Method for preparing bone marrow organoid and composition and culture medium used in method

Abstract

The invention discloses a method for preparing bone marrow organoid and a composition and a culture medium used by the method. The method provided by the invention comprises the following steps: (1) inducing differentiation of embryonic stem cells to form a cell population containing stromal cells, endothelial cells and hematopoietic stem progenitor cells, namely a cell population 1; (2) dividing the cell population 1 into a CD34 positive cell subset and a CD34 negative cell subset through sorting, and then mixing the CD34 positive cell subset and the CD34 negative cell subset according to the cell number of 1: 1 to obtain a cell population 2; (3) culturing the cell population 2 into a cell aggregate, and then mixing the cell aggregate with matrigel to obtain a mixture; and (4) culturing the mixture and the vascular endothelial cells in a device with a bone marrow blood barrier to obtain the bone marrow organ. The bone marrow organ can be used in the fields of organ differentiation and development mechanism research, disease injury model construction, new drug screening and safety evaluation, personalized medical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for preparing bone marrow organoids and a composition and culture medium used therein. Background Art

[0002] Organoid technology is a new in vitro cell culture model that has emerged in recent years. This technology uses stem cells or precursor cells from various sources, such as pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells), tissue adult stem cells, tumor stem cells, etc., to proliferate, differentiate and self-assemble in semi-solid culture media such as matrix gel, collagen, and hydrogel, thereby forming cell aggregates with specific tissue structural characteristics. Organoids show a high degree of similarity with the tissues from which they are derived in terms of cell composition type, tissue structure and physiological function, and can simulate and reproduce the complex biological processes of tissues and organs in vitro to a certain extent. On this basis, if the organoid preparation technology is combined with an organ chip culture device with microfluidic function, a microphysiological system with a more complex culture structure and more bionic physiological function can be further constructed. With the help of its internal environmental sensing control device or the unique three-dimensional culture structure of the culture device, the microphysiological system can more accurately simulate and control the complex microenvironmental regulatory factors in the process of realizing tissue physiological functions, such as material diffusion, fluid shear force, physiological signal stimulation feedback, etc. At present, in vitro construction technologies for organoids and microphysiological systems of various human tissues and organs, such as the small intestine, kidney, brain, and lungs, have been developed one after another, and have been widely used in research in the fields of organ differentiation and development, disease damage model construction, new drug screening and safety evaluation, personalized medicine, etc., showing good application trends and development prospects.

[0003] Although significant progress has been made in the cultivation methods of organoids and microphysiological systems for solid tissue organs, the development of bone marrow hematopoietic system organoids and microphysiological system construction technology still faces many challenges. The two major difficulties in the construction of bone marrow organoids and microphysiological systems are to simulate the complex and orderly physiological structure and unique and rich physiological functions of bone marrow in vitro. On the one hand, unlike solid tissues that usually perform a single physiological function, bone marrow performs complex and diverse physiological functions. As the core part for maintaining human hematopoietic and immune functions, bone marrow continuously generates various types of blood cells throughout the life cycle to meet the metabolism and renewal needs of various mature blood cells in peripheral blood; and can quickly sense stress signals and respond adaptively after pathological changes such as infection and injury occur in the body. On the other hand, the structural basis for the bone marrow to achieve long-lasting and orderly hematopoietic function lies in its complex three-dimensional microenvironment. In the bone marrow, hematopoietic stem cells and their differentiated hematopoietic progenitor cells and hematopoietic precursor cells (as "seed cells" for maintaining hematopoietic function) are combined with the hematopoietic microenvironment composed of microenvironment cells (such as endothelial cells, stromal cells, osteoblasts, etc.), bioactive components (such as extracellular matrix proteins, cytokines, microvesicles, etc.) and physical and chemical factors (such as dissolved oxygen content, fluid force, etc.), together forming the structural and functional units of the bone marrow hematopoietic system. The bone marrow microenvironment precisely regulates the self-renewal and lineage differentiation fate of hematopoietic stem cells through intercellular communication and remote signal transmission. It is precisely because of this structural and functional complexity that it is difficult to reproduce the unique phenotypic functions of the bone marrow through co-culture of one or several cells in an in vitro culture environment. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing bone marrow organoids and a composition and culture medium used therein.

[0005] The present invention provides a method for preparing bone marrow organoids (named as method I), comprising the following steps: (1) inducing embryonic stem cells to differentiate into a cell population containing stromal cells, endothelial cells and hematopoietic stem and progenitor cells, named as cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) The cell population 1 is divided into CD34 positive cell subpopulations (CD34 + cell subsets) and CD34-negative cell subsets (CD34 - Cell subsets), then the CD34-positive cell subsets and the CD34-negative cell subsets were mixed at a cell number of 1:1 to obtain a cell population, which was named cell population 2; (3) culturing the cell population 2 into cell aggregates, and then mixing with matrix gel to obtain a mixture; (4) culturing the mixture and vascular endothelial cells in a device having a bone marrow-blood barrier to obtain bone marrow organoids; The device with bone marrow blood barrier has more than one unit structure; the unit structure is composed of a cavity (orgamoid chamber) for accommodating simulated bone marrow and a channel (perfusion channel) for accommodating simulated blood vessels, a sample addition hole (reservior) is provided at the end of the channel, and a bone marrow blood barrier is provided at the intersection of the cavity and the channel; the bone marrow blood barrier is a membrane with a pore size of 5µm-10µm; The method for culturing the mixture and vascular endothelial cells in a device with a bone marrow-blood barrier is: culturing the mixture in the cavity to form simulated bone marrow, and culturing the vascular endothelial cells in the channel to form simulated blood vessels.

[0006] Specifically, the mixture is cultured using culture medium 6.

[0007] Specifically, the bone marrow-blood barrier is a polycarbonate membrane with a pore size of 5µm-10µm.

[0008] Specifically, “pore diameter of 5µm-10µm” may mean pore diameter of 8µm or pore diameter of 10µm.

[0009] The present invention also provides a method for preparing bone marrow organoids (named as method II), comprising the following steps: (1) inducing embryonic stem cells to differentiate into a cell population containing stromal cells, endothelial cells and hematopoietic stem and progenitor cells, named as cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) The cell population 1 is divided into CD34 positive cell subpopulations (CD34 + cell subsets) and CD34-negative cell subsets (CD34 - Cell subsets), then the CD34-positive cell subsets and the CD34-negative cell subsets were mixed at a cell number of 1:1 to obtain a cell population, which was named cell population 2; (3) culturing the cell population 2 into cell aggregates, and then mixing with matrix gel to obtain a mixture; (4) The mixture obtained in step (3) is cultured using culture medium 6 to obtain bone marrow organoids.

[0010] Specifically, the method for preparing cell population 1 comprises the following steps: ① Collect the digested embryonic stem cells and resuspend the cell pellet in medium 1 to obtain a cell suspension; ② Take a Matrigel-coated 6-well plate, inoculate the cell suspension prepared in step ①, culture for 24 hours, then discard the supernatant, add PBS buffer to wash the cells, and then discard the supernatant; ③ Take the 6-well plate that has completed step ②, add medium 2, culture for 48 hours, then aspirate and discard the supernatant, add PBS buffer to wash the cells, and then aspirate and discard the supernatant; ④ Take the 6-well plate prepared in step ③, add culture medium 3, and culture for 72 hours; during the culture process, replace new culture medium 3 every 24 hours.

[0011] Specifically, the method for preparing cell population 1 comprises the following steps: ① Collect the digested embryonic stem cells and resuspend the cell pellet in medium 1 to obtain a cell suspension (0.4×10 5 viable cells / mL); ② Take a Matrigel-coated 6-well plate, inoculate the cell suspension prepared in step ① (3 mL per well), culture for 24 hours, then discard the supernatant, add PBS buffer to wash the cells, and then discard the supernatant; ③Take the 6-well plate that has completed step ②, add medium 2 (3 mL per well), culture for 48 hours, then aspirate the supernatant, add PBS buffer to wash the cells, and then aspirate the supernatant; ④ Take the 6-well plate that has completed step ③, add culture medium 3 (3 mL per well), and culture for 72 hours; during the culture process, replace new culture medium 3 every 24 hours.

