Thymus organoid culture kit and application thereof

Abstract

The invention provides a thymus organ culture kit and application thereof. Specifically, the invention provides a culture reagent / kit for organoid culture, and the culture reagent / kit comprises a culture medium selected from the following groups: (A) an R-B27 culture medium which is an RPMI 1640 culture medium supplemented with B-27, ascorbic acid and Primocin; and / or (B) an organ-like (ATO) complete culture medium, wherein the ATO complete culture medium is an RPMI 1640 culture medium in which B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L are supplemented. The organoid culture reagent / kit provided by the invention has the advantages of high success rate of organoid culture, short time required by primary cell passage and capability of reducing the time for constructing thymus organoids.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a thymus organoid culture kit and applications thereof. Background Art

[0002] The thymus is a very fragile organ. Environmental damage, aging, genetic variation, viral infection, radiation and anticancer drug treatment can cause irreversible damage to the function of the thymus, which is manifested as a decrease in thymic cells, increased interstitial fibrosis and a decrease in the production of initial T cells. Thymic dysfunction can lead to impaired immune surveillance and induce autoimmune deficiency diseases and malignant tumors. In an organism infected with pathogens, the thymus will atrophy, the CD4+CD8+ double-positive cells in the thymus will undergo apoptosis, and the output of initial T lymphocytes will be reduced. Therefore, studying the mechanism of thymic aging and developing effective means to promote thymic regeneration are of great significance for delaying aging and improving and enhancing the health level of the elderly population.

[0003] Organoids are cell clusters formed by three-dimensional culture of cells with stem cell potential in vitro. They have the ability to self-renew and self-assemble, and exhibit structures and functions similar to the source tissues. In addition, they can not only be cultured for a long time, but also have stable phenotypes and genetic characteristics. Researchers can use organoids to simulate human development and diseases because organoids are grown from induced pluripotent stem cells generated from human stem cells or adult cells, their composition and structure are also similar to primary tissues, and they are easy to manipulate and cryopreserved.

[0004] This means that organoids can be used to study human tissues derived from stem cells and to analyze human diseases that are difficult to simulate with animal models, and researchers only need a small amount of starting material to culture organoids. Secondly, advances in stem cell bioengineering technology have improved the ability to control cell types, organization, and interactions, and organoid engineering requires manipulating each structural layer by directly modifying stem cells or controlling the microenvironment.

[0005] Now, scientists have developed more precise synthetic environments that can better control the activity of stem cells by modifying biologically inert areas of the matrix with signaling proteins. Organoid engineering technology is particularly useful for developmental studies where the composition of the in vivo environment is complex and requires precise modeling. Organoids have great therapeutic potential. Using biopsy technology, organoid models with genetic similarities to patients can be cultivated. It also means that organoid systems derived from patient stem cells can be used to conduct personalized drug efficacy tests, providing patients with more precise treatments.

[0006] Existing thymic organoid culture methods are based on the construction of cells from cell lines, or use thymus tissue from animal models for in vitro culture. The whole process is relatively complicated. More than a dozen cytokines and regulatory factors need to be added to the culture medium used, all of which need to be dissolved and prepared when used. On the one hand, it is easy to cause contamination and preparation errors. On the other hand, with the development of cell biology technology and the discovery of new cell growth regulatory signal pathways, the original culture medium formula needs to be continuously improved. In addition, the general culture medium has a low success rate (less than 70%), a long culture time (6 to 8 weeks), and cumbersome operation steps.

[0007] Therefore, there is an urgent need to develop a reagent / kit for serum-free culture of thymic organoids that is easy to operate and has simple components. Summary of the invention

[0008] The present invention provides a reagent / kit for culturing thymic organoids in a serum-free manner with simple operation and simple components.

[0009] In a first aspect of the present invention, a culture reagent / kit for organoid culture is provided, comprising a culture medium selected from the group consisting of:

[0010] (A) R-B27 medium, which is RPMI1640 medium supplemented with B-27, ascorbic acid and Primocin;

[0011] (B) Organoid (ATO) complete medium: The ATO complete medium is RPMI 1640 medium supplemented with B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L;

[0012] or a combination thereof.

[0013] In another preferred embodiment, the culture reagent / kit for organoid culture is a culture reagent / kit for thymic organoid culture.

[0014] In another preferred embodiment, the basal culture medium of the culture medium (A) and (B) is RPMI 1640 culture medium.

