Methods for preparing bone marrow organoids, compositions and culture media used therefor

Abstract

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

Description

Technical Field

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

[0002] Organoids technology is a new type of in vitro cell culture model that has emerged in recent years. This technology uses stem cells or progenitor cells from multiple sources, such as pluripotent stem cells (e.g., embryonic stem cells and induced pluripotent stem cells), tissue adult stem cells, and tumor stem cells, to proliferate, differentiate, and self-assemble in semi-solid culture media such as Matrigel, collagen, and hydrogel, thereby forming cell aggregates with specific tissue structure characteristics. Organoids are highly similar to the tissues from which they are derived in terms of cell composition type, tissue structure, and physiological function, and can to a certain extent simulate and reproduce the complex biological processes of tissues and organs in vitro. On this basis, if the organoid preparation technology is combined with an organ-on-a-chip culture device with microfluidic functions, a microphysiological system with a more complex culture structure and more biophysiological functions can be further constructed. With the help of its internal environmental sensing and 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 during the realization of tissue physiological functions, such as substance diffusion, fluid shear stress, and physiological signal stimulation feedback. Currently, organoid and microphysiological system in vitro construction technologies for various human tissues and organs, such as the small intestine, kidney, brain, and lung, have been successively developed and widely used in research in the fields of organ differentiation and development, disease injury model construction, new drug screening and safety evaluation, and personalized medicine, showing good application prospects and development trends.

[0003] Although significant progress has been made in the culturing methods of organoids and microphysiological systems for solid tissues and organs, the development of construction techniques for bone marrow hematopoietic system organoids and microphysiological systems still faces many challenges. Two major difficulties in constructing bone marrow organoids and microphysiological systems lie in mimicking the complex and orderly physiological structure and unique and rich physiological functions of bone marrow in vitro. On the one hand, different from solid tissues that usually perform a single physiological function, bone marrow performs complex and diverse physiological functions. As the core site for maintaining human hematopoiesis and immune functions, bone marrow continuously generates various types of blood cells throughout the life cycle to meet the metabolic and renewal needs of various mature blood cells in peripheral blood; and can quickly sense stress signals and make adaptive responses after pathological changes such as infection and injury in the body. On the other hand, the structural basis for bone marrow to achieve long-term and orderly hematopoiesis function is its complex three-dimensional microenvironment. In bone marrow, hematopoietic stem cells and their differentiated hematopoietic progenitor cells and hematopoietic precursor cells (as the "seed cells" for maintaining hematopoiesis 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, fluid force, etc.) to jointly form the structural and functional units of the bone marrow hematopoietic system. The bone marrow microenvironment precisely regulates the self-renewal and lineage differentiation fates of hematopoietic stem cells through cell-cell communication and distal signal transduction. It is precisely because of this complexity of structure and function that it is difficult to reproduce the unique phenotypic functions of bone marrow through co-culture of one or several types of 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, as well as the compositions and culture media used therefor.

[0005] The present invention provides a method for preparing bone marrow organoids (named Method I), comprising the following steps:

[0006] (1) Inducing the differentiation of embryonic stem cells to form a cell population containing stromal cells, endothelial cells, and hematopoietic stem and progenitor cells, named cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells;

[0007] (2) Sorting the cell population 1 into a CD34-positive cell subset (CD34 + cell subset) and a CD34-negative cell subset (CD34 - cell subset), and then mixing the CD34-positive cell subset and the CD34-negative cell subset according to a cell number ratio of 1:1 to obtain a cell population, named cell population 2;

[0008] (3) Culturing the cell population 2 into cell aggregates, and then mixing with Matrigel to obtain a mixture;

[0009] (4) Culturing the mixture and vascular endothelial cells in a device with a bone marrow-blood barrier to obtain bone marrow organoids;

[0010] The device with a bone marrow-blood barrier has more than one unit structure; the unit structure consists of a cavity (orgamoid chamber) for accommodating simulated bone marrow and a channel (perfusion channel) for accommodating simulated blood vessels, a sampling hole (reservior) is provided at the end of the channel, and a bone marrow-blood barrier is present at the intersection of the cavity and the channel; the bone marrow-blood barrier is a membrane with a pore size of 5 µm - 10 µm;

[0011] 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.

[0012] Specifically, medium 6 is used to culture the mixture.

[0013] Specifically, the bone marrow-blood barrier is a polycarbonate membrane with a pore size of 5 µm - 10 µm.

[0014] Specifically, "with a pore size of 5 µm - 10 µm" can be a pore size of 8 µm or a pore size of 10 µm.

[0015] The present invention also provides a method for preparing bone marrow organoids (named Method II), which includes the following steps:

[0016] (1) Inducing the differentiation of embryonic stem cells to form a cell population containing stromal cells, endothelial cells, and hematopoietic stem and progenitor cells, named cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells;

[0017] (2) Sorting the cell population 1 into a CD34-positive cell subset (CD34 + cell subset) and a CD34-negative cell subset (CD34 - cell subset), and then mixing the CD34-positive cell subset and the CD34-negative cell subset according to a cell number ratio of 1:1 to obtain a cell population, named cell population 2;

[0018] (3) Culturing the cell population 2 into cell aggregates, and then mixing with Matrigel to obtain a mixture;

[0019] (4) Culturing the mixture obtained in step (3) with medium 6 to obtain bone marrow organoids.

[0020] Specifically, the method for preparing cell population 1 includes the following steps:

[0021] ①Collect the digested embryonic stem cells, resuspend the cell pellet with Medium 1 to obtain a cell suspension.

[0022] ②Take a 6-well plate coated with Matrigel, inoculate the cell suspension prepared in step ①, culture for 24 hours, then aspirate the supernatant, add PBS buffer to wash the cells, and then aspirate the supernatant.

[0023] ③Take the 6-well plate completed in step ②, add Medium 2, culture for 48 hours, then aspirate the supernatant, add PBS buffer to wash the cells, and then aspirate the supernatant.

[0024] ④Take the 6-well plate completed in step ③, add Medium 3, culture for 72 hours; during the culture process, replace the fresh Medium 3 every 24 hours.

[0025] Specifically, the method for preparing Cell Population 1 includes the following steps:

[0026] ①Collect the digested embryonic stem cells, resuspend the cell pellet with Medium 1 to obtain a cell suspension (0.4×10 5 viable cells / mL).

[0027] ②Take a 6-well plate coated with Matrigel, inoculate the cell suspension prepared in step ① (3 mL per well), culture for 24 hours, then aspirate the supernatant, add PBS buffer to wash the cells, and then aspirate the supernatant.

[0028] ③Take the 6-well plate completed in 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.

[0029] ④Take the 6-well plate completed in step ③, add Medium 3 (3 mL per well), culture for 72 hours; during the culture process, replace the fresh Medium 3 every 24 hours.

[0030] Specifically, the method for preparing Cell Population 2 includes the following steps:

[0031] ①Digest the Cell Population 1 and then perform sorting operation using a CD34 magnetic sorting kit to separately collect CD34 + cells and CD34 - cells.

[0032] ②Mix the CD34 + cells obtained in step ①, the CD34 - cells obtained in step ① and Medium 4, which is the cell suspension containing Cell Population 2.

[0033] Specifically, the method for preparing Cell Population 2 includes the following steps:

[0034] ① Digest the cell population 1 and then perform sorting operation using a CD34 magnetic sorting kit to separately collect CD34 + cells and CD34 - cells;

[0035] ② Mix the CD34 + cells obtained in step ①, the CD34 - cells obtained in step ① and the culture medium 4 so that the content of CD34 + cells is 0.5×10 5 viable cells / ml and the content of CD34 - cells is 0.5×10 5 viable cells / ml, thus obtaining a cell suspension containing cell population 2.