[0012] Specifically, the method for preparing cell population 2 comprises the following steps: ① After digesting the cell population 1, a CD34 magnetic separation kit was used to separate the cells and collect CD34 + Cells and CD34 - cell; ② The CD34 + cells, CD34 obtained in step ① - The cells and the culture medium 4 are mixed to obtain a cell suspension containing the cell population 2.

[0013] Specifically, the method for preparing cell population 2 comprises the following steps: ① After digesting the cell population 1, a CD34 magnetic separation kit was used to separate the cells and collect CD34 + Cells and CD34 - cell; ② The CD34 + cells, CD34 obtained in step ① - Mix the cells and medium 4 to make CD34 + The cell content is 0.5×105 viable cells / ml and CD34 - The cell content is 0.5×10 5 The cell suspension contains cell population 2.

[0014] Specifically, the method for culturing the cell group 2 into cell aggregates comprises the following steps: taking an Aggrewell plate, inoculating a cell suspension containing the cell group 2 (1 mL per well), first incubating for half an hour, and then culturing for 18-24 hours.

[0015] Specifically, the method of "mixing with matrix gel to obtain a mixture" includes the following steps: mixing 6500-7500 cell aggregates (specifically 7200 cell aggregates) with 0.5-1 mL Matrigel to obtain a mixture (also called a cell aggregate / matrigel mixture).

[0016] Specifically, the method of "mixing with matrix gel to obtain a mixture" includes the following steps: blowing out all cell aggregates (about 7,200) formed in one Angrewell plate and collecting them in a centrifuge tube pre-cooled in an ice bath, discarding the supernatant after centrifugation, continuing to place the centrifuge tube on ice and adding 0.5 mL of Matrigel, mixing, and obtaining a mixture (also called a cell aggregate / matrigel mixture).

[0017] Specifically, step (4) of method I comprises the following steps in sequence: ① Coating the cavity and channels of the device with bone marrow-blood barrier with collagen; ② Add the cell aggregate / matrigel mixture to the chamber, incubate, and then add medium 6; then place on a shaker (tilt angle set to 30°, speed set to 3-5 cycles / min) and culture for 96 hours; ③Discard the liquid in the cavity and channel, add endothelial cells to the channel, incubate upside down, then add culture medium 6 to the cavity and EGM2 culture medium to the channel, culture for 24 hours, and then place on a shaker (tilt angle set to 30°, speed set to 3-5 cycles / min) and culture for 96 hours.

[0018] Specifically, step (4) of method I comprises the following steps in sequence: ① Coating the cavity and channel of the device with bone marrow-blood barrier with collagen and then washing with PBS buffer; ② Aspirate the PBS buffer in the chamber, then add 10 μL of the cell aggregate / matrigel mixture to the chamber, incubate at 37°C for 1 hour, and then add 100 μL of medium 6 to the chamber; then, aspirate the PBS buffer in the channel, and add 200 μL of medium 6 to the channel; then, place the device on a shaker (tilt angle set to 30°, speed set to 3-5 cycles / min), and culture for 96 hours; ③After completing step ②, aspirate the liquid in the cavity and channel, add 20 μL of HUVEC cell suspension to the channel, incubate upside down for 1 hour, then put it upright, add 100 μL of culture medium 6 to the cavity, add 200 μl of EGM2 culture medium to the channel, and culture for 24 hours; ④After completing step ③, place the device on a shaker (tilt angle set to 30°, speed set to 3-5 cycles / min) and culture for 96 hours.

[0019] Preparation of HUVEC cell suspension: Resuspend the digested HUVEC cells in EGM2 medium to a cell concentration of (1-3) × 10 6 viable cells / mL.

[0020] Specifically, the device having a bone marrow-blood barrier is a commercially available barrier function organ chip.

[0021] Specifically, the device with bone marrow-blood barrier is a high-throughput barrier function organ chip of Beijing Daxiang Technology Co., Ltd.

[0022] Specifically, the device with bone marrow-blood barrier is the IBAC M1 chip of Beijing Daxiang Technology Co., Ltd.

[0023] Step (4) of method II may specifically be as follows: take a cell culture plate, add the cell aggregate / matrigel mixture, culture for 30 minutes, then add culture medium 6, and culture for 192 hours. During the culture process, culture medium 6 is added after 48 hours, supernatant is discarded after 96 hours and culture medium 6 is added, and culture medium 6 is added after 144 hours.

[0024] Step (4) of the method II may specifically be as follows: take a 24-well cell culture plate, add the cell aggregate / matrigel mixture (50 μl / well), culture for 30 minutes, then add culture medium 6 (0.6 ml / well), and culture for 192 hours. During the culture process, culture medium 6 (0.6 ml / well) is added after 48 hours, the supernatant is aspirated and culture medium 6 (0.6 ml / well) is added after 96 hours, and culture medium 6 (0.6 ml / well) is added after 144 hours.

[0025] Bone marrow organoids prepared by any of the above methods also fall within the protection scope of the present invention.

[0026] The present invention also protects a composition consisting of the following ten compounds: SCF, TPO, IL-3, Flt31, GM-CSF, Y-27632, SB431542, Forskolin, PGE2 and FK506; The ratio of the ten compounds is as follows: 50-100µg SCF: 50-100µg TPO: 50-100µg IL-3: 50-100µg Flt3l: 20-50µg GM-CSF: 1-5µmol Y-27632: 2-10µmol SB431542: 1-5µmol Forskolin: 0.3-2µmol PGE2: 1-5µmol FK506; The function of the composition is to promote cell aggregates to form bone marrow organoids.

[0027] The present invention also protects a culture medium, which contains the composition; the function of the culture medium is to promote cell aggregates to form bone marrow organoids.

[0028] Specifically, the culture medium is culture medium 6.

[0029] The present invention also protects the use of the composition or the culture medium in preparing a kit; the functions of the kit are as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

[0030] The present invention also protects a kit, which includes the composition or the culture medium; the functions of the kit are as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

[0031] Specifically, the kit further includes culture medium 1 and / or culture medium 2 and / or culture medium 3 and / or culture medium 4.

[0032] Specifically, the kit also includes EGM2 culture medium.

[0033] The present invention also protects the use of the kit, which is as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

[0034] The present invention also protects a compound combination consisting of the following five compounds: Y-27632, SB431542, Forskolin, PGE2 and FK506; The five compounds are as follows: 1-5μmol Y-27632: 2-10μmol SB431542: 1-5μmol Forskolin: 0.3-2μmol PGE2: 1-5μmol FK506; The function of the compound combination is to promote the formation of hematopoietic stem and progenitor cells and the formation of endothelial cell network structures when the cell aggregates are cultured into bone marrow organoids; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

[0035] The present invention also protects the following application of the compound combination: when culturing cell aggregates into bone marrow organoids, promoting the formation of hematopoietic stem and progenitor cells and promoting the formation of endothelial cell network structures; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

[0036] Specifically, the cell aggregates are cell aggregates formed by cell groups differentiated from embryonic stem cells.

[0037] Specifically, the cell aggregate is a cell aggregate formed by cell group 2.

[0038] The preparation method of cell population 2 is as described above.

[0039] Medium 1 is a medium named Medium 1.

[0040] The supplements required to prepare the medium 1 per liter are as follows: 3-10 μmol Y-27632.

[0041] The supplements required to prepare the medium 1 per liter are as follows: 10 μmol Y-27632.

[0042] The culture medium 1 consists of the additives and a basal culture medium.

[0043] Specifically, the basic culture medium is mTeSR1 culture medium.

[0044] Medium 2 is a medium named Medium 2.

[0045] The following supplements are required to prepare each liter of the culture medium 2: 10-50 µg BMP-4, 10-50 µg Activin A, 5-30 µg bFGF, and 1-5 µmol (CHIR-99021) HCl.

[0046] The following supplements were used to prepare each liter of the culture medium 2: 25 µg BMP-4, 25 µg Activin A, 25 µg bFGF, and 2 µmol (CHIR-99021) HCl.

[0047] The culture medium 2 consists of the additives and a basal culture medium.

[0048] Specifically, the basal culture medium is BEL culture medium.