[0015] In another preferred embodiment, the R-B27 medium can be prepared by adding B-27, ascorbic acid and Primocin to RPMI 1640 medium.

[0016] In another preferred embodiment, the ATO complete medium can be prepared by adding RhIL-7 and RhFLT3L to the R-B27 medium.

[0017] In another preferred embodiment, the RhIL-7 concentration is 2-10 ng / ml.

[0018] In another preferred embodiment, the RhFLT3L concentration is 2-10 ng / ml.

[0019] In another preferred embodiment, the RhIL-7 concentration is 5 ng / ml.

[0020] In another preferred embodiment, the RhFLT3L concentration is 5 ng / ml.

[0021] In another preferred embodiment, the organoid is a thymic organoid.

[0022] In a second aspect of the present invention, a method for culturing an in vitro organoid is provided, comprising the steps of:

[0023] (S1) culturing trophoblast cells and hematopoietic stem cells in two dimensions respectively; and

[0024] (S2) performing three-dimensional culture on the cells after the two-dimensional culture to form in vitro organoids;

[0025] Wherein, in step (S1), two-dimensional culture is performed using R-B27 medium, wherein the R-B27 medium is RPMI 1640 medium supplemented with B-27, ascorbic acid and Primocin; and / or

[0026] In step (S2), three-dimensional culture is performed using ATO complete medium, wherein the ATO complete medium is RPMI 1640 medium supplemented with B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L.

[0027] In another preferred embodiment, in step (S1), the culture is carried out using R-B27 medium during the entire period of two-dimensional culture.

[0028] In another preferred embodiment, in step (S2), culture is carried out with R-B27 medium in the early stage of three-dimensional culture (such as 1-3 days, preferably 1-2 days), and culture is carried out with ATO complete medium in the middle and late stages of three-dimensional culture (i.e. the rest of the time after the early stage).

[0029] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0030] In another preferred embodiment, the hematopoietic stem cells are extracted from body fluids.

[0031] In another preferred embodiment, the body fluid is selected from the following group: umbilical cord blood, peripheral blood, bone marrow, or a combination thereof.

[0032] In another preferred embodiment, the hematopoietic stem cells are hematopoietic stem cells that can cause blood malignancies.

[0033] In another preferred embodiment, the hematopoietic stem cells are tumor cells.

[0034] In another preferred embodiment, the method for extracting tumor cells from body fluids comprises the steps of:

[0035] Trasnswells are used to enrich large tumor cells or cell clusters.

[0036] In another preferred embodiment, the two-dimensional culture conditions include hypoxia.

[0037] In another preferred embodiment, the oxygen content during hypoxia is 2-6%.

[0038] In a third aspect of the present invention, an organoid cultured by the in vitro organ culture method according to the second aspect of the present invention is provided.

[0039] In another preferred embodiment, the organoid is a thymic organoid.

[0040] In a fourth aspect of the present invention, there is provided a use of the organoid as described in the third aspect of the present invention as a drug evaluation model, characterized in that it is used to prepare an anti-tumor drug evaluation model or evaluation reagent.

[0041] In another preferred embodiment, the organoid is a thymic organoid.

[0042] In a fifth aspect of the present invention, a method for evaluating an anti-tumor drug is provided, comprising the steps of:

[0043] (N1) providing an organoid prepared by the method according to the second aspect of the present invention; and

[0044] (N2) Using the organoids to evaluate anti-tumor drug sensitivity.

[0045] In another preferred embodiment, the organoid is a thymic organoid.

[0046] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0048] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a reagent combination and a kit that are particularly suitable for culturing thymic organoids. The organoid culture reagents of the present invention include (A) R-B27 medium, which is an RPMI 1640 medium supplemented with B-27, ascorbic acid and Primocin; and / or (B) organoid (ATO) complete medium: the ATO complete medium is an RPMI 1640 medium supplemented with B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L.

[0049] The thymic organoid culture kit provided by the present invention can improve the success rate of organoid culture (>90%), shorten the time required for primary cell passage, and reduce the time for thymic organoid construction, which is conducive to the next step of research work. On this basis, the present invention was completed.

[0050] the term

[0051] As used herein, the terms "organoid tissue", "organoid" and "ATO" are used interchangeably to refer to an organoid prepared by in vitro culture using the method of the present invention.

[0052] As used herein, the term "two-dimensional culture" refers to immersing stem cells in a culture medium and allowing them to grow along a two-dimensional plane on the surface of a culture bottle made of ordinary glass or plastic.