[0036] Specifically, the method for culturing the cell population 2 into cell aggregates includes the following steps: Take an Aggrewell plate, inoculate the cell suspension containing cell population 2 (1 mL per well), first incubate statically for half an hour, and then culture for 18 - 24 hours.

[0037] Specifically, the method for "mixing with Matrigel to obtain a mixture" includes the following steps: Mix 6500 - 7500 cell aggregates (specifically, 7200 cell aggregates) with 0.5 - 1 mL of Matrigel to obtain a mixture (also known as cell aggregate / Matrigel mixture).

[0038] Specifically, the method for "mixing with Matrigel to obtain a mixture" includes the following steps: Blow out and collect all the cell aggregates (about 7200) formed in one Angrewell plate into 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 mixture (also known as cell aggregate / Matrigel mixture).

[0039] Specifically, step (4) of method Ⅰ successively includes the following steps:

[0040] ① Collagen - coat the cavity and channels of the device with a bone marrow - blood barrier;

[0041] ② Add the cell aggregate / Matrigel mixture into the cavity, incubate, and then add the culture medium 6; then place it on a shaker (with an inclination angle set at 30° and a rotation speed set at 3 - 5 cycles per minute) and culture for 96 hours;

[0042] ③Discard the liquid in the cavity and the channel, add vascular endothelial cells to the channel, incubate in an inverted position, then add medium 6 to the cavity and EGM2 medium to the channel, culture for 24 hours, and then place it on a shaker (the inclination angle is set to 30°, and the rotation speed is set to 3 - 5 cycles / minute), and culture for 96 hours.

[0043] Specifically, step (4) of method I successively includes the following steps:

[0044] ①Coat the cavity and the channel of the device with a blood - brain barrier with collagen, and then wash with PBS buffer solution;

[0045] ②Aspirate and discard the PBS buffer solution in the cavity, then add 10 μL of cell aggregate / matrix gel mixture to the cavity, incubate at 37 °C for 1 hour, and then add 100 μL of medium 6 to the cavity; then, aspirate and discard the PBS buffer solution in the channel, add 200 μL of medium 6 to the channel; then, place the device on a shaker (the inclination angle is set to 30°, and the rotation speed is set to 3 - 5 cycles / minute), and culture for 96 hours;

[0046] ③After completing step ②, aspirate and discard the liquid in the cavity and the channel, add 20 μL of HUVEC cell suspension to the channel, incubate in an inverted position for 1 hour, then place it upright, add 100 μL of medium 6 to the cavity, add 200 μl of EGM2 medium to the channel, and culture for 24 hours;

[0047] ④After completing step ③, place the device on a shaker (the inclination angle is set to 30°, and the rotation speed is set to 3 - 5 cycles / minute), and culture for 96 hours.

[0048] Method for preparing HUVEC cell suspension: Resuspend the digested HUVEC cells in EGM2 medium so that the cell concentration is (1 - 3)×10 6 viable cells / mL.

[0049] Specifically, the device with a blood - brain barrier is a commercially available barrier - function organ chip.

[0050] Specifically, the device with a blood - brain barrier is a high - throughput barrier - function organ chip of Beijing Daxiang Technology Co., Ltd.

[0051] Specifically, the device with a blood - brain barrier is the IBAC M1 chip of Beijing Daxiang Technology Co., Ltd.

[0052] Step (4) of the above-described Method II may specifically be as follows: Take a cell culture plate, add the cell aggregate / matrix gel mixture, incubate for 30 min, and then add Medium 6 and incubate for 192 hours. During the incubation process, add Medium 6 after 48 hours, aspirate the supernatant and add Medium 6 after 96 hours, and add Medium 6 after 144 hours.

[0053] Step (4) of the above-described Method II may specifically be as follows: Take a 24-well cell culture plate, add the cell aggregate / matrix gel mixture (50 μl / well), incubate for 30 min, and then add Medium 6 (0.6 ml / well) and incubate for 192 hours. During the incubation process, add Medium 6 (0.6 ml / well) after 48 hours, aspirate the supernatant and add Medium 6 (0.6 ml / well) after 96 hours, and add Medium 6 (0.6 ml / well) after 144 hours.

[0054] The bone marrow organoids prepared by any of the above-described methods also fall within the protection scope of the present invention.

[0055] The present invention also protects a composition, which is composed of the following ten compounds: SCF, TPO, IL-3, Flt3l, GM-CSF, Y-27632, SB431542, Forskolin, PGE2, and FK506;

[0056] The ratios of the ten compounds are as follows:

[0057] 50 - 100 μg SCF: 50 - 100 μg TPO: 50 - 100 μg IL-3: 50 - 100 μg Flt3l: 20 - 50 μg GM-CSF: 1 - 5 μmol Y-27632: 2 - 10 μmol SB431542: 1 - 5 μmol Forskolin: 0.3 - 2 μmol PGE2: 1 - 5 μmol FK506;

[0058] The function of the above-described composition is: to promote the formation of bone marrow organoids from cell aggregates.

[0059] The present invention also protects a culture medium, which contains the above-described composition; the function of the culture medium is: to promote the formation of bone marrow organoids from cell aggregates.

[0060] Specifically, the culture medium is Medium 6.

[0061] The present invention also protects the use of the above-described composition or the above-described culture medium in the preparation of a kit; the functions of the kit are as follows (a) or (b):

[0062] (a) To promote the formation of bone marrow organoids from cell aggregates;

[0063] (b) To prepare bone marrow organoids.

[0064] The present invention also protects a kit, which comprises the composition or the culture medium; the functions of the kit are as follows (a) or (b):

[0065] (a) Promote the formation of bone marrow organoids from cell aggregates;

[0066] (b) Prepare bone marrow organoids.

[0067] Specifically, the kit further comprises culture medium 1 and / or culture medium 2 and / or culture medium 3 and / or culture medium 4.

[0068] Specifically, the kit further comprises EGM2 culture medium.

[0069] The present invention also protects the application of the kit, which is as follows (a) or (b):

[0070] (a) Promote the formation of bone marrow organoids from cell aggregates;

[0071] (b) Prepare bone marrow organoids.

[0072] The present invention also protects a compound combination, which is composed of the following five compounds: Y-27632, SB431542, Forskolin, PGE2 and FK506;

[0073] The ratio of the five compounds is as follows:

[0074] 1 - 5 μmol of Y-27632: 2 - 10 μmol of SB431542: 1 - 5 μmol of Forskolin: 0.3 - 2 μmol of PGE2: 1 - 5 μmol of FK506;

[0075] The function of the compound combination is: when culturing cell aggregates into bone marrow organoids, it promotes the formation of hematopoietic stem and progenitor cells and promotes the formation of endothelial cell network structures; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

[0076] The present invention also protects the following application of the compound combination: when culturing cell aggregates into bone marrow organoids, it promotes the formation of hematopoietic stem and progenitor cells and promotes the formation of endothelial cell network structures; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells.

[0077] Specifically, the cell aggregates are cell aggregates formed by a cell population differentiated from embryonic stem cells.

[0078] Specifically, the cell aggregates are cell aggregates formed by cell population 2.

[0079] The preparation method of cell population 2 is as described in any of the above.

[0080] Culture medium 1 is a culture medium named culture medium 1.

[0081] The additives required for preparing each liter of the culture medium 1 are as follows: 3-10 μmol Y-27632.

[0082] The additives required for preparing each liter of the culture medium 1 are as follows: 10 μmol Y-27632.

[0083] The culture medium 1 is composed of the additives and a basal medium.

[0084] Specifically, the basal medium is mTeSR1 medium.