[0049] Medium 3 is a medium named as Medium 3.

[0050] The following supplements were used to prepare each liter of the medium 3: 50-100 µg VEGF, 10-20 µg bFGF, and 2-5 µmol SB431542.

[0051] The following supplements were used to prepare each liter of the medium 3: 100 µg VEGF, 20 µg bFGF, and 5 µmol SB431542.

[0052] The culture medium 3 consists of the additives and a basal culture medium.

[0053] Specifically, the basal culture medium is BEL culture medium.

[0054] Medium 4 is a medium named Medium 4.

[0055] The following supplements are required to prepare each liter of the medium 4: 50-100 µg VEGF, 10-20 µg bFGF, 2-5 µmol SB431542, and 2-10 µmol Y-27632.

[0056] The following supplements were used to prepare each liter of the medium 4: 100 µg VEGF, 20 µg bFGF, 5 µmol SB431542, and 10 µmol Y-27632.

[0057] The culture medium 4 consists of the additives and a basal culture medium.

[0058] Specifically, the basal culture medium is BEL culture medium.

[0059] Medium 6 is a medium named Medium 6.

[0060] The supplements required to prepare each liter of the culture medium 6 are as follows: 50-100µg SCF, 50-100µg TPO, 50-100µg IL-3, 50-100µg Flt31, 20-50µg GM-CSF, 1-5µmol Y-27632, 2-10µmol SB431542, 1-5µmol Forskolin, 0.3-2µmol PGE2 and 1-5µmol FK506.

[0061] The supplements required to prepare the medium 6 per liter are as follows: 50 µg SCF, 50 µg TPO, 50 µg IL-3, 50 µg Flt31, 20 µg GM-CSF, 5 µmol Y-27632, 10 µmol SB431542, 2 µmol Forskolin, 1 µmol PGE2 and 2 µmol FK506.

[0062] The culture medium 6 consists of the additives and a basal culture medium.

[0063] Specifically, the basal culture medium is EGM2 culture medium.

[0064] Specifically, the bone marrow organoid is a human bone marrow organoid.

[0065] Specifically, the embryonic stem cells are human embryonic stem cells or human embryonic stem cell lines.

[0066] Specifically, the embryonic stem cells are WA09 cells.

[0067] Specifically, the vascular endothelial cells are human vascular endothelial cells.

[0068] Specifically, the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC cells).

[0069] The term "organoid" refers to a three-dimensional tissue culture, a small organ-like object produced or synthesized by culturing one or more types of cells on a substrate. As the cells differentiate, the organoid has anatomical features similar to mammalian organs, such as simulated blood vessels and simulated bone marrow.

[0070] The bone marrow organoids can be used in the fields of studying organ differentiation and development mechanisms, constructing disease injury models, screening new drugs and evaluating their safety, and personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is an example graph of flow cytometry in step 6 of Example 3.

[0072] Figure 2 Exemplary photographs of microscopic cell morphology in steps 5 and 7 of Example 3. The bar represents 200 µm.

[0073] Figure 3 The results of cell survival rate in step 3 of Example 4 and the relative value of the number of live cells in bone marrow organoids.

[0074] Figure 4 The percentages and numbers of various cells in step 3 of Example 4.

[0075] Figure 5Exemplary photographs of observations under a microscope in step 5 of Example 4 (bar represents 50 µm) and the diameters of cell aggregates.

[0076] Figure 6 The figures are exemplary photographs observed under a microscope in step 7 of Example 4 and the number of buds of cell aggregates.

[0077] Figure 7 This is an exemplary photograph of immunofluorescence staining (using CD144 antibody) in step 7 of Example 4. The bar represents 100 µm.

[0078] Figure 8 These are exemplary photographs of immunofluorescence staining in step 7 of Example 4 (using CD45 antibodies, CD271 antibodies, and CD144 antibodies).

[0079] Fig. 9 This is an exemplary picture of flow cytometry output in step 7 of Example 4.

[0080] Fig.10 Exemplary photographs of Wright-Giemsa staining and immunofluorescence staining (using CD41 antibodies, CD14 antibodies, CD71 antibodies, CD66b antibodies, and CD3 antibodies).

[0081] Fig.11 Schematic diagram of bone marrow organoids in Example 6.

[0082] Fig.12 This is an exemplary photograph of immunofluorescence staining (using CD45 antibody, CD144 antibody and CD271 antibody) in Example 6. The bar represents 30 µm.

[0083] Fig.13 This is the result diagram in Example 7. DETAILED DESCRIPTION

[0084] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0085] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are all repeated three times, and the results are averaged. Unless otherwise specified, the cultures in the examples are all cultured under the following conditions: placed in a cell culture incubator (37°C, 5% CO2) for static culture. In the examples, the total number of cells was counted using a cell counting plate, and the cell survival rate was identified using AO / PI staining. The total number of cells multiplied by the cell survival rate is the number of live cells. Matrigel, used to prepare a cell aggregate / matrigel mixture, is the full name of Corning ® Matrigel ® Growth factor reduced (GFR) basement membrane matrix (phenol red free, LDEV free), Corning, catalog number 356231. "Hematopoietic stem / progenitor cells" or "hematopoietic stem / progenitor cells" refers to "hematopoietic stem cells and / or hematopoietic progenitor cells".

[0086] WA09 cells (human embryonic stem cells), also known as H9 cells, WiCell (American hESC cell bank), number WAe009-A. HUVEC cells (human umbilical vein endothelial cells): EallBio / E-Bio, product catalog number PC.00241.

[0087] CD34 MicroBead Kit: Miltenyi Biotec, product catalog number 130-046-702.

[0088] Aggrewell™ 800 24-well plate: STEMCELL Technologies. Each Aggrewell plate has 24 wells, each of which contains 300 microwells (which can form 300 spherical cell aggregates), so each Aggrewell plate forms approximately 7,200 cell aggregates.

[0089] Preparation method of Matrigel-coated 6-well plate: Take a 6-well plate, add Matrigel working solution (1 mL / well), and incubate at room temperature for 30-60 minutes to obtain a Matrigel-coated 6-well plate. Dilute Matrigel to 100 times the volume with DMEM / F-12 medium to obtain Matrigel working solution. Matrigel used to prepare Matrigel working solution is called Corning ® Matrigel ®Matrix (for hESCs, LDEV-free), Corning, catalog number 354277.

[0090] TrypLE Select enzyme (1×): Thermo Fisher Scientific, product catalog number 12563029, brand Gibco™. mTeSR1 medium (mTeSR™1 Basal Medium): Stemcell, product catalog number 05850. DMEM / F-12 medium: Thermo Fisher Scientific, product catalog number 11330032, brand Gibco™.

[0091] EGM2 medium (Lonza EGM-2 endothelial cell medium, CC-3156+CC-4176): Mix the components provided in the Lonza EGM-2 BulletKit (CC-3156 & CC-4176) according to the instructions to obtain EGM2 medium. The seller is Zepin Technology Co., Ltd., the product catalog number is CC-3162, and the brand (manufacturer) is Lonza.

[0092] Y-27632 (CAS: 146986-50-7): Selleck, catalog number S6390. SB431542 (CAS: 301836-41-9): Selleck, catalog number S1067. (CHIR-99021) HCl, also known as CHIR-99021 hydrochloride (CAS: 1797989-42-4): Selleck, catalog number S2924. Forskolin (CAS: 66575-29-9): MCE, catalog number HY-15371. PGE2 (Prostaglandin E2) (CAS: 363-24-6): MCE, catalog number HY-101952. FK506 (Tacrolimus) (CAS: 104987-11-3): MCE, catalog number HY-13756.