[0053] As used herein, the term "three-dimensional culture" refers to culturing stem cells together with a three-dimensional structured scaffold material to form a three-dimensional cell and carrier complex, allowing it to grow, proliferate and migrate in a three-dimensional space.

[0054] Thymic organoids

[0055] Thymic organoids reproduce the microstructure of the thymic stroma to a certain extent, making the in vitro differentiation of T cells closer to the in vivo differentiation process. Thymic organoids can not only be cultured for a long time, but also their phenotype and genetic characteristics are more stable than those of two-dimensional culture products, and have outstanding advantages in simulating the occurrence process and physiological and pathological states of organ tissues.

[0056] Thymus organoid culture kit of the present invention

[0057] The present invention provides a thymic organoid culture kit, comprising three independently packaged components: an organoid culture medium A, an organoid culture medium B, and a sample preservation solution:

[0058] (Z1) the organoid culture medium A is RPMI 1640 culture medium containing 100 μg / mL Primocin and 1% Gluta Max 100X solution;

[0059] (Z2) the organoid culture medium B is RPMI 1640 culture medium containing growth factors such as recombinant human IL-7 RhIL-7 and recombinant human FLT3 ligand RhFLT3L, 4% B-27, and ascorbic acid; and

[0060] (Z3) The thymic organoid culture kit further comprises a sample preservation solution, wherein the sample preservation solution is 0.5% BSA containing 100 μg / mL Primocin and 2 mM EDTA.

[0061] In another preferred embodiment, the thymic organoid refers to a special cell tissue structure similar to the normal or tumor tissue structure and genetic characteristics in vivo formed by hematopoietic stem cells from normal people or patients with blood malignancies after being cultured in vitro.

[0062] In another preferred embodiment, the organoid culture medium A is sterile, 500 ml independently packaged, stored at 4° C., and has a shelf life of about 90 days or longer.

[0063] In another preferred embodiment, the organoid culture medium B is sterile, 50 ml independently packaged, stored at 4° C., and has a shelf life of about 30 days or longer.

[0064] In another preferred embodiment, the sample preservation solution is sterile, 500 ml is individually packaged, stored at 4° C., and has a shelf life of about 90 days or longer.

[0065] In another preferred embodiment, the thymic organoid culture kit is mainly used in the culture of thymic organoids.

[0066] B27 is a multi-component supplement suitable for the culture of neuronal and embryonic stem cells.

[0067] R-B27 medium can be stored at 4°C for approximately 2 weeks. Using medium older than 2 weeks may adversely affect T cell differentiation. Ascorbic acid powder is first dissolved in PBS to make a 30mM (1000×) stock solution. This stock solution can be stored at 4°C for up to 2 days, but it is best to use freshly made ascorbic acid solution each time you make R-B27 medium.

[0068] R-B27 medium and ATO complete medium have some components in common, so ATO complete medium can be prepared by adding a predetermined amount of RhIL-7 (such as 5ng / ml) and RhFLT3L (such as 5ng / ml) to R-B27 medium. The inventors unexpectedly discovered that after adding FMS-related tyrosine kinase 3 (FLT3) ligand and interleukin (IL)-7 to R-B27 medium, a particularly advantageous ATO complete medium can be formed. When cultured in the ATO complete medium, T cells in thymic organoids will not mutate, thereby making each batch of thymic organoids high in quality and stable.

[0069] Lyophilized stem cell factor was dissolved in sterile PBS / 0.1% BSA to 50 μg / mL and stored at -80°C in 20-50 μL aliquots.

[0070] Once thawed, samples can be stored at 4°C for up to 10 days. It is recommended that cytokines be added to the required amount of R-B27 each time ATO complete medium is prepared, but it is not recommended to store cytokine-supplemented medium for future use.

[0071] The main advantages of the present invention include:

[0072] (a) Effectively reduce the complexity of organoid culture: The original more than ten reagents and dozens of preparation processes were simplified into three independently packaged prepared test kits, which greatly simplified the operation process. At the same time, it reduced the contamination and confusion that may occur in the original preparation process, making the organoid culture process simpler.

[0073] (b) The stability of cell culture is effectively improved, and the obtained thymic organoids can be cultured, passaged or cryopreserved for a long time.

[0074] (c) The difficulty of manual operation is reduced, and the operator can complete the organoid culture process by following the operating steps of the kit.

[0075] (d) Thymic organoids can be cultured on a large scale, providing an ideal in vitro cell model for constructing a thymic biobank, studying the mechanisms of T cell maturation and differentiation, and developing thymic drugs.