[0085] Culture medium 2 is a culture medium named culture medium 2.

[0086] The additives required for preparing each liter of the culture medium 2 are as follows: 10-50 μg BMP-4, 10-50 μg Activin A, 5-30 μg bFGF, and 1-5 μmol (CHIR-99021)HCl.

[0087] The additives required for preparing each liter of the culture medium 2 are as follows: 25 μg BMP-4, 25 μg Activin A, 25 μg bFGF, and 2 μmol (CHIR-99021)HCl.

[0088] The culture medium 2 is composed of the additives and a basal medium.

[0089] Specifically, the basal medium is BEL medium.

[0090] Culture medium 3 is a culture medium named culture medium 3.

[0091] The additives required for preparing each liter of the culture medium 3 are as follows: 50-100 μg VEGF, 10-20 μg bFGF, and 2-5 μmol SB431542.

[0092] The additives required for preparing each liter of the culture medium 3 are as follows: 100 μg VEGF, 20 μg bFGF, and 5 μmol SB431542.

[0093] The culture medium 3 is composed of the additives and a basal medium.

[0094] Specifically, the basal medium is BEL medium.

[0095] Culture medium 4 is a culture medium named culture medium 4.

[0096] The additives required for preparing each liter of the medium 4 are as follows: 50 - 100 µg of VEGF, 10 - 20 µg of bFGF, 2 - 5 μmol of SB431542, and 2 - 10 μmol of Y-27632.

[0097] The additives required for preparing each liter of the medium 4 are as follows: 100 µg of VEGF, 20 µg of bFGF, 5 μmol of SB431542, and 10 μmol of Y-27632.

[0098] The medium 4 is composed of the additives and a basal medium.

[0099] Specifically, the basal medium is BEL medium.

[0100] The medium 6 is a medium named medium 6.

[0101] The additives required for preparing each liter of the medium 6 are as follows: 50 - 100 µg of SCF, 50 - 100 µg of TPO, 50 - 100 µg of IL-3, 50 - 100 µg of Flt3l, 20 - 50 µg of GM-CSF, 1 - 5 μmol of Y-27632, 2 - 10 μmol of SB431542, 1 - 5 μmol of Forskolin, 0.3 - 2 μmol of PGE2, and 1 - 5 μmol of FK506.

[0102] The additives required for preparing each liter of the medium 6 are as follows: 50 µg of SCF, 50 µg of TPO, 50 µg of IL-3, 50 µg of Flt3l, 20 µg of GM-CSF, 5 μmol of Y-27632, 10 μmol of SB431542, 2 μmol of Forskolin, 1 μmol of PGE2, and 2 μmol of FK506.

[0103] The medium 6 is composed of the additives and a basal medium.

[0104] Specifically, the basal medium is EGM2 medium.

[0105] Specifically, the bone marrow organoids are human bone marrow organoids.

[0106] Specifically, the embryonic stem cells are human embryonic stem cells or human embryonic stem cell lines.

[0107] Specifically, the embryonic stem cells are WA09 cells.

[0108] Specifically, the vascular endothelial cells are human vascular endothelial cells.

[0109] Specifically, the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC cells).

[0110] The term "organoid" refers to a three-dimensional tissue culture or mini-organoid 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 mimicking blood vessels and mimicking bone marrow.

[0111] The bone marrow organoids can be used in fields such as the study of organ differentiation and development mechanisms, the construction of disease injury models, the screening and safety evaluation of new drugs, and personalized medicine. Description of the Drawings

[0112] Figure 1 It is an exemplary atlas of flow cytometry in step 6 of Example 3.

[0113] Figure 2 Exemplary photos of the cell morphology under the microscope in steps 5 and 7 of Example 3, where bar represents 200 µm.

[0114] Figure 3 It is the result of cell viability and the relative value of the number of live cells in the bone marrow organoids in step 3 of Example 4.

[0115] Figure 4 It is the percentage of various cells and the number of various cells in step 3 of Example 4.

[0116] Figure 5 Exemplary photos observed under the microscope in step 5 of Example 4 (bar represents 50 µm) and the diameter of cell aggregates.

[0117] Figure 6 Exemplary photos observed under the microscope and the number of buds of cell aggregates in step 7 of Example 4.

[0118] Figure 7 Exemplary photos of immunofluorescence staining (using CD144 antibody) in step 7 of Example 4, where bar represents 100 µm.

[0119] Figure 8 Exemplary photos of immunofluorescence staining (using CD45 antibody, CD271 antibody and CD144 antibody) in step 7 of Example 4.

[0120] Figure 9 Exemplary pictures output by flow cytometry in step 7 of Example 4.

[0121] Figure 10Exemplary photographs of Wright-Giemsa staining and immunofluorescence staining (using CD41 antibody, CD14 antibody, CD71 antibody, CD66b antibody, and CD3 antibody).

[0122] Figure 11 Schematic diagram of the bone marrow organoid in Example 6.

[0123] Figure 12 Exemplary photograph of immunofluorescence staining (using CD45 antibody, CD144 antibody, and CD271 antibody) in Example 6, bar represents 30 µm.

[0124] Figure 13 Result graph in Example 7. Detailed implementation manners

[0125] The present invention will be further described in detail below in combination with the detailed implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0126] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, 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 cell number is counted using a cell counting plate, and the cell viability is identified using AO / PI staining. The total cell number multiplied by the cell viability is the number of live cells. Matrigel used for preparing the cell aggregate / matrix gel mixture, the full name is Corning ® Matrigel ® Growth Factor Reduced (GFR) Basement Membrane Matrix (phenol red-free, LDEV-free), Corning, product catalog number 356231. "Hematopoietic stem cells / hematopoietic progenitor cells" or "hematopoietic stem / progenitor cells" refers to "hematopoietic stem cells and / or hematopoietic progenitor cells".

[0127] 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 / Yi'aobang Co., Ltd., product catalog number PC.00241.

[0128] CD34 Magnetic Sorting Kit (CD34 MicroBead Kit): Miltenyi Biotec, catalog number 130-046-702.

[0129] Aggrewell Plate (AggreWell™800 24-well plate): STEMCELL Technologies. Each Aggrewell plate has 24 wells, and each well contains 300 micro-wells (which can form 300 spherical cell aggregates), so each Aggrewell plate forms approximately 7,200 cell aggregates.

[0130] Method for preparing 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 its volume with DMEM / F-12 medium to obtain the Matrigel working solution. The Matrigel used for preparing the Matrigel working solution is fully named Corning ® Matrigel ® Matrix (suitable for hESC, free of LDEV), Corning, catalog number 354277.

[0131] TrypLE Select Enzyme (TrypLE™ Select Enzyme, 1×): Thermo Fisher Scientific, catalog number 12563029, brand Gibco™. mTeSR1 Medium (mTeSR™1 Basal Medium): stemcell, catalog number 05850. DMEM / F-12 Medium: Thermo Fisher Scientific, catalog number 11330032, brand Gibco™.

[0132] EGM2 Medium (Lonza EGM-2 Endothelial Cell Medium, CC-3156 + CC-4176): Mix each component provided in Lonza EGM-2 BulletKit (CC-3156&CC-4176) according to the instructions to obtain EGM2 medium. The seller is Zeping Technology Company, catalog number CC-3162, brand (manufacturer) is Lonza.

[0133] 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 the hydrochloride salt of CHIR-99021 (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.