[0093] BMP-4 (Recombinant Human BMP-4 Protein): R&D Company, product catalog number is 314-BP, product description is "human BMP-4 protein Ser293-Arg408", which is the amino acid residues 293-408 in UniProtKB / Swiss-Prot: Q53XC5 (31-OCT-2006). Activin A (Recombinant Human / Mouse / RatActivin A Protein): R&D Company, product catalog number is 338-AC, product description is "Activin A proteinGly311-Ser426", which is the amino acid residues 311 to 426 in UniProtKB / Swiss-Prot: P08476.2 (05-FEB-2025). VEGF (Recombinant Human VEGF 165 Protein): R&D company, product catalog number 293-VE, product description "human VEGF protein Ala27-Arg191", which is the amino acid residues 27-191 in NCBI Reference Sequence: NP_001165097.1 (24-OCT-2024). bFGF (Human FGF-basic, PeproTech ®): Thermo Fisher Scientific, catalog number AF-100-18C, brand Gibco™, NCBI Reference Sequence: NP_001348594.1 (29-OCT-2024) amino acid residues 10-155. SCF (Recombinant Human SCF Protein): R&D company, catalog number BT-SCF, product description "human SCF / c-kit Ligand protein Glu26-Ala189, with a N-terminal Met", human SCF / c-kit Ligand protein Glu26-Ala189 is the amino acid residues 26-189 in UniProtKB / Swiss-Prot: P21583.1 (05-FEB-2025). TPO (Recombinant Human Thrombopoietin): R&D company, product catalog number is 288-TPN, product description is "human Thrombopoietin / Tpo protein Ser22-Gly353", which is the amino acid residues 22-353 in UniProtKB / Swiss-Prot: P40225.1 (05-FEB-2025). IL-3 (Recombinant Human IL-3 Protein): R&D company, product catalog number is 203-IL, product description is "human IL-3 protein Ala20-Phe152", which is the amino acid residues 20-152 in GenBank: AAC08706.1 (31-MAR-1998). Flt3l (Recombinant Human Flt-3 Ligand Protein): R&D Company, product catalog number is 308-FKHB, product description is "human Flt-3 Ligand / FLT3L protein Thr27-Pro185", which is the amino acid residues 27-185 in GenBank: AAA17999.1 (11-MAY-1994). GM-CSF (Recombinant Human GM-CSF Protein): R&D Company, product catalog number is 7954-GM, product description is "human GM-CSF protein Ala18-Glu144", which is the amino acid residues 18-144 in UniProtKB / Swiss-Prot: P04141.1 (05-FEB-2025).

[0094] PEcy7-labeled CD34 antibody (BD Pharmingen™ PE-Cy™7 Mouse Anti-Human CD34): BD Company, catalog number 560710. BV421-labeled CD43 antibody (BD Horizon™ BV421 Mouse Anti-Human CD43): BD Company, catalog number 562916. PerCPcy5.5-labeled CD144 antibody (BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD144): BD Company, catalog number 561566. PE-labeled CD140a antibody (BD Pharmingen™ PE Mouse Anti-Human CD140a): BD Company, catalog number 556002. APC-labeled LEPR antibody (BD Pharmingen™ Alexa Fluor ® 647 Mouse Anti-HumanLeptin Receptor): BD Company, catalog number 564376. PE-labeled CD144 antibody (BD Pharmingen™ PE Mouse anti-Human CD144): BD Company, catalog number 560410. BV786-labeled CD45 antibody (BD Horizon™ BV786 Mouse Anti-Human CD45): BD Company, catalog number 563716. PE-labeled CD44 antibody (BD Pharmingen™ PE Mouse Anti-Human CD44): BD Company, catalog number 555479.

[0095] CD144 antibody (Human VE-Cadherin Antibody) for cell immunofluorescence: R&D Company, product catalog number is AF938. LEPR antibody (Human Leptin R Antibody) for cell immunofluorescence: R&D Company, product catalog number is MAB867. CD45 antibody (Anti-CD45 antibody, rabbit polyclonal antibody) for cell immunofluorescence: abcam Company, product catalog number is ab10558. CD41 antibody (Anti-CD41 antibody, rabbit monoclonal antibody) for cell immunofluorescence: abcam Company, product catalog number is ab134131. CD271 antibody (Anti-NGFRantibody produced in rabbit) for cell immunofluorescence: MERCK Company, product catalog number is HPA004765. RUNX1 antibody (Anti-RUNX1 rabbit antibody) for cell immunofluorescence: MERCK Company, product catalog number is HPA004176. CXCL12 antibody (CXCL12 Rabbit pAb) used for cell immunofluorescence: Ibotek Biotech Co., Ltd., product catalog number A18225. CD14 antibody (CD14 Monoclonal Antibody) used for cell immunofluorescence: Thermo Fisher Scientific, product catalog number 12-0149-42. CD71 antibody (CD71 MonoclonalAntibody) used for cell immunofluorescence: Thermo Fisher Scientific, product catalog number 17-0719-42. CD66b antibody (CD66b Monoclonal Antibody) used for cell immunofluorescence: Thermo Fisher Scientific, product catalog number 11-0666-42. CD3 antibody (CD3 Monoclonal Antibody) used for cell immunofluorescence: Thermo Fisher Scientific, product catalog number 11-0032-82.

[0096] BEL medium consists of additives and basal medium; the basal medium is obtained by mixing equal volumes of "IMDM + GlutaMAX" and "F12 + GlutaMAX"; the additives and their addition amounts per liter of BEL medium are as follows: 2mmolGlutaMAX, 2.5g bovine serum albumin, 2ml cholesterol solution, 100µg linolenic acid, 100µg linoleic acid, 450µmol 1-thioglycerol, 50ml PFHM-Ⅱ, 50mg AA2P and 10ml ITS-G.

[0097] IMDM + GlutaMAX (IMDM, GlutaMAX™ Supplement) (1X): Gibco™ Company, catalog number is 31980030. F12 + GlutaMAX (Ham's F-12 Nutrient Mixture, GlutaMAX™ Supplement) (1X): Gibco™ Company, catalog number is 31765035.

[0098] GlutaMAX (GlutaMAX™ Supplement) (containing 200 mM L-alanyl-L-glutamine dipeptide) (100×): Thermo Fisher Scientific, product catalog number 35050061. Bovine serum albumin (BSA): MERCK, product catalog number A3311. Cholesterol solution (SyntheChol ® NS0 supplement) (500×): MERCK, product catalog number S5442. Linolenic acid (CAS: 463-40-1): MERCK, product catalog number L2376. Linoleic acid (CAS: 60-33-3): MERCK, product catalog number L1012. 1-Thioglycerol (CAS: 96-27-5): MERCK, product catalog number M6145. PFHM-Ⅱ (PFHM-II protein-free hybridoma cell culture medium): Thermo Fisher Scientific, product catalog number 12040077. AA2P (CAS: 1713265-25-8): MERCK, product catalog number A8960. ITS-G (insulin-transferrin-selenium complex) (100×): Thermo Fisher Scientific, product catalog number 41400045.

[0099] Example 1: Cultivation and passage of human embryonic stem cells 1. Suspend WA09 cells in mTeSR1 medium to obtain a cell suspension (1×10 5 -2×10 5 living cells).

[0100] 2. Take a Matrigel-coated 6-well plate, inoculate the cell suspension obtained in step 1 (3 mL per well), and culture until the cell confluence reaches 80%. During the culture process, replace the mTeSR1 medium every 24 hours.

[0101] 3. After completing step 2, discard the supernatant, add TrypLE Select enzyme (0.3-0.5mL per well), incubate for 3-5 minutes, and then add mTeSR1 medium (3mL per well) to terminate digestion.

[0102] 4. After completing step 3, transfer the entire liquid phase system in the well to a centrifuge tube, centrifuge at 900 rpm for 4 min, then discard the supernatant and resuspend the cell pellet with mTeSR1 medium to obtain a cell suspension (1×10 cells per 3 mL). 5 -2×10 5 living cells).

[0103] 5. Take a Matrigel-coated 6-well plate, inoculate the cell suspension obtained in step 4 (3 mL per well), and culture until the cell confluence reaches 80%. During the culture process, replace the mTeSR1 medium every 24 hours.

[0104] The cells can be subcultured continuously during the culture process (follow steps 3, 4, and 5 in sequence), and cells subcultured for less than 60 generations can be used.

[0105] Example 2. Preparation of culture medium Culture medium 1 consists of additives and basal culture medium; the basal culture medium is mTeSR1 culture medium; the additives and their addition amounts per liter of culture medium 1 are as follows: 10 μmol Y-27632.