[0076] (e) The thymic organoid culture kit provided by the present invention can improve the success rate of organoid culture (>90%), shorten the time required for primary cell passaging (3 to 4 weeks), and reduce the time for thymic organoid construction (about 4 weeks), which is conducive to the next step of research work.

[0077] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0078] Example 1

[0079] Organoid culture medium preparation method

[0080] 1. Preparation of sample preservation solution: Add the prepared 20mM EDTA to a final concentration of 10%. Sterilize the prepared solution by filtration using 0.22 micron and 0.45 micron filter membranes. Store at 4°C after preparation and use within 90 days.

[0081] 2. Preparation of organoid culture medium A: Preparation of R-B27 culture medium; suitable for the organoid culture described in the present invention, store at 4°C after preparation and use within 90 days.

[0082] 3. Preparation of organoid culture medium B: Add fresh RhIL-7 (5 ng / ml) and RhFLT3L (5 ng / ml) to R-B27 culture medium to prepare complete ATO culture medium.

[0083] Lyophilized stem cell factor was dissolved in sterile 1×PBS / 0.1% BSA to 50 μg / mL and stored at -80°C in 20-50 μL aliquots.

[0084] Once thawed, samples can be stored at 4°C for up to 10 days. It is therefore recommended to add cytokines to the required R-B27 each time before preparing ATO complete medium, but it is not recommended to store cytokine-supplemented medium for future use.

[0085] 4. Recombinant human IL-7: Dissolve in phosphate buffered saline (PBS buffer) to 100 μg / ml, dispense into 2 ml centrifuge tubes, and store at -20°C.

[0086] 5. Recombinant human FLT3 ligand RhFLT3L: Dissolve in phosphate buffered saline (PBS buffer) to 100 μg / ml, dispense into 2 ml centrifuge tubes, and store at -20°C.

[0087] 6. Serum-free additive B-27: Aliquot into 2 ml centrifuge tubes and store at -20 degrees.

[0088] 7. Packaging of components: The kit for culturing thymic organoids consists of the following independently packaged components: 1 bottle of sample preservation solution (500 ml), 1 bottle of organoid culture medium A (500 ml), and 1 bottle of organoid culture medium B (50 ml).

[0089] Example 2

[0090] Culture of thymic organoids

[0091] (1) Prepare a single-cell suspension from CB, BM, MPB, or PB by diluting the sample 4× with sterile PBS. Isolate mononuclear cells (MNCs) by Ficoll-Paque gradient centrifugation (this step is not required for thymus samples).

[0092] PB and CB CD8 + T cells were isolated from the above monocyte fractions. + T cell isolation kit (Miltenyi Biotec) for CD8 + SP T cells were enriched by magnetic beads to separate CD8 + T cells. In some experiments, thymic T cells were further purified by flow cytometry to deplete CD4 ISP or DP precursors, and PB T cells were further purified by flow cytometry to isolate naive T cells (CD45RO-CCR7+).

[0093] (2) Enrichment of CD34 in MNCs using CD34 MicroBead Kit UltraPure and magnetic-assisted cell sorting (MACS) (CD34 MicroBead Kit UltraPure, Miltenyi) + cell.

[0094] Enrichment of CD34 from different sources + Cells can be cryopreserved after magnetic separation in FBS / 10% DMSA and stored in liquid nitrogen. + CD3 - Freezing of the entire MNC fraction prior to CD34 enrichment by MACS is not recommended.

[0095] (3) When cells are needed, they can be thawed and resuspended in MACS buffer (PBS / 0.5% BSA / 2mM EDTA), stained with anti-CD34 and anti-CD3 antibodies and DAPI to elute CD34. + The CD34+ CD3 - Cells. If lentivirally transduced CD34 cells are to be used in ATO, lentiviral transduction is performed immediately after the FACS purification step. HSPCs are typically activated for 12-18 h and then transduced for 24 h before use in ATO. Harvest transduced cells, wash at least twice to remove free viral particles, and resuspend in R-B27 complete medium.

[0096] (4) Resuspend the purified cells in R-B27 complete medium to 1-2×10 5 cells / mL, and store the suspension on ice until ATO needs to be cultured.

[0097] Time: The time required depends on the type and amount of sample, but typically it takes about 2 hours to complete the Ficoll and MACS steps. FACS purification of CD34 cells typically takes 2 hours (including antibody staining and cell sorting). Thus, preparations enriched for hematopoietic stem and progenitor cells (HSPCs) can be completed in 4 hours (or only 2 hours if CD34 cells were previously MACS-enriched and frozen).