[0134] BMP-4 (Recombinant Human BMP-4 Protein): R&D, catalog number 314-BP, product description "human BMP-4 protein Ser293-Arg408", which is the amino acid residues at positions 293-408 in UniProtKB / Swiss-Prot: Q53XC5 (31-OCT-2006). Activin A (Recombinant Human / Mouse / Rat Activin A Protein): R&D, catalog number 338-AC, product description "Activin A protein Gly311-Ser426", which is the amino acid residues at positions 311 to 426 in UniProtKB / Swiss-Prot: P08476.2 (05-FEB-2025). VEGF (Recombinant Human VEGF 165 Protein): R&D, catalog number 293-VE, product description "human VEGF protein Ala27-Arg191", which is the amino acid residues at positions 27-191 in NCBI Reference Sequence: NP_001165097.1 (24-OCT-2024). bFGF (Human FGF-basic, PeproTech ®): Thermo Fisher Scientific Inc., catalog number AF-100-18C, brand Gibco™, amino acid residues 10 - 155 in NCBI Reference Sequence: NP_001348594.1 (29 - OCT - 2024). SCF (Recombinant Human SCF Protein): R&D Systems, 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 amino acid residues 26 - 189 in UniProtKB / Swiss-Prot: P21583.1 (05 - FEB - 2025). TPO (Recombinant Human Thrombopoietin): R&D Systems, catalog number 288-TPN, product description "human Thrombopoietin / Tpo protein Ser22 - Gly353", which is amino acid residues 22 - 353 in UniProtKB / Swiss-Prot: P40225.1 (05 - FEB - 2025). IL-3 (Recombinant Human IL-3 Protein): R&D Systems, catalog number 203-IL, product description "human IL-3 protein Ala20 - Phe152", which is amino acid residues 20 - 152 in GenBank: AAC08706.1 (31 - MAR - 1998). Flt3l (Recombinant Human Flt-3 Ligand Protein): R&D Systems, catalog number 308-FKHB, product description "human Flt-3 Ligand / FLT3L protein Thr27 - Pro185", which is amino acid residues 27 - 185 in GenBank: AAA17999.1 (11 - MAY - 1994). GM-CSF (Recombinant Human GM-CSF Protein): R&D Systems, catalog number 7954-GM, product description "human GM-CSF protein Ala18 - Glu144", which is amino acid residues 18 - 144 in UniProtKB / Swiss-Prot: P04141.1 (05 - FEB - 2025).

[0135] PE-Cy7-labeled CD34 antibody (BD Pharmingen™ PE-Cy™7 Mouse Anti-Human CD34): BD Biosciences, catalog number 560710. BV421-labeled CD43 antibody (BD Horizon™ BV421 Mouse Anti-Human CD43): BD Biosciences, catalog number 562916. PerCP-Cy5.5-labeled CD144 antibody (BD Pharmingen™ PerCP-Cy™5.5 Mouse Anti-Human CD144): BD Biosciences, catalog number 561566. PE-labeled CD140a antibody (BD Pharmingen™ PE Mouse Anti-Human CD140a): BD Biosciences, catalog number 556002. APC-labeled LEPR antibody (BD Pharmingen™ Alexa Fluor ® 647 Mouse Anti-Human Leptin Receptor): BD Biosciences, catalog number 564376. PE-labeled CD144 antibody (BD Pharmingen™ PE Mouse anti-Human CD144): BD Biosciences, catalog number 560410. BV786-labeled CD45 antibody (BD Horizon™ BV786 Mouse Anti-Human CD45): BD Biosciences, catalog number 563716. PE-labeled CD44 antibody (BD Pharmingen™ PE Mouse Anti-Human CD44): BD Biosciences, catalog number 555479.

[0136] CD144 antibody for cellular immunofluorescence (Human VE-Cadherin Antibody): R&D Company, product catalog number AF938. LEPR antibody for cellular immunofluorescence (Human Leptin R Antibody): R&D Company, product catalog number MAB867. CD45 antibody for cellular immunofluorescence (Anti-CD45 antibody, rabbit polyclonal antibody): abcam Company, product catalog number ab10558. CD41 antibody for cellular immunofluorescence (Anti-CD41 antibody, rabbit monoclonal antibody): abcam Company, product catalog number ab134131. CD271 antibody for cellular immunofluorescence (Anti-NGFR antibody produced in rabbit): MERCK Company, product catalog number HPA004765. RUNX1 antibody for cellular immunofluorescence (anti-RUNX1 rabbit antibody): MERCK Company, product catalog number HPA004176. CXCL12 antibody for cellular immunofluorescence (CXCL12 Rabbit pAb): Abbkine Scientific Co., Ltd., product catalog number A18225. CD14 antibody for cellular immunofluorescence (CD14 Monoclonal Antibody): Thermo Fisher Scientific, product catalog number 12-0149-42. CD71 antibody for cellular immunofluorescence (CD71 Monoclonal Antibody): Thermo Fisher Scientific, product catalog number 17-0719-42. CD66b antibody for cellular immunofluorescence (CD66b Monoclonal Antibody): Thermo Fisher Scientific, product catalog number 11-0666-42. CD3 antibody for cellular immunofluorescence (CD3 Monoclonal Antibody): Thermo Fisher Scientific, product catalog number 11-0032-82.

[0137] BEL medium consists of additives and a basal medium; the basal medium is obtained by mixing "IMDM + GlutaMAX" and "F12 + GlutaMAX" in equal volumes; the additives in each liter of BEL medium and their addition amounts are as follows: 2 mmol GlutaMAX, 2.5 g bovine serum albumin, 2 ml cholesterol solution, 100 µg linolenic acid, 100 µg linoleic acid, 450 µmol 1-thioglycerol, 50 ml PFHM-II, 50 mg AA2P, and 10 ml ITS-G.

[0138] IMDM + GlutaMAX (IMDM, GlutaMAX™ Supplement) (1X): Gibco™, Catalog No. 31980030. F12 + GlutaMAX (Ham’s F-12 Nutrient Mixture, GlutaMAX™ Supplement) (1X): Gibco™, Catalog No. 31765035.

[0139] GlutaMAX (GlutaMAX™ Supplement) (containing 200 mM L-alanyl-L-glutamine dipeptide) (100X): Thermo Fisher Scientific, Catalog No. 35050061. Bovine Serum Albumin (BSA): MERCK, Catalog No. A3311. Cholesterol Solution (SyntheChol ® NS0 Supplement) (500X): MERCK, Catalog No. S5442. Linolenic Acid (CAS: 463-40-1): MERCK, Catalog No. L2376. Linoleic Acid (CAS: 60-33-3): MERCK, Catalog No. L1012. 1-Thioglycerol (CAS: 96-27-5): MERCK, Catalog No. M6145. PFHM-II (Protein-Free Hybridoma Medium-II): Thermo Fisher Scientific, Catalog No. 12040077. AA2P (CAS: 1713265-25-8): MERCK, Catalog No. A8960. ITS-G (Insulin-Transferrin-Selenium Complex) (100X): Thermo Fisher Scientific, Catalog No. 41400045.

[0140] Example 1: Culture and Passage of Human Embryonic Stem Cells

[0141] 1. Suspend WA09 cells in mTeSR1 medium to obtain a cell suspension (containing 1×10 5 -2×10 5 viable cells per 3 mL).

[0142] 2. Take a Matrigel-coated 6-well plate and 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 fresh mTeSR1 medium every 24 hours.

[0143] 3. After completing step 2, aspirate 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.

[0144] 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 (containing 1×10 5 -2×10 5 viable cells per 3 mL).

[0145] 5. Take a 6-well plate coated with Matrigel, 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 fresh mTeSR1 medium every 24 hours.

[0146] Subculture can be carried out continuously during the culture process (operate according to Step 3, Step 4, and Step 5 in sequence), and cells within 60 passages can be used.