[0106] Medium 2 consists of supplements and basal medium; the basal medium is BEL medium; the supplements and their amounts per liter of Medium 2 are as follows: 25µg BMP-4, 25µg Activin A, 25µg bFGF and 2μmol (CHIR-99021) HCl.

[0107] Medium 3 consisted of supplements and basal medium; the basal medium was BEL medium; the supplements and their amounts per liter of Medium 3 were as follows: 100 µg VEGF, 20 µg bFGF, and 5 μmol SB431542.

[0108] Medium 4 consisted of supplements and basal medium; the basal medium was BEL medium; the supplements and their amounts per liter of Medium 4 were as follows: 100 µg VEGF, 20 µg bFGF, 5 µmol SB431542, and 10 µmol Y-27632.

[0109] Medium 5 consists of additives and basal medium; the basal medium is EGM2 medium; the additives and their amounts per liter of Medium 5 are as follows: 50µg SCF, 50µg TPO, 50µg IL-3, 50µg Flt31 and 20µg GM-CSF.

[0110] Medium 6 consists of additives and basal medium; the basal medium is EGM2 medium; the additives and their amounts per liter of medium 6 are as follows: 50µg SCF, 50µg TPO, 50µg IL-3, 50µg Flt3l, 20µg GM-CSF, 5μmol Y-27632, 10μmol SB431542, 2μmol Forskolin, 1μmol PGE2 and 2μmol FK506.

[0111] The culture medium prepared in Example 2 was used in subsequent examples.

[0112] Example 3: First stage cultivation and identification The purpose of the first stage of culture is to induce embryonic stem cells to differentiate and form a cell population containing "stromal cells", "endothelial cells" and "hematopoietic stem cells / hematopoietic progenitor cells", namely cell population 1.

[0113] 1. After completing step 5 of Example 1, discard the supernatant, add TrypLE Select enzyme (0.3-0.5 mL per well), incubate for 3-5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate the digestion.

[0114] 2. After completing step 1, transfer the entire liquid phase system in the well to a centrifuge tube, centrifuge at 900 rpm for 4 min, then discard the supernatant and resuspend the cell pellet with medium 1 to obtain a cell suspension (0.4×10 5 viable cells / mL).

[0115] 3. Take a Matrigel-coated 6-well plate, inoculate the cell suspension prepared in step 2 (3 mL per well), culture for 24 hours, then discard the supernatant, add PBS buffer to wash the cells, and then discard the supernatant.

[0116] 4. Take the 6-well plate that has completed step 3, add culture medium 2 (3 mL per well), culture for 48 hours, then discard the supernatant, add PBS buffer to wash the cells, and then discard the supernatant.

[0117] 5. Take the 6-well plate that has completed step 4, add culture medium 3 (3 mL per well), and culture for 72 hours. During the culture process, replace new culture medium 3 every 24 hours. After the culture is completed, observe the cell morphology under a microscope. See the exemplary photos for details. Figure 2 Left picture of A.

[0118] 6. Flow cytometry identification After completing step 5, take a sample and perform cell ratio analysis using flow cytometry (the antibodies used in flow cytometry are: CD34 antibody labeled with PEcy7, CD43 antibody labeled with BV421, CD144 antibody labeled with PerCPcy5.5, and CD140a antibody labeled with PE).

[0119] Example images of flow cytometry output are shown in Figure 1 The left graph (the horizontal axis is the CD34 signal intensity). Extract the CD34 in the output graph + The cells also show their CD43 signal intensity (abscissa) and CD144 signal intensity (ordinate). For example, see Figure 1 Extract CD34 from the output image - The cells also show their CD140a signal intensity (abscissa) and CD144 signal intensity (ordinate), and exemplary images are shown in Figure 1 Right image. Characteristics of stromal cells: CD34 - CD140a + CD144 - . Characteristics of endothelial cells: CD34 + CD43 - CD144 + Characteristics of hematopoietic stem cells / hematopoietic progenitor cells: CD34 + CD43 + CD144 + In the cell population that completed step 5 (statistical results of 7 repeated samples, mean ± standard deviation), the percentage of stromal cells in the total number of living cells was 17.04% ± 4.57%, the percentage of endothelial cells in the total number of living cells was 43.56% ± 8.08%, and the percentage of hematopoietic stem cells / hematopoietic progenitor cells in the total number of living cells was 18.03% ± 3.97%. The results showed that after completing the culture of step 5, WA09 cells can efficiently differentiate into a cell population containing "stromal cells", "endothelial cells" and "hematopoietic stem cells / hematopoietic progenitor cells".

[0120] 7. Identification of differentiation potential ①After completing step 5, discard the supernatant, add TrypLE Select enzyme (0.3-0.5mL per well), incubate for 3-5 minutes, and then add mTeSR1 medium (3mL per well) to terminate digestion.

[0121] ②After completing step ①, transfer the entire liquid phase system in the well to a centrifuge tube, centrifuge at 900 rpm for 4 min, then discard the supernatant, add PBS buffer to wash the cells, then aspirate and discard the supernatant, resuspend the cells with PBS buffer, and then use the CD34 magnetic separation kit and perform separation operations according to the instructions to collect CD34+ Cells and CD34 - cell.

[0122] ③ CD34 obtained in sampling step ② + Cells and CD34 - The cells were cultured in medium 5 for 7 days (new medium 5 was replaced every day). After the culture was completed, the cell morphology was observed under a microscope. CD34 + Exemplary photographs of cells are shown in Figure 2 Middle image of A, CD34 - Exemplary photographs of cells are shown in Figure 2 Right picture of A.

[0123] ④After completing step ③, two samples were taken from each culture system for immunofluorescence analysis (one group used CD144 antibody, CD45 antibody and RUNX1 antibody; the other group used CXCL12 antibody and LEPR antibody). Figure 2 B (CD34 in the above picture) + Samples were taken after cell culture. The picture below shows CD34 - Samples were taken after cell culture) and Figure 2 C (CD34 in the above figure) + Samples were taken after cell culture. The picture below shows CD34 - Samples were taken after cell culture). Due to the 7-day culture, hematopoietic stem cells / hematopoietic progenitor cells further differentiated into mature hematopoietic cells. CD34 + Cell differentiation mainly forms CD144 + cells (judged to be endothelial cells) and CD45 + RUNX1 + Cells (judged to be hematopoietic cells). CD34 - Cell differentiation mainly forms CXCL12 + LEPR + Cells (judged to be stromal cells). Endothelial cells, hematopoietic cells, and stromal cells are the three key cell types that make up bone marrow organoids.

[0124] Example 4: Optimization of cell composition in the second stage of culture and optimization of culture medium in the third stage of culture 1. After completing step 5 of Example 3, discard the supernatant, add TrypLE Select enzyme (0.3-0.5 mL per well), incubate for 3-5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate the digestion.

[0125] 2. After completing step 1, transfer the entire liquid phase system in the well to a centrifuge tube, centrifuge at 900 rpm for 4 minutes, then discard the supernatant, add PBS buffer to wash the cells, then aspirate and discard the supernatant, resuspend the cells with PBS buffer, and then use the CD34 magnetic separation kit and perform separation operations according to the instructions to collect CD34 + Cells and CD34 - cell.

[0126] 3. Optimization of cell ratio (1) Preparation of cell suspension Cell suspension 1 (8:2 ratio): CD34 + cells, CD34 obtained in step 2 - Mix the cells and medium 4 to make CD34 + The cell content is 0.8×10 5 viable cells / ml and CD34 - The cell content is 0.2×10 5 The cell suspension is 1.

[0127] Cell suspension 2 (5:5 ratio): CD34 + cells, CD34 obtained in step 2 - Mix the cells and medium 4 to make CD34 + The cell content is 0.5×10 5 viable cells / ml and CD34 - The cell content is 0.5×10 5 The cell suspension is composed of 2 living cells / ml.

[0128] Cell suspension 3 (2:8 ratio): CD34 + cells, CD34 obtained in step 2 - Mix the cells and medium 4 to make CD34 + The cell content is 0.2×10 5 viable cells / ml and CD34 - The cell content is 0.8×10 5 viable cells / ml, that is, cell suspension 3.