[0098] Example 3

[0099] Culture and expansion of MS5-hDLL1 cells

[0100] (1) To generate MS5-hdll1 cells, MS5 cells should be transduced with enhanced green fluorescent protein (eGFP) mediated by a lentiviral vector encoding the human DLL1 gene. The top 5% of OP9-DL1 cells expressing GFP were sorted by flow cytometry (FACS) and passaged in DMEM medium (DMEM; Cellgro) supplemented with 10% FCS (Gemini). After several weeks of culture, stable GFP expression was detected by flow cytometry, and DLL1 gene expression was detected by qRT-PCR and DNA sequencing. The cells were then allowed to continue the culture process if there was no mycoplasma infection.

[0101] (2) MS5-hDLL1 or OP9-DL1 cells were cultured and expanded in standard T75 or T150 tissue culture flasks containing DMEM / 10% FBS. When the cells reached 70-90% confluence, they were trypsinized twice a week for 5 minutes at 37°C. Splitting cells were usually passaged at a ratio of cells: medium = 1:10. Cells were cultured in a standard cell culture incubator with a humidified environment at 37°C and 5% CO2.

[0102] (3) Expand the MS5-hDLL1 or OP9-DL1 cells to the desired number by seeding them in multiple T75 or T150 flasks. Ideally, the MS5-hDLL1 or OP9-DL1 cells should be stored in the flasks for at least 2 days before being used for ATO culture to ensure that the interval between trypsin treatments is >24h. Ideally, the confluence of MS5-hDLL1 or OP9-DL1 cells should reach 70-90% on the day of ATO culture.

[0103] (4) Harvest MS5-hDLL1 or OP9-DL1 cells with trypsin and quench with DMEM / 10% FBS. Gently pipette the cells to obtain a single cell suspension and filter with a 70 μm filter if there are still visible clumps. Resuspend the cells in R-B27 complete medium and count. Store the single cell suspension on ice and take it out when you need to culture ATO.

[0104] Timing: Harvesting MS5-hDLL1 or OP9-DL1 cells for ATOs typically takes 30 min to 1 h, depending on the number of cell culture flasks used, and can be performed while isolating HSPCs.

[0105] Example 4. Cultivation of organoids (ATO)

[0106] (1) Prepare a 6-well culture plate as required and add 1 mL of ATO complete medium (R-B27 complete medium + cytokines) to each well.

[0107] (2) Using small blunt forceps, insert a single cell culture pool into each well. Only the membrane at the bottom of the cell culture pool should be in contact with the culture medium. Place it in a biosafety cabinet until use. Combine the required number of CD34+CD3- cells and MS5-hDLL1 or OP9-DL1 cells in a microcentrifuge tube. It is recommended that the amount of cells loaded into each tube can culture 12 ATOs. A ratio of HSPC: stromal cells = 1:20 is used with a lower starting cell number (for example, use 7500 CD34+CD3- cells and 1.5×10 cells per ATO). 5 MS5-hDLL1 or OP9-DL1 cells) are most useful for T cell expansion and maturation using CB CD34+CD3- cells. The cytosol is very thick, and to avoid losing cells that stick to the sides of the pipette tip, pipette the medium up and down to coat the tip before aspirating the cells.

[0108] (3) Centrifuge the cells at 300 g for 5 min using a swinging bucket centrifuge (1.5 and 1.7 mL microcentrifuge tubes fit into the top of a standard 5 mL FACS tube swinging bucket). Only a swinging bucket centrifuge can ensure that the cells form a pellet at the bottom of the centrifuge tube, so using a standard fixed-angle rotor microcentrifuge is not recommended.

[0109] (4) Since the cell slurry is relatively viscous, the step of adjusting its volume may be challenging. It is recommended to combine the cells required for culturing 12 ATOs into one tube, carefully aspirate the supernatant using a vacuum flask and a glass pipette, briefly vortex the tube at medium-low speed with short pulses to resuspend the cell pellet, add 10 μL of ATO complete medium, and gently mix the cell pellet by pipetting. This will make it easier to measure the exact volume with a pipette, and then add enough complete R-B27 medium to obtain a final volume of 60 μL (5 μL per ATO).