[0147] Example 2. Preparation of culture medium

[0148] Culture medium 1 consists of additives and a basal medium; the basal medium is mTeSR1 medium; the additives and their addition amounts per liter of culture medium 1 are as follows: 10 μmol Y-27632.

[0149] Culture medium 2 consists of additives and a basal medium; the basal medium is BEL medium; the additives and their addition amounts per liter of culture medium 2 are as follows: 25 μg BMP-4, 25 μg Activin A, 25 μg bFGF, and 2 μmol (CHIR-99021)HCl.

[0150] Culture medium 3 consists of additives and a basal medium; the basal medium is BEL medium; the additives and their addition amounts per liter of culture medium 3 are as follows: 100 μg VEGF, 20 μg bFGF, and 5 μmol SB431542.

[0151] Culture medium 4 consists of additives and a basal medium; the basal medium is BEL medium; the additives and their addition amounts per liter of culture medium 4 are as follows: 100 μg VEGF, 20 μg bFGF, 5 μmol SB431542, and 10 μmol Y-27632.

[0152] Culture medium 5 consists of additives and a basal medium; the basal medium is EGM2 medium; the additives and their addition amounts per liter of culture medium 5 are as follows: 50 μg SCF, 50 μg TPO, 50 μg IL-3, 50 μg Flt3l, and 20 μg GM-CSF.

[0153] The culture medium 6 consists of additives and a basal medium; the basal medium is EGM2 medium; the additives in each liter of the culture medium 6 and their addition amounts are as follows: 50 μg of SCF, 50 μg of TPO, 50 μg of IL-3, 50 μg of Flt3l, 20 μg of GM-CSF, 5 μmol of Y-27632, 10 μmol of SB431542, 2 μmol of Forskolin, 1 μmol of PGE2, and 2 μmol of FK506.

[0154] The culture medium prepared in Example 2 was used in the subsequent examples.

[0155] Example 3, Cultivation and Identification in the First Stage

[0156] Purpose of the first-stage cultivation: To induce the differentiation of embryonic stem cells to form a cell population containing "stromal cells", "endothelial cells", and "hematopoietic stem cells / hematopoietic progenitor cells", namely cell population 1.

[0157] 1. After completing step 5 of Example 1, aspirate the supernatant, add TrypLE Select enzyme (0.3 - 0.5 mL per well), incubate statically for 3 - 5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate digestion.

[0158] 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 culture medium 1 to obtain a cell suspension (0.4×10 5 viable cells / mL).

[0159] 3. Take a 6-well plate coated with Matrigel, inoculate the cell suspension prepared in step 2 (3 mL per well), culture for 24 hours, then aspirate the supernatant, add PBS buffer to wash the cells, and then aspirate the supernatant.

[0160] 4. Take the 6-well plate completed in step 3, add culture 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.

[0161] 5. Take the 6-well plate completed in step 4, add culture medium 3 (3 mL per well), and culture for 72 hours. During the culture process, replace the fresh culture medium 3 every 24 hours. After the culture is completed, observe the cell morphology under a microscope. An exemplary photo is shown in the left figure of A in Figure 2 ...

[0162] 6. Identification by flow cytometry

[0163] After completing Step 5, samples were taken and flow cytometry was used to analyze the proportion of cells (the antibodies used in flow cytometry were: CD34 antibody labeled with PEcy7, CD43 antibody labeled with BV421, CD144 antibody labeled with PerCPcy5.5, CD140a antibody labeled with PE).

[0164] An exemplary picture output by flow cytometry is shown in Figure 1 the left figure (the abscissa is the CD34 signal intensity). CD34 in the output figure was extracted + cells and their CD43 signal intensity (abscissa) and CD144 signal intensity (ordinate) were shown. An exemplary picture is shown in Figure 1 the middle figure. CD34 in the output figure was extracted - cells and their CD140a signal intensity (abscissa) and CD144 signal intensity (ordinate) were shown. An exemplary picture is shown in Figure 1 the right figure. 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 + . Among the cell population after completing Step 5 (statistical results of 7 repeated samples, mean ± standard deviation), the percentage of stromal cell number in the total number of live cells was 17.04% ± 4.57%, the percentage of endothelial cell number in the total number of live cells was 43.56% ± 8.08%, and the percentage of hematopoietic stem cell / hematopoietic progenitor cell number in the total number of live cells was 18.03% ± 3.97%. The results showed that after the culture in Step 5 was completed, WA09 cells could be efficiently differentiated into a cell population containing "stromal cells", "endothelial cells" and "hematopoietic stem cells / hematopoietic progenitor cells".

[0165] 7. Identification of differentiation potential

[0166] ① After completing Step 5, the supernatant was aspirated and discarded, TrypLE Select enzyme (0.3 - 0.5 mL per well) was added, and it was left to incubate for 3 - 5 minutes, and then mTeSR1 medium (3 mL per well) was added to terminate digestion.

[0167] 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 a CD34 magnetic sorting kit and perform sorting operations according to the instructions to collect CD34 + cells and CD34 - cells respectively.

[0168] ③Respectively take samples of the CD34 + cells and CD34 - cells obtained in step ②, and culture them with medium 5 for 7 days (replace the fresh medium 5 every day). After the culture is completed, observe the cell morphology under a microscope. Exemplary photos of CD34 + cells are shown in the middle figure of Figure 2 A, and exemplary photos of CD34 - cells are shown in the right figure of Figure 2 A.

[0169] ④After completing step ③, take two samples from each culture system and perform immunofluorescence respectively (one group uses CD144 antibody, CD45 antibody and RUNX1 antibody; the other group uses CXCL12 antibody and LEPR antibody). Exemplary photos are shown in Figure 2 B (the upper figure is the sample taken after culturing CD34 + cells, and the lower figure is the sample taken after culturing CD34 - cells) and Figure 2 C (the upper figure is the sample taken after culturing CD34 + cells, and the lower figure is the sample taken after culturing CD34 - cells). Since the culture lasts for 7 days, hematopoietic stem cells / hematopoietic progenitor cells further differentiate into mature hematopoietic cells. The differentiation of CD34 + cells mainly forms CD144 + cells (judged as endothelial cells) and CD45 + RUNX1 + cells (judged as hematopoietic cells). The differentiation of CD34 - cells mainly forms CXCL12 + LEPR + cells (judged as stromal cells). Endothelial cells, hematopoietic cells and stromal cells are three key cell types that make up the bone marrow organoids.

[0170] Example 4, Optimization of cell composition in the second-stage culture and optimization of culture medium in the third-stage culture

[0171] 1. After completing step 5 of Example 3, aspirate and discard the supernatant, add TrypLE Select enzyme (0.3 - 0.5 mL per well), incubate statically for 3 - 5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate digestion.

[0172] 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, add PBS buffer to wash the cells, then aspirate and discard the supernatant, resuspend the cells with PBS buffer, and then use a CD34 magnetic sorting kit and perform sorting operations according to the instructions to collect CD34 + cells and CD34 - cells.

[0173] 3. Optimization of cell ratio

[0174] (1) Preparation of cell suspension

[0175] Cell suspension 1 (8:2 ratio): Mix the CD34 + cells obtained in step 2, the CD34 - cells obtained in step 2, and medium 4, so that the content of CD34 + cells is 0.8×10 5 viable cells / ml and the content of CD34 - cells is 0.2×10 5 viable cells / ml, which is cell suspension 1.

[0176] Cell suspension 2 (5:5 ratio): Mix the CD34 + cells obtained in step 2, the CD34 - cells obtained in step 2, and medium 4, so that the content of CD34 + cells is 0.5×10 5 viable cells / ml and the content of CD34 - cells is 0.5×10 5 viable cells / ml, which is cell suspension 2.

