The results show that the NAC-Linker membrane modification of the application exhibits excellent biocompatibility, and almost no negative effects on cell activity are observed even at a high concentration of 50 nanomolar NAC-Linker.

[0082] To better demonstrate the advantage of NAC-Linker in facilitating cell aggregation, the hanging drop method was used to establish organoid culture without the use of ultra-low adherent cell culture plates. First, 2 mL of PBS was added to each well of a 6-well plate to keep it moist. Then, the mixed cells incubated with NAC-Linker were supplemented with complete medium, with a volume of 20 μL for each drop, the plate cover was turned over, 20 μL of cell suspension was added to each drop position on the plate cover, the distance between two drops was kept more than 0.5 cm, finally, the plate cover was turned over, and the cells were aggregated by gravity to form organoids at the center of each drop. After the formation of organoids, 50% of the medium was replaced with complete medium every day.

[0083] Based on the NAC-Linker facilitated cell aggregation into microspheres, as shown in Figure 8, liver cells (cell A) and immune cells (cell B) formed representative fluorescence images of assembled organoids cultured for 24 h (A cells: Calcein-AM, green; B cells: CMTPX, red). In contrast, unmodified cells and cells modified with only one NAC-Linker aggregate showed rough edges and holes (Figure 9). Representative images of organoids of different initial cell numbers (50, 100, 250, 500, 1 x 10^3, 2.5 x 10^3, 5 x 10^3, and 1 x 10^4) cultured for 1 day or 7 days are shown in Figure 10, and the significant increase in microsphere diameter reflects the high proliferation capacity of cells within the microspheres. Phase contrast images of organoids generated using the same batch of different initial cell numbers (10^3, 10^4, 10^5, and 10^6) cultured for 7 days are shown in Figure 11, and the diameter results are shown in Figure 12, indicating that the method of the present application can produce highly uniform microspheres on a large scale, up to millions of cells (1,000-1,000,000).

[0084] Example 3

[0085] Transcriptomic analysis of drug metabolizing enzymes (DMEs) and transporter genes in liver organoids and transcriptional profiling of liver functions.

[0086] The experimental subjects include freshly isolated mouse liver tissue (fresh liver) (the specific process is that primary mouse hepatocytes (mH) and non-parenchymal cells (mNPCs) are isolated by two-step perfusion. After perfusion, the liver is added with an appropriate amount of DMEM medium, and the liver is gently torn, filtered through a 100 μm filter membrane and collected into a centrifuge tube. Then, centrifugation at 50xg for 2 min obtains liver parenchymal cells. The supernatant is transferred to another test tube, and centrifugation at 630xg for 10 min obtains non-parenchymal cells. After isolation of liver parenchymal cells and non-parenchymal cells, they are respectively resuspended in liver physiological medium. ) 2D culture of primary mouse hepatocytes is inoculated in a 50 μg / mL rat tail collagen coated 6-well plate at 40,000 cells per square centimeter. Primary mouse 3D NAC-Liver organoids (the construction method includes incubating 1x10^4 liver parenchymal cells and non-parenchymal cells with 20 nM NAC-Linker A or NAC-Linker B, incubating at 25°C for 5 min, mixing all the cells, and adding an appropriate amount of liver physiological medium to each well of a 96-well plate.

[0087] The results of transcriptome analysis of drug metabolizing enzymes (DMEs) and transporter protein genes in liver organoids are shown in FIG. 13A and FIG. 13B, wherein FIG. 13A is a heat map showing the expression differences of DMEs and transporter genes between freshly isolated mouse liver tissue (fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4), and 3D NAC-liver constructed from primary mouse hepatocytes at day 1 and day 4 (3D day 1, 3D day 4), and the genes are sorted according to their tissue expression levels from high to low, excluding genes with very low expression; FIG. 13B is a principal component analysis (PCA) showing the changes of DMEs and transporters in freshly isolated mouse liver tissue (fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4), and 3D NAC-liver at day 1 and day 4 (3D day 1, 3D day 4), and the gray arrow indicates the time change of the marker of the 2D primary mouse hepatocyte transcriptome, and the red arrow indicates the change of the NAC-liver. The above results show that compared with freshly isolated liver tissue, NAC-Liver organoids can effectively maintain the expression levels of genes of phase I / II DMEs and phase III transporters.

[0088] The results of the transcriptional profiling of liver function are shown in Figure 14, wherein a) is a heatmap showing the expression of liver function genes in freshly isolated mouse liver tissue (Fresh liver), 2D primary mouse hepatocytes at day 1 and day 4 (2D day 1, 2D day 4) and 3D NAC-livers constructed from primary mouse hepatocytes at day 1 and day 4 (3D day 1, 3D day 4); b) is a principal component analysis of liver function expression, with grey arrows indicating the temporal changes in the transcriptome of 2D primary mouse hepatocytes and red arrows indicating the changes in NAC-livers, DME, drug metabolizing enzymes. The results show that NAC-livers can effectively maintain the expression levels of liver function genes compared to freshly isolated liver tissue.

[0089] In summary, the present application designs specific DNA origami, uses self-assembly DNA origami to decorate living cells, mediates cell aggregation self-assembly to generate designed spheres with programmable three-dimensional structures, can quickly generate feasible and functionally normal spheres with reproducible and uniform size, and can achieve precise spatial arrangement, simulate natural microscopic anatomical structures, and realize the combination of tissue-like complexity and drug screening throughput.

[0090] The applicant states that the present application is illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed methods, i.e. it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A DNA origami for 3D culturing, characterized in that, The DNA origami comprises a scaffold strand and staple strands, which are hybridized by base complementary pairing principle; The staple strands comprise cell staple strands, connection staple strands and fixation staple strands; The nucleic acid sequence of the cell staple strands comprises sequences shown in SEQ ID NO. 1-8, and the 5' end of the cell staple strands is modified with cholesterol; The nucleic acid sequence of the connection staple strands comprises sequences shown in SEQ ID NO. 9-16, or sequences shown in SEQ ID NO. 17-24; The nucleic acid sequence of the fixation staple strands comprises sequences shown in SEQ ID NO. 25-242; The DNA origami has a cross structure; The scaffold strand is M13mp18 single-stranded DNA.

2. The DNA origami of claim 1, wherein, The DNA origami has a cross structure; 3. The method for preparing a DNA origami according to claim 1 or 2, wherein The preparation method comprises: Mixing and incubating the scaffold strand and the staple strands to obtain the DNA origami.

4. The method of claim 3, wherein the DNA origami is prepared by, The preparation method comprises: Mixing the scaffold strand, the cell staple strands, the connection staple strands and the fixation staple strands with a buffer solution and incubating; The buffer solution contains Tris, acetic acid, EDTA and magnesium acetate; The concentration of the scaffold strand is 10-30 nM; The concentration ratio of the scaffold strand, the cell staple strands, the connection staple strands and the fixation staple strands is 1:(3-6):(3-6):(3-6).

5. The method of claim 3 or 4, wherein the DNA origami is prepared by The incubation program comprises: Incubating at 93-96℃ for 3-6 min, and then reducing by 0.1-0.2℃ every 10 seconds until 18-21℃.

6. The DNA origami of claim 1 or 2 is used in constructing an organoid model.

7. A method of constructing an organoid model, characterized by, The method comprises: Incubating the DNA origami of claim 1 or 2 with cells, modifying the cells, mixing the modified cells and culturing to obtain the organoid model.

8. The method of constructing an organoid model of claim 7, wherein, The cells comprise any one or a combination of at least two of liver cells, immune cells or cancer cells.

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