[0129] (2) Cultivating cell suspension 1 to prepare bone marrow organoids ① Cultivate the cell suspension 1 to form cell aggregates.

[0130] Take an Aggrewell plate and inoculate the cell suspension 1 prepared in step ① (1 mL per well), first incubate for half an hour, and then culture for 24 hours.

[0131] ②Preparation of cell aggregate / matrigel mixture After completing step ①, all cell aggregates (about 7,200) formed in one Angrewell plate were gently blown out and collected in a centrifuge tube pre-cooled in an ice bath. After centrifugation, the supernatant was discarded, the centrifuge tube was placed on ice again, 1 mL of Matrigel was added, and the mixture was mixed to obtain a cell aggregate / matrigel mixture.

[0132] ③Preparation of organoids Take a 24-well cell culture plate, add the cell aggregate / matrigel mixture prepared in step ② (50μl / well), culture for 30 minutes, then slowly add culture medium 6 (0.6ml / well) along the edge, and culture for 192 hours. During the culture process, add culture medium 6 (0.6ml / well) after 48 hours, discard the supernatant and add new culture medium 6 (0.6ml / well) after 96 hours, and add culture medium 6 (0.6ml / well) after 144 hours. After completing this step, the content in each well is one bone marrow organoid.

[0133] (3) Cultivating cell suspension 2 to prepare bone marrow organoids Replace cell suspension 1 with cell suspension 2. Other steps are the same as step (2).

[0134] (4) Cultivating cell suspension 3 to prepare bone marrow organoids Replace cell suspension 1 with cell suspension 3. Other steps are the same as step (2).

[0135] (5) Identification of cell survival rate and number of living cells After completing step (2) or step (3) or step (4), dissociate the organoids and prepare a single cell suspension, then count the total number of cells and identify the cell viability, and calculate the number of live cells. The results of cell viability are shown in Figure 3 The left figure (statistical results of 3 repeated samples) shows that there is no significant difference in the cell survival rate of bone marrow organoids obtained by inoculating the three cell suspensions. Taking the number of viable cells in the bone marrow organoids obtained by inoculating cell suspension 2 as a reference, the relative values ​​of the number of viable cells in the bone marrow organoids obtained by inoculating the three cell suspensions are shown in Figure 3 Right figure (statistical results of 3 repeated samples).

[0136] (6) Identification of cell ratios After completing step (2) or step (3) or step (4), dissociate the organoids and prepare a single cell suspension, then take samples and use flow cytometry to perform cell ratio analysis (the antibodies used in flow cytometry are: PE-labeled CD144 antibody, PEcy7-labeled CD34 antibody, APC-labeled LEPR antibody, BV786-labeled CD45 antibody). According to the output results of flow cytometry, count the CD45 +Cells (judged to be hematopoietic cells), CD45 - CD144 - LEPR + Cells (judged to be stromal cells), CD45 - CD144 + cells (judged to be endothelial cells) and CD45 + CD34 + The percentage of cells (judged to be hematopoietic stem cells / hematopoietic progenitor cells) in the total number of living cells is shown in Figure 4 The above figure (statistical results of 3 repeated samples). The number of various cells was calculated based on the number of living cells and the percentage of various cells. The results are shown in Figure 4 The following figure shows the statistical results of 3 repeated samples.

[0137] The results showed that CD34 + Cells and CD34 - The cell number ratio has an important influence on the final formation of bone marrow organoids. + When the cell ratio is too high, hematopoietic cells and endothelial cells in bone marrow organoids are dominant, but stromal cells are too few. Stromal cells, as important supporting cells of bone marrow organoids, can secrete a variety of cytokines and extracellular matrix components to form a microenvironment support for hematopoietic and endothelial cells. - When the cell ratio is too high, the proportion of stromal cells in bone marrow organoids is too high. Since the proliferation ability of these cells is stronger than that of endothelial cells and blood cells, they consume a large amount of nutrients, inhibit the differentiation of endothelial cells and hematopoietic cells, and cannot output enough hematopoietic cells. + Cells and CD34 - The cells are mixed in a 5:5 ratio (i.e., a 1:1 ratio), and the resulting bone marrow organoids have a high cell viability, a large total cell number, and the ratio of the three structural cells, endothelial cells, hematopoietic cells, and stromal cells, is the most balanced. The ratio of hematopoietic stem cells / hematopoietic progenitor cells is high, which is suitable for producing more mature blood cells.

[0138] 4. The CD34 obtained in step 2 + cells, CD34 obtained in step 2 - Mix the cells and medium 4 to make CD34 + The cell content is 0.5×10 5 viable cells / ml and CD34 - The cell content is 0.5×10 5 The cell suspension is composed of viable cells / ml.

[0139] 5. Take the Aggrewell plate and inoculate the cell suspension prepared in step 4 (1 mL per well). Incubate for half an hour and then incubate for 24 hours. Observe under a microscope before incubation and after 24 hours of incubation. See the exemplary photos for details. Figure 5 The left and middle pictures (the left picture is before static incubation, and the middle picture is after 24 hours of culture). It can be observed that the cells self-assemble into spherical cell aggregates in the wells of the Aggrewell plate. The diameters of 25 cell aggregates were randomly measured. Figure 5 The right picture shows that the size of the cells is 113.53±13.83μm, which is uniform and of appropriate size, and can ensure that they will be placed in the matrix gel later to maintain a good cell growth state and form a vascular network structure.

[0140] 6. After completing step 5, gently blow out all the cell aggregates (about 7,200) formed in one Angrewell plate and collect them in a centrifuge tube pre-cooled in an ice bath. After centrifugation, discard the supernatant, keep the centrifuge tube on ice, add 1 mL of Matrigel, mix well, and obtain a cell aggregate / matrigel mixture.

[0141] 7. Optimization of culture medium The test culture medium was medium 5 or medium 6.

[0142] Take a 24-well cell culture plate, add the cell aggregate / matrigel mixture prepared in step 6 (50 μl / well), culture for 30 minutes, then slowly add the test medium (0.6 ml / well) along the edge, and culture for 192 hours. During the culture process, add the test medium (0.6 ml / well) after 48 hours, discard the supernatant and add new test medium (0.6 ml / well) after 96 hours, and add the test medium (0.6 ml / well) after 144 hours.

[0143] After 48 hours of culture, the samples were observed under a microscope. See the photo for an example. Figure 6 The left picture (the right side is a partial enlargement of the left side). The number of buds of 100 cell aggregates was randomly counted, and the results are shown in Figure 6 After 192 hours of culture, immunofluorescence staining was performed (using CD144 antibody, CD144 is an endothelial cell marker protein). See the exemplary photo for Figure 7. The results showed that the cells in the cell aggregates gradually sprouted and formed a vascular network structure in the matrix gel. Compared with culture medium 5, the cell aggregates using culture medium 6 sprouted more and formed a denser endothelial cell network. The vascular niche structure is the main site for bone marrow hematopoiesis. In this structure, endothelial cells form a sinusoidal structure with gaps, where hematopoietic stem cells can proliferate and differentiate to form blood cells and release them into the peripheral blood. Compared with culture medium 5, culture medium 6 can better simulate the structure of the bone marrow vascular niche in vitro.

[0144] After 192 hours of culture, samples were taken for immunofluorescence staining (using CD45 antibodies, CD271 antibodies, and CD144 antibodies). Figure 8 Cell aggregates can differentiate in Matrigel to produce CD45 + Cells (judged to be hematopoietic cells), CD144 + Cells (judged to be endothelial cells) and CD271 + Cells (judged to be stromal cells) indicate that bone marrow organoids with hematopoietic function have been successfully constructed. Compared with medium 5, the endothelial cell network structure of bone marrow organoids formed using medium 6 is richer and more complete, and the number of hematopoietic cells formed in the endothelial cell network is greater, indicating that it has a stronger bone marrow hematopoietic output capacity.