[0177] Cell suspension 3 (2:8 ratio): Mix the CD34 + cells obtained in step 2, the CD34 - cells obtained in step 2, and medium 4, so that the content of CD34 + cells is 0.2×10 5 viable cells / ml and the content of CD34 - cells is 0.8×10 5 viable cells / ml, which is cell suspension 3.

[0178] (2) Culture cell suspension 1 to prepare bone marrow organoids

[0179] ① Culture cell suspension 1 to form cell aggregates.

[0180] Take an Aggrewell plate and inoculate the cell suspension 1 prepared in step ① (1 mL per well). First, incubate statically for half an hour, and then culture for 24 h.

[0181] ② Prepare the cell aggregate / matrix gel mixture

[0182] After completing step ①, gently blow out and collect all the cell aggregates formed in one Angrewell plate (about 7,200) into a centrifuge tube pre-cooled in an ice bath. After centrifugation, aspirate and discard the supernatant. Keep the centrifuge tube on ice and add 1 mL of Matrigel, and mix well to obtain the cell aggregate / matrix gel mixture.

[0183] ③ Prepare organoids

[0184] Take a 24-well cell culture plate and add the cell aggregate / matrix gel mixture prepared in step ② (50 μL per well). Culture for 30 min, and then slowly add medium 6 (0.6 mL per well) along the edge. Culture for 192 hours. During the culture process, add medium 6 (0.6 mL per well) after 48 hours, aspirate and discard the supernatant and add new medium 6 (0.6 mL per well) after 96 hours, and add medium 6 (0.6 mL per well) after 144 hours. After completing this step, the content in each well is 1 bone marrow organoid.

[0185] (3) Prepare bone marrow organoids by culturing cell suspension 2

[0186] Use cell suspension 2 to replace cell suspension 1, and the others are the same as in step (2).

[0187] (4) Prepare bone marrow organoids by culturing cell suspension 3

[0188] Use cell suspension 3 to replace cell suspension 1, and the others are the same as in step (2).

[0189] (5) Identification of cell survival rate and number of living cells

[0190] After completing step (2) or step (3) or step (4), dissociate the organoids and prepare a single-cell suspension, and then count the total number of cells and identify the cell survival rate, and calculate the number of living cells. The results of the cell survival rate are shown in Figure 3 the left figure (statistical results of 3 repeated samples). There is no significant difference in the cell survival rate of the bone marrow organoids obtained by inoculating the three cell suspensions. Taking the number of living cells of the bone marrow organoids obtained by inoculating cell suspension 2 as a reference, the relative values of the number of living cells of the bone marrow organoids obtained by inoculating the three cell suspensions are shown in Figure 3 the right figure (statistical results of 3 repeated samples).

[0191] (6) Identification of cell ratio

[0192] After completing step (2) or step (3) or step (4), dissociate the organoids and prepare a single-cell suspension, then sample and perform cell ratio analysis using flow cytometry (the antibodies used in flow cytometry are: CD144 antibody labeled with PE, CD34 antibody labeled with PEcy7, LEPR antibody labeled with APC, CD45 antibody labeled with BV786). According to the output results of flow cytometry, count CD45 + cells (judged as hematopoietic cells), CD45 - CD144 - LEPR + cells (judged as stromal cells), CD45 - CD144 + cells (judged as endothelial cells) and CD45 + CD34 + cells (judged as hematopoietic stem cells / hematopoietic progenitor cells), and calculate the percentage of the number of these cells in the total number of live cells. The results are shown in Figure 4 the upper figure above (statistical results of 3 repeated samples). Calculate the number of each type of cell based on the number of live cells and the percentage of each type of cell. The results are shown in Figure 4 the lower figure below (statistical results of 3 repeated samples).

[0193] The results show that the cell number ratio of CD34 + cells and CD34 - cells has an important impact on the finally formed bone marrow organoids. When the proportion of CD34 + cells is too high, the differentiation of hematopoietic cells and endothelial cells in the bone marrow organoids is dominant but the stromal cells are too few. Stromal cells, as important supporting cells in the bone marrow organoids, can secrete various cytokines and extracellular matrix components to support the formation of a microenvironment for hematopoiesis and endothelial cells. When the proportion of CD34 - cells is too high, the proportion of stromal cells in the bone marrow organoids is too high. Since the proliferation ability of this type of cell is stronger than that of endothelial cells and blood cells, it consumes a large amount of nutrient components, inhibits the differentiation of endothelial cells and hematopoietic cells, and cannot output enough hematopoietic cells. When CD34 + cells and CD34 - cells are mixed in a 5:5 cell number ratio (i.e., a 1:1 cell number ratio), the formed bone marrow organoids have a high cell viability, a large total cell number, and the most balanced ratio of the three constituent structure cells of endothelial cells, hematopoietic cells, and stromal cells, and a high proportion of hematopoietic stem cells / hematopoietic progenitor cells, which is suitable for producing more mature blood cells.

[0194] 4. Take the CD34 obtained in step 2+ cells, CD34 obtained in step 2 - Mix the cells and medium 4 so that the content of CD34 + cells is 0.5×10 5 viable cells / ml and the content of CD34 - cells is 0.5×10 5 viable cells / ml, which is the cell suspension.

[0195] 5. Take an Aggrewell plate, inoculate the cell suspension prepared in step 4 (1 mL per well), incubate statically for half an hour first, and then culture for 24 hours. Observe under a microscope before static incubation and after 24 hours of culture. Exemplary photos are shown in Figure 5 the left and middle figures (the left figure is before static incubation, and the middle figure 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. Randomly measure the diameters of 25 cell aggregates, as shown in Figure 5 the right figure, which is 113.53±13.83 μm, with uniform and appropriate sizes, which can ensure that they can maintain a good cell growth state and form a vascular network structure after being placed in Matrigel subsequently.

[0196] 6. After completing step 5, gently blow out and collect all the cell aggregates (about 7200) formed in one Angrewell plate into a centrifuge tube pre-cooled in an ice bath. After centrifugation, aspirate and discard the supernatant. Keep the centrifuge tube on ice and add 1 mL of Matrigel, and mix well to obtain a cell aggregate / Matrigel mixture.

[0197] 7. Optimization of the medium

[0198] The test media are: medium 5 or medium 6.

[0199] Take a 24-well cell culture plate, add the cell aggregate / Matrigel mixture prepared in step 6 (50 μl / well), culture for 30 min, and then slowly add the test medium (0.6 ml / well) along the edge, and culture for 192 hours. During the culture process, supplement the test medium (0.6 ml / well) after 48 hours, aspirate and discard the supernatant and add a new test medium (0.6 ml / well) after 96 hours, and supplement the test medium (0.6 ml / well) after 144 hours.

[0200] Observe under a microscope after 48 hours of culture. Exemplary photos are shown in Figure 6 the left figure (the right side is a partial magnification of the left side). Randomly count the number of buds of 100 cell aggregates, and the results are shown in Figure 6The right figure. After culturing for 192 hours, immunofluorescence staining was performed (using CD144 antibody, and CD144 is a marker protein for endothelial cells). An exemplary photograph is shown in Figure 7 . The results showed that cells within the cell aggregates would gradually sprout and form a vascular network structure within the Matrigel. Compared with using Medium 5, the cell aggregates cultured with Medium 6 had more sprouts and could form a denser endothelial cell network. The vascular niche structure is the main site of 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 using Medium 5, culturing with Medium 6 could better simulate the structure of the bone marrow vascular niche in vitro.