[0110] (5) Use forceps to remove the cell culture pool from the medium and place it at the edge of the well so that it drains slightly immediately before inoculating the ATO. Use a 10 or 20 μL pipette tip to draw 5 μL of cell slurry for a single ATO and gently release the cell slurry at the end of the tip. Gently touch the middle of the membrane dropped to the bottom of the cell culture pool to release the cell slurry. The cell slurry will form a small sphere on the surface of the cell culture pool. If multiple ATOs are inoculated to increase T cell yield, up to 2 ATOs can be placed on a single cell culture pool, approximately 1 cm apart and as close to the center of the pool as possible. It is not recommended to inoculate more than 2 ATOs per well, as T cell differentiation may be adversely affected.

[0111] (6) Use forceps to gently place the cell culture pool back into the well containing the culture medium. The culture medium should not directly contact the upper surface of the cell organoid. Once each well has generated an organoid (ATO), replace the lid of the cell culture plate and place it in a cell culture incubator at 37°C and 5% CO2.

[0112] Among them, the steps for replacing ATO culture medium are as follows:

[0113] (1) Replace the ATO complete medium (R-B27 complete medium + cytokines) every 3-4 days. Tilt the culture plate to allow the medium to flow to one side of the well, and be careful not to let the medium flow to the surface of the cell culture pool. Do not damage ATO during this process.

[0114] (2) Connect a glass pipette to a vacuum aspirator and aspirate the culture medium from around the cell culture pool. Leave 100-200 μL of culture medium under the cell culture pool to prevent it from drying out.

[0115] (3) Using a P-1000 micropipette and a 1 mL pipette, add 1 mL of fresh, warm ATO complete medium between the cell culture reservoir and the well, allowing the medium to diffuse under the cell culture reservoir without coming into direct contact with the ATO.

[0116] discuss

[0117] The early stage of T cell commitment in the organoid culture kit of the present invention is very similar to that in the thymus, and all known progenitor cells undergo human CD3 + TCRαβ + The thymic precursors differentiate into T cells, which are then positively selected to eventually make the initial CD3 + TCRαβ + Single positive CD8 + and CD4 + T cell maturation.

[0118] Maintaining the survival of thymic organoids only requires replacing the culture medium, without the need to supplement stromal cells. Thymic organoids can directly culture bioengineered T cells derived from stem cells. The organoid culture kit of the present invention can culture a large number of tumor-specific T cells, and is easy to perform genetic manipulation, which can effectively reduce the risk of T cells attacking healthy tissues of the body.

[0119] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A culture reagent / kit for organoid culture, It is characterized in that A culture medium comprising: (A) R-B27 medium, which is RPMI 1640 medium supplemented with B-27, ascorbic acid and Primocin; (B) Organoid (ATO) complete medium: The ATO complete medium is RPMI 1640 medium supplemented with B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L; or a combination thereof.

2. The method according to claim 1, It is characterized in that The basic culture medium of the culture medium (A) and (B) is RPMI1640 culture medium.

3. The method according to claim 1, It is characterized in that The ATO complete medium can be prepared by adding RhIL-7 and RhFLT3L to the R-B27 medium.

4. The method according to claim 1, It is characterized in that The organoid is a thymic organoid.

5. A method for culturing organoids in vitro, It is characterized in that Includes steps: (S1) culturing trophoblast cells and hematopoietic stem cells in two dimensions respectively; and (S2) performing three-dimensional culture on the cells after the two-dimensional culture to form in vitro organoids; Wherein, in step (S1), two-dimensional culture is performed using R-B27 medium, wherein the R-B27 medium is RPMI 1640 medium supplemented with B-27, ascorbic acid and Primocin; and / or In step (S2), three-dimensional culture is performed using ATO complete medium, wherein the ATO complete medium is RPMI 1640 medium supplemented with B-27, ascorbic acid, Primocin, RhIL-7 and RhFLT3L.

6. The method according to claim 5, It is characterized in that The hematopoietic stem cells are extracted from body fluids.

7. The method according to claim 6, It is characterized in that The body fluid is selected from the group consisting of umbilical cord blood, peripheral blood, bone marrow, or a combination thereof.

8. An organoid cultured by the in vitro organ culture method according to claim 5.

9. Use of the organoid as claimed in claim 8 as a drug evaluation model, It is characterized in that Used to prepare anti-tumor drug evaluation models or evaluation reagents.

10. A method for evaluating antitumor drugs, It is characterized in that Includes steps: (N1) providing an organoid prepared by the method of claim 5; and (N2) Using the organoids to evaluate anti-tumor drug sensitivity.