[0145] After 192 hours of culture, samples were taken and flow cytometry was used to analyze the cell ratio (the antibodies used in flow cytometry were: CD43 antibody labeled with BV421, CD44 antibody labeled with PE, and CD34 antibody labeled with PEcy7). An exemplary image of flow cytometry output is shown in Fig. 9 . Extract CD34 from the output image + The cells and their CD43 signal intensity and CD44 signal intensity are shown in the exemplary pictures. Fig. 9 . Statistics CD34 + CD43 + CD44 + The ratio of the number of cells (judged to be hematopoietic stem cells / hematopoietic progenitor cells) to the total number of living cells. The results are shown in Fig. 9 (The average value of five repeated samples was calculated). Compared with culture medium 5, the ratio of hematopoietic stem cells / hematopoietic progenitor cells in the bone marrow organoids formed by culture medium 6 treatment was significantly increased, thus having stronger hematopoietic colony formation ability and proliferation potential. Therefore, compared with culture medium 5, the bone marrow organoids produced by culture medium 6 treatment have stronger hematopoietic cell output ability and a longer maintenance period of hematopoietic cell output.

[0146] After 192 hours of culture, the suspended cells in the wells cultured with medium 6 were collected and subjected to Wright-Giemsa staining and immunofluorescence staining (using CD41 antibody, CD14 antibody, CD71 antibody, CD66b antibody and CD3 antibody). An exemplary photograph of Wright-Giemsa staining is shown in Fig.10 A. The results showed that the suspended cells had typical morphological characteristics of granulocytes, erythroid cells, lymphocytes, monocytes and megakaryocytes. An exemplary photo of immunofluorescence staining is shown in Fig.10 B. The results showed that CD41 + Cells (judged to be megakaryocytes), CD14 + Cells (judged to be monocytes), CD71 + Cells (identified as erythroid cells), CD66b + cells (judged to be granulocytes) and CD3 + cells (judged to be T lymphocytes), demonstrating that the differentiated cells produced by bone marrow organoids do have multi-lineage blood cell phenotypic characteristics.

[0147] Example 5: Second stage of cultivation The purpose of the second stage of culture is to prepare cell population 2 by CD34 sorting, culture cell population 2 into cell aggregates, and prepare a cell aggregate / matrigel mixture.

[0148] 1. After completing step 5 of Example 3, discard the supernatant, add TrypLE Select enzyme (0.3-0.5 mL per well), incubate for 3-5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate the digestion.

[0149] 2. After completing step 1, transfer the entire liquid phase system in the well to a centrifuge tube, centrifuge at 900 rpm for 4 minutes, then discard the supernatant, add PBS buffer to wash the cells, then aspirate and discard the supernatant, resuspend the cells with PBS buffer, and then use the CD34 magnetic separation kit and perform separation operations according to the instructions to collect CD34 + Cells and CD34 - cell.

[0150] 3. The CD34 obtained in step 2 + cells, CD34 obtained in step 2 - Mix the cells and medium 4 to make CD34 + The cell content is 0.5×10 5 viable cells / ml and CD34 - The cell content is 0.5×10 5 The cell suspension is composed of viable cells / ml.

[0151] 4. Take the Aggrewell plate and inoculate the cell suspension prepared in step 3 (1 mL per well). Incubate for half an hour and then culture for 24 hours (in actual operation, 18-24 hours is acceptable).

[0152] 5. After completing step 4, gently blow out all the cell aggregates (about 7,200) formed in one Angrewell plate and collect them in a centrifuge tube pre-cooled in an ice bath. After centrifugation, aspirate and discard the supernatant. Keep the centrifuge tube on ice and add 0.5 mL of Matrigel. Mix well to obtain a cell aggregate / matrigel mixture.

[0153] Example 6: Third stage cultivation and identification The purpose of the third stage culture is to prepare bone marrow organoids using the cell aggregate / matrigel mixture and vascular endothelial cells.

[0154] The device with bone marrow-blood barrier used in this embodiment is a high-throughput barrier function organ chip of Beijing Daxiang Technology Co., Ltd., model IBAC M1, 24 flux (i.e., it has 24 unit structures and can prepare 24 organ models). The chip is referred to as IBAC M1 chip. Each unit structure is composed of a cavity (orgamoid chamber) for accommodating simulated bone marrow and a channel (perfusion channel) for accommodating simulated blood vessels. A sample loading hole (reservior) is provided at each end of the channel. The intersection of the cavity and the channel has a bone marrow-blood barrier (i.e., a polycarbonate membrane with a pore size of 8µm). The orgamoid chamber is a cavity with an open top, and samples are loaded directly from the top. The perfusion channel is loaded through the sample loading hole.

[0155] 1. Coating Take the IBAC M1 chip, add collagen working solution to the orgamoid chamber and perfusion channel (12 µL collagen working solution to the orgamoid chamber, 100 µL collagen working solution to the perfusion channel), let it stand at 37°C for 1 hour, then discard the liquid and wash with PBS buffer.

[0156] Preparation of collagen working solution: Mix 1 volume of collagen solution with 20 volumes of PBS buffer. Collagen solution (CellAdhere™ Type I Collagen, Bovine, Solution): STEMCELL, product catalog number 07001.

[0157] 2. After completing step 1, the PBS buffer in the orgamoid chamber was discarded, and then 10 μL of the cell aggregate / matrigel mixture prepared in step 5 of Example 5 was added to the orgamoid chamber, and the mixture was allowed to stand at 37°C for 1 hour. Then, 100 μL of culture medium 6 was added to the orgamoid chamber; then, the PBS buffer in the perfusion channel was discarded, and 200 μL of culture medium 6 was added to the perfusion channel; then, the IBAC M1 chip was placed on a shaker (the inclination angle was set to 30° and the speed was set to 3-5 cycles / min) and cultured for 96 hours. During the culture process, new culture medium 6 was replaced after 48 hours (both in the orgamoid chamber and the perfusion channel).

[0158] 3. After completing step 2, discard the liquid in the orgamoid chamber and perfusion channel, add 20 μL of HUVEC cell suspension to the perfusion channel, and after slight shaking, quickly flip the IBAC M1 chip 180° and invert it for 1 hour. Then, place the IBAC M1 chip upright, and then add 100 μL of culture medium 6 to the orgamoid chamber and 200 μl of EGM2 culture medium to the perfusion channel, and culture for 24 hours.

[0159] Preparation of HUVEC cell suspension: Resuspend the HUVEC cells digested with TrypLE Select enzyme in EGM2 medium to a cell concentration of (1-3) × 10 6 viable cells / mL.

[0160] 4. After completing step 3, place the IBAC M1 chip on a shaker (tilt angle set to 30°, speed set to 3-5 cycles / min) and culture for 96 hours. During the culture process, replace the culture medium after 48 hours (replace culture medium 6 in the orgamoid chamber and replace EGM2 culture medium in the perfusion channel).

[0161] After completing step 4, bone marrow organoids are obtained.

[0162] Schematic diagram of bone marrow organoids can be found in Fig.11The orgamoid chamber contains simulated bone marrow formed by a mixture of cell aggregates / matrigel, and the perfusion channel contains simulated blood vessels formed by HUVEC cells (HUVEC cells adhere to the inner wall of the perfusion channel to form simulated blood vessels). The intersection of the orgamoid chamber and the perfusion channel is the bone marrow blood barrier (a polycarbonate membrane with a pore size of 8µm). By swinging up and down at a fixed angle in a shaker, the culture medium flows back and forth in the perfusion channel to simulate the blood flow state in the bone marrow, and can reflect the physiological state of blood and bone marrow cytokines, small molecule compounds and other substances communicating and exchanging through the endothelial cell barrier under specific physiological conditions.

[0163] 5. After completing step 4, perform immunofluorescence staining (using CD45 antibody, CD144 antibody and CD271 antibody). See the exemplary photos for Fig.12 The three main cell types in bone marrow organoids are CD45 + Cells (judged to be hematopoietic cells), CD144 + Cells (judged to be endothelial cells) and CD271 + Cells (judged to be matrix cells). In the orgamoid chamber, endothelial cells form a network-like vascular network structure, and the three types of cells are intertwined in the matrix gel, reproducing the natural positional relationship of the three types of cells in natural bone marrow. In the perfusion channel, the densely arranged single-layer cell layer formed by endothelial cells can be mainly observed, simulating the barrier formed by the vascular endothelium.