[0201] After culturing for 192 hours, samples were taken for immunofluorescence staining (using CD45 antibody, CD271 antibody, and CD144 antibody). An exemplary photograph is shown in Figure 8 . The cell aggregates could differentiate into CD45 + cells (judged as hematopoietic cells), CD144 + cells (judged as endothelial cells), and CD271 + cells (judged as stromal cells), indicating that the bone marrow organoids with hematopoietic function had been successfully constructed. Compared with using Medium 5, the endothelial cell network structure of the bone marrow organoids formed with Medium 6 was richer and more perfect, and the number of hematopoietic cells formed in this endothelial cell network was larger, indicating that it had a stronger bone marrow hematopoietic output ability.

[0202] After culturing for 192 hours, samples were taken and flow cytometry was used for cell proportion analysis (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 picture output by flow cytometry is shown in Figure 9 . CD34 + cells were extracted from the output figure and their CD43 signal intensity and CD44 signal intensity were shown. An exemplary picture is shown in Figure 9 . The proportion of the number of CD34 + CD43 + CD44 + cells (judged as hematopoietic stem cells / hematopoietic progenitor cells) in the total number of live cells was statistically analyzed. The results are shown in Figure 9 (the average value of five repeated samples was statistically analyzed). Compared with Medium 5, in the bone marrow organoids formed by the treatment with Medium 6, the proportion of hematopoietic stem cells / hematopoietic progenitor cells was significantly increased, thus having a stronger hematopoietic colony-forming ability and proliferation potential. Therefore, compared with Medium 5, the bone marrow organoids produced by the treatment with Medium 6 had a stronger hematopoietic cell output ability and a longer maintenance period for hematopoietic cell output.

[0203] After culturing for 192 hours, the suspended cells in the wells cultured with medium 6 were collected, and Wright-Giemsa staining and immunofluorescence staining (using CD41 antibody, CD14 antibody, CD71 antibody, CD66b antibody and CD3 antibody) were performed respectively. Exemplary photos of Wright-Giemsa staining are shown in Figure 10 A of. The results showed that the suspended cells had typical morphological characteristics of granulocytic cells, erythroid cells, lymphocytic cells, monocytes and megakaryocytes. Exemplary photos of immunofluorescence staining are shown in Figure 10 B of. The results showed that CD41 + cells (judged as megakaryocytes), CD14 + cells (judged as monocytes), CD71 + cells (judged as erythroid cells), CD66b + cells (judged as granulocytic cells) and CD3 + cells (judged as T lymphocytes) were present in the suspended cells, demonstrating that the differentiated cells produced by the bone marrow organoids indeed had multi-lineage hematopoietic cell phenotypic characteristics.

[0204] Example 5, Culturing in the Second Stage

[0205] Purpose of culturing in the second stage: Prepare cell population 2 by CD34 sorting, culture cell population 2 into cell aggregates, and prepare a cell aggregate / matrix gel mixture.

[0206] 1. After completing step 5 of Example 3, aspirate the supernatant, add TrypLE Select enzyme (0.3 - 0.5 mL per well), incubate statically for 3 - 5 minutes, and then add mTeSR1 medium (3 mL per well) to terminate digestion.

[0207] 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, add PBS buffer to wash the cells, then aspirate the supernatant, resuspend the cells with PBS buffer, and then perform sorting operation using a CD34 magnetic sorting kit according to the instructions, and collect CD34 + cells and CD34 - cells respectively.

[0208] 3. Mix the CD34 + cells obtained in step 2, the CD34 - cells obtained in step 2 and medium 4, so that the content of CD34 + cells is 0.5×10 5 viable cells / ml and the content of CD34 - cells is 0.5×10 5 viable cells / ml, which is the cell suspension.

[0209] 4. Take an Aggrewell plate and inoculate the cell suspension prepared in step 3 (1 mL per well). First, let it stand and incubate for half an hour, and then culture for 24 hours (in actual operation, 18 - 24 hours are both acceptable).

[0210] 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, and mix well to obtain a cell aggregate / Matrigel mixture.

[0211] Example 6. Third-stage culture and identification

[0212] Purpose of the third-stage culture: Prepare bone marrow organoids using the cell aggregate / Matrigel mixture and vascular endothelial cells.

[0213] The device with a bone marrow blood barrier used in this example is the high-throughput barrier function organ chip of Beijing Daxiang Technology Co., Ltd., model IBAC M1, with 24 fluxes (i.e., having 24 unit structures and can prepare 24 organ models). This chip is simply referred to as the IBAC M1 chip. Each unit structure consists of a cavity (orgamoid chamber) for accommodating simulated bone marrow and a channel (perfusion channel) for accommodating simulated blood vessels. There is a sampling hole (reservior) 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 8 µm). The orgamoid chamber is a cavity with an open top and samples are added directly from the top. The perfusion channel is sampled through the sampling hole.

[0214] 1. Coating

[0215] Take the IBAC M1 chip and add collagen working solution to the orgamoid chamber and perfusion channel (add 12 µL of collagen working solution to the orgamoid chamber and 100 µL of collagen working solution to the perfusion channel). Let it stand at 37 °C for 1 hour, then aspirate and discard the liquid, and then wash with PBS buffer.

[0216] Preparation method of the collagen working solution: Mix 1 volume of collagen solution with 20 volumes of PBS buffer. Collagen solution (CellAdhere™ Type I Collagen, Bovine, Solution): STEMCELL company, product catalog number 07001.

[0217] 2. After completing Step 1, aspirate and discard the PBS buffer in the orgamoid chamber, then add 10 μL of the cell aggregate / matrix gel mixture prepared in Step 5 of Example 5 into the orgamoid chamber, let it stand at 37°C for 1 hour, and then add 100 μL of Medium 6 into the orgamoid chamber; then, aspirate and discard the PBS buffer in the perfusion channel, and add 200 μL of Medium 6 into the perfusion channel; then, place the IBAC M1 chip on a shaker (tilt angle set to 30°, rotation speed set to 3 - 5 cycles / minute), and culture for 96 hours. During the culture process, replace the fresh Medium 6 after 48 hours (both in the orgamoid chamber and the perfusion channel).

[0218] 3. After completing Step 2, aspirate and discard the liquid in the orgamoid chamber and the perfusion channel, add 20 μL of HUVEC cell suspension into the perfusion channel, gently shake it, then quickly flip the IBAC M1 chip 180° and invert it for 1 hour, then place the IBAC M1 chip upright, then add 100 μL of Medium 6 into the orgamoid chamber, and add 200 μL of EGM2 medium into the perfusion channel, and culture for 24 hours.

[0219] Preparation method of HUVEC cell suspension: Resuspend the HUVEC cells digested by TrypLE Select enzyme in EGM2 medium to make the cell concentration (1 - 3)×10 6 viable cells / mL.

[0220] 4. After completing Step 3, place the IBAC M1 chip on a shaker (tilt angle set to 30°, rotation speed set to 3 - 5 cycles / minute), and culture for 96 hours. During the culture process, replace the fresh medium after 48 hours (replace Medium 6 in the orgamoid chamber and replace EGM2 medium in the perfusion channel).

[0221] After completing Step 4, bone marrow organoids are obtained.

[0222] The schematic diagram of the bone marrow organoids is shown in Figure 11The orgamoid chamber contains a simulated bone marrow formed by a cell aggregate / matrix gel mixture, and the perfusion channel contains a simulated blood vessel formed by HUVEC cells (the HUVEC cells adhere to the inner wall of the perfusion channel to form a simulated blood vessel). The intersection of the orgamoid chamber and the perfusion channel is the bone marrow-blood barrier (a polycarbonate membrane with a pore size of 8 µm). 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 the communication and exchange of substances such as cytokines and small molecule compounds between the blood and the cells in the bone marrow through the endothelial cell barrier under specific physiological conditions.