[0164] Example 7: Application of bone marrow organoids in screening of drugs against radiation-induced hematopoietic damage 1. After completing step 4 of Example 6, take the IBAC M1 chip, discard the liquid in the orgamoid chamber and the liquid in the perfusion channel, add EGM2 medium to the orgamoid chamber, add EGM2 medium containing the test drug to the perfusion channel, and culture for 2 hours. When the test drug is thrombopoietin, the drug concentration is 50 ng / ml. When the test drug is romistine, the drug concentration is 50 ng / ml. When the test drug is eltrombopag, the drug concentration is 1 µM.

[0165] 2. After completing step 1, place the IBAC M1 chip that has completed step 1 in an X-ray irradiator and irradiate it with a dose of 4 Gy.

[0166] 3. After completing step 2, culture for 24 hours.

[0167] 4. After completing step 3, dissociate the organoids and prepare a single-cell suspension, then take a sample to detect the number of live cells.

[0168] 5. After completing step 3, dissociate the organoids and prepare a single cell suspension, and then use flow cytometry to detect the relative fluorescence intensity of reactive oxygen species and the percentage of activated Caspase3-positive cells in the total number of cells. The fluorescent probe used to detect the relative fluorescence intensity of reactive oxygen species is CellROX™ Green reagent, Thermo Fisher Scientific, product catalog number C10444. The antibody used to detect activated Caspase3-positive cells is Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Alexa Fluor ® 488 Conjugate), CST Company, catalog number 9603S.

[0169] A control treatment group without step 2 and without adding the test drug is set up, which is called the non-irradiated group.

[0170] A control treatment without adding the test drug is set up, called the irradiation group.

[0171] Results Fig.13 .

[0172] Note: The method for dissociating organoids and preparing single cell suspension in the embodiment is as follows: Take the container (24-well cell culture plate or chip) containing the organoids, discard the liquid in the wells or orgamoid chamber, and add PBS buffer to rinse; then, add pre-cooled Recovery solution (500µl / well or 100µl / orgamoid chamber), and then place the container on ice for 20 minutes; then transfer the contents of the wells or orgamoid chamber to a centrifuge tube, centrifuge at 4℃ and 1200rpm for 5 minutes, aspirate the supernatant, and the remaining cell pellet in the centrifuge tube; then, add 200µl of 37℃ preheated digestion enzyme solution to the centrifuge tube and resuspend the cell pellet, incubate at 37℃ for 10-15min (invert the centrifuge tube upside down every 5min to resuspend the cell pellet), then gently pipette with a pipette tip and add 2ml of PBS buffer containing 10% FBS to stop digestion; then, centrifuge at 1200rpm for 5min, aspirate the supernatant, add PBS buffer to wash, and then resuspend with PBS buffer to obtain a single cell suspension.

[0173] Preparation of digestive enzyme solution: Mix 1.6mL Liberase™ TH solution, 3mL Dispase II solution, and 500µL DNase I solution, then add D-PBS buffer to make the total volume of the system reach 10mL.

[0174] Recovery solution (Products for Cell Release and Recovery from ECMs): Corning, product catalog number 354253. Liberase™ TH: MERCK, product catalog number 5401135001. Dissolve 5 mg of Liberase™ TH in 2 ml of D-PBS buffer to obtain Liberase™ TH solution. DNaseI Solution (1 mg / mL): Stemcell, product catalog number 07900. Dispase II: MERCK, product catalog number D4693. Dissolve Dispase II in D-PBS buffer to a concentration of 10 mg / mL to obtain Dispase II solution. D-PBS buffer (without Ca ++ and Mg ++ ): MERCK Company, product catalog number is BSS-1006.

[0175] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for preparing bone marrow organoids, comprising the following steps: (1) inducing embryonic stem cells to differentiate into a cell population containing stromal cells, endothelial cells and hematopoietic stem and progenitor cells, named as cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) dividing the cell population 1 into a CD34-positive cell subpopulation and a CD34-negative cell subpopulation by sorting, and then mixing the CD34-positive cell subpopulation and the CD34-negative cell subpopulation at a cell number ratio of 1:1 to obtain a cell population, which is named cell population 2; (3) culturing the cell population 2 into cell aggregates, and then mixing with matrix gel to obtain a mixture; (4) culturing the mixture and vascular endothelial cells in a device having a bone marrow-blood barrier to obtain bone marrow organoids; The device with bone marrow blood barrier has more than one unit structure; the unit structure is composed of a cavity for accommodating simulated bone marrow and a channel for accommodating simulated blood vessels, the end of the channel is provided with a sample addition hole, and the intersection of the cavity and the channel has a bone marrow blood barrier; the bone marrow blood barrier is a membrane with a pore size of 5µm-10µm; The method of culturing the mixture and vascular endothelial cells in a device having a bone marrow-blood barrier is as follows: culturing the mixture in the cavity to form simulated bone marrow, and culturing the vascular endothelial cells in the channel to form simulated blood vessels; Cultivating the mixture using medium 6; The additives required to prepare each liter of the culture medium 6 are as follows: 50-100µg SCF, 50-100µg TPO, 50-100µg IL-3, 50-100µg Flt31, 20-50µg GM-CSF, 1-5µmol Y-27632, 2-10µmol SB431542, 1-5µmol Forskolin, 0.3-2µmol PGE2 and 1-5µmol FK506.

2. A method for preparing bone marrow organoids, comprising the following steps: (1) inducing embryonic stem cells to differentiate into a cell population containing stromal cells, endothelial cells and hematopoietic stem and progenitor cells, named as cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) dividing the cell population 1 into a CD34-positive cell subpopulation and a CD34-negative cell subpopulation by sorting, and then mixing the CD34-positive cell subpopulation and the CD34-negative cell subpopulation at a cell number ratio of 1:1 to obtain a cell population, which is named cell population 2; (3) culturing the cell population 2 into cell aggregates, and then mixing with matrix gel to obtain a mixture; (4) culturing the mixture obtained in step (3) using culture medium 6 to obtain bone marrow organoids; The additives required to prepare each liter of the culture medium 6 are as follows: 50-100µg SCF, 50-100µg TPO, 50-100µg IL-3, 50-100µg Flt31, 20-50µg GM-CSF, 1-5µmol Y-27632, 2-10µmol SB431542, 1-5µmol Forskolin, 0.3-2µmol PGE2 and 1-5µmol FK506.

3. Bone marrow organoids prepared by the method of claim 1 or 2.

4. A composition consisting of the following ten compounds: SCF, TPO, IL-3, Flt3l, GM-CSF, Y-27632, SB431542, Forskolin, PGE2 and FK506; The ratio of the ten compounds is as follows: 50-100µg SCF: 50-100µg TPO: 50-100µg IL-3: 50-100µg Flt3l: 20-50µg GM-CSF: 1-5µmol Y-27632: 2-10µmol SB431542: 1-5µmol Forskolin: 0.3-2µmol PGE2: 1-5μmolFK506; The function of the composition is to promote cell aggregates to form bone marrow organoids.

5. A culture medium comprising the composition according to claim 4; the function of the culture medium is to promote cell aggregates to form bone marrow organoids.

6. Use of the composition of claim 4 or the culture medium of claim 5 in preparing a kit; the functions of the kit are as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

7. A kit comprising the composition of claim 4 or the culture medium of claim 5; the functions of the kit are as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

8. The use of the kit according to claim 7, which is as follows (a) or (b): (a) Promoting cell aggregates to form bone marrow organoids; (b) Preparation of bone marrow organoids.

9. A compound combination consisting of the following five compounds: Y-27632, SB431542, Forskolin, PGE2 and FK506; The five compounds are as follows: 1-5μmol Y-27632: 2-10μmol SB431542: 1-5μmol Forskolin: 0.3-2μmol PGE2: 1-5μmol FK506; The function of the compound combination is to promote the formation of hematopoietic stem and progenitor cells and the formation of endothelial cell network structures when the cell aggregates are cultured into bone marrow organoids; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

10. The following application of the compound combination according to claim 9: when culturing cell aggregates into bone marrow organoids, promoting the formation of hematopoietic stem and progenitor cells and promoting the formation of endothelial cell network structures; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

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