[0223] 5. After completing step 4, perform immunofluorescence staining (using CD45 antibody, CD144 antibody, and CD271 antibody). Exemplary photos are shown in Figure 12 。The three main types of cells in the bone marrow organoid are CD45 + cells (judged as hematopoietic cells), CD144 + cells (judged as endothelial cells), and CD271 + cells (judged as stromal cells). In the orgamoid chamber, the endothelial cells form a network-like simulated blood vessel network structure, and the three types of cells are intertwined and distributed within the matrix gel, reproducing the natural positional relationship of these three types of cells in the natural bone marrow. In the perfusion channel, a dense monolayer cell layer mainly formed by endothelial cells can be observed, simulating the barrier formed by the vascular endothelium.

[0224] Example 7. Application of bone marrow organoids in screening anti-radiation hematopoietic injury drugs

[0225] 1. After completing step 4 of Example 6, take an IBAC M1 chip, aspirate the liquid in the orgamoid chamber and the liquid in the perfusion channel, add EGM2 medium to the orgamoid chamber, and 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 romiplostim, the drug concentration is 50 ng / ml. When the test drug is eltrombopag, the drug concentration is 1 µM.

[0226] 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.

[0227] 3. After completing step 2, incubate for 24 hours.

[0228] 4. After completing step 3, dissociate the organoids and prepare a single-cell suspension, and then sample to detect the number of live cells.

[0229] 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, product catalog number 9603S.

[0230] Set up a control treatment group that does not perform step 2 and does not add the test drug, called the non-irradiated group.

[0231] Set up a control treatment without adding the test drug, called the irradiated group.

[0232] The results are shown in Figure 13 .

[0233] Note: The method for dissociating organoids and preparing a single-cell suspension in the examples is as follows:

[0234] Take a container (24-well cell culture plate or chip) containing organoids, discard the liquid in the well or orgamoid chamber, and add PBS buffer for rinsing; then, add pre-cooled Recovery solution (500 µl / well or 100 µl / orgamoid chamber), and then place the container on ice for 20 min; then transfer the contents of the well or orgamoid chamber to a centrifuge tube, centrifuge at 4°C and 1200 rpm for 5 min, aspirate the supernatant, and the remaining cell pellet in the centrifuge tube; then, add 200 µl of digestion enzyme solution preheated to 37°C to the centrifuge tube and resuspend the cell pellet, incubate at 37°C for 10 - 15 min (invert the centrifuge tube up and down every 5 min to resuspend the cell pellet), and then gently pipette and add 2 ml of PBS buffer containing 10% FBS to terminate the digestion; then, centrifuge at 1200 rpm for 5 min, aspirate the supernatant, add PBS buffer for washing, and then resuspend with PBS buffer to obtain a single-cell suspension.

[0235] Preparation method of digestive enzyme solution: Mix 1.6 mL of Liberase™ TH solution, 3 mL of Dispase II solution, and 500 μL of DNase I Solution, and then add D-PBS buffer to make the total volume of the system reach 10 mL.

[0236] Recovery solution (Products for Cell Release and Recovery from ECMs): Corning, catalog number 354253. Liberase™ TH: MERCK, 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, catalog number 07900. Dispase II: MERCK, catalog number D4693. Dissolve Dispase II in D-PBS buffer to make its concentration 10 mg / mL to obtain Dispase II solution. D-PBS buffer (without Ca ++ and Mg ++ ): MERCK, catalog number BSS-1006.

[0237] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A method for preparing bone marrow organoids, comprising the following steps: (1) Induce the differentiation of embryonic stem cells to form a cell population containing stromal cells, endothelial cells, and hematopoietic stem and progenitor cells, named cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) Sort cell population 1 into a CD34-positive cell subset and a CD34-negative cell subset, and then mix the CD34-positive cell subset and the CD34-negative cell subset in a cell number ratio of 1:1 to obtain a cell population, named cell population 2; (3) Culture cell population 2 into cell aggregates, and then mix with Matrigel to obtain a mixture; (4) Culture the mixture and vascular endothelial cells in a device with a bone marrow blood barrier to obtain bone marrow organoids; The device with a bone marrow blood barrier has more than one unit structure; the unit structure consists of a cavity for accommodating simulated bone marrow and a channel for accommodating simulated blood vessels, a sampling hole is provided at the end of the channel, and a bone marrow blood barrier is present at the intersection of the cavity and the channel; the bone marrow blood barrier is a membrane with a pore size of 5 µm - 10 µm; The method for culturing the mixture and vascular endothelial cells in a device with a bone marrow blood barrier is: culture the mixture in the cavity to form simulated bone marrow, and culture the vascular endothelial cells in the channel to form simulated blood vessels; Culture the mixture using medium 6; The additives required for preparing each liter of medium 6 are as follows: 50 - 100 µg SCF, 50 - 100 µg TPO, 50 - 100 µg IL-3, 50 - 100 µg Flt3l, 20 - 50 µg GM-CSF, 1 - 5 μmol Y-27632, 2 - 10 μmol SB431542, 1 - 5 μmol Forskolin, 0.3 - 2 μmol PGE2, and 1 - 5 μmol FK506.

2. A method for preparing bone marrow organoids, comprising the following steps: (1) Induce the differentiation of embryonic stem cells to form a cell population containing stromal cells, endothelial cells, and hematopoietic stem and progenitor cells, named cell population 1; the hematopoietic stem and progenitor cells are hematopoietic stem cells and / or hematopoietic progenitor cells; (2) Sort cell population 1 into a CD34-positive cell subset and a CD34-negative cell subset, and then mix the CD34-positive cell subset and the CD34-negative cell subset in a cell number ratio of 1:1 to obtain a cell population, named cell population 2; (3) Culture cell population 2 into cell aggregates, and then mix with Matrigel to obtain a mixture; (4) Culture the mixture obtained in step (3) using medium 6 to obtain bone marrow organoids; The additives required for preparing each liter of the medium 6 are as follows: 50 - 100 μg of SCF, 50 - 100 μg of TPO, 50 - 100 μg of IL-3, 50 - 100 μg of Flt3l, 20 - 50 μg of GM-CSF, 1 - 5 μmol of Y-27632, 2 - 10 μmol of SB431542, 1 - 5 μmol of Forskolin, 0.3 - 2 μmol of PGE2, and 1 - 5 μmol of FK506.

3. The bone marrow organoids prepared by the method according to 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; 3. The mixing ratios of the ten compounds are as follows: 50 - 100 μg of SCF: 50 - 100 μg of TPO: 50 - 100 μg of IL-3: 50 - 100 μg of Flt3l: 20 - 50 μg of GM-CSF: 1 - 5 μmol of Y-27632: 2 - 10 μmol of SB431542: 1 - 5 μmol of Forskolin: 0.3 - 2 μmol of PGE2: 1 - 5 μmol of FK506; The function of the composition is: to promote the formation of bone marrow organoids from cell aggregates.

5. A medium having the composition according to claim 4; the function of the medium is: to promote the formation of bone marrow organoids from cell aggregates.

6. The use of the composition according to claim 4 or the medium according to claim 5 in the preparation of a kit; the function of the kit is as follows (a) or (b): (a) To promote the formation of bone marrow organoids from cell aggregates; (b) To prepare bone marrow organoids.

7. A kit comprising the composition according to claim 4 or the medium according to claim 5; the function of the kit is as follows (a) or (b): (a) To promote the formation of bone marrow organoids from cell aggregates; (b) To prepare bone marrow organoids.

8. The use of the kit according to claim 7 is as follows (a) or (b): (a) To promote the formation of bone marrow organoids from cell aggregates; (b) To prepare bone marrow organoids.

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