Human bone marrow organoid and its construction method and application

Abstract

The present invention provides a human bone marrow organoid and its construction method and application. The construction method includes the following steps: extracting microvascular fragments from human adipose tissue by enzymatic digestion; preparing pre-vascularized cartilage tissue by dynamic culture: mechanically preparing human micro-adipocyte tissue, adding human micro-adipocyte tissue and microvascular fragments to a bioreactor in a predetermined ratio, adding culture medium for dynamic culture to obtain pre-vascularized self-aggregated adipose tissue; transplanting the pre-vascularized cartilage tissue subcutaneously into a target animal; after a predetermined period of time, the pre-vascularized cartilage tissue develops into humanized bone marrow organoids with specific functions in the target organism. The human bone marrow organoids obtained by the construction method provided by the present invention have three complete structural components: the endosteal niche, the vascular niche, and the hematopoietic cell niche, and maintain the physiological function of human hematopoietic stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a human bone marrow organoid and a construction method and application thereof. Background Art

[0002] All cells in the blood system differentiate from hematopoietic stem cells. As a crucial hematopoietic organ, the bone marrow provides a spatial environment for the growth and development of hematopoietic stem cells, maintaining their physiological functions of self-renewal and differentiation. The complex and delicate structure of the bone marrow niche ensures the fulfillment of these functions. Structurally, the bone marrow can be divided into three major components: the endosteal niche, the vascular niche, and the hematopoietic niche. The endosteal niche is the microenvironment lining the inner surface of bone tissue and contains osteoblasts, osteoclasts, and the endosteal membrane. The vascular niche is the complex network of vascular structures within the bone marrow, including sinusoids, arterioles, and their endothelial cells and pericytes. The hematopoietic niche is the rich cellular composition of the bone marrow, primarily containing hematopoietic elements, namely various hematopoietic cells (hematopoietic stem cells, hematopoietic progenitor cells, lymphocytes, myeloid cells, megakaryocytes, erythrocytes, etc.) and supporting hematopoietic cells, such as mesenchymal stem cells. Disruption of the dynamic balance of these components within the bone marrow microenvironment is a key mechanism in the development and progression of both benign and malignant hematological diseases. Studying the interactions between components of the bone marrow microenvironment and hematopoietic stem cells can provide valuable insights into important medical fields, including normal physiological hematopoiesis, blood diseases, and bone metastasis of solid tumors. However, in vitro environments struggle to simulate the complex physiological structure and function of bone marrow components, including the vascular system, immune cells, and cytokines. Therefore, the creation of human bone marrow organoids is considered an optimal and urgently needed approach to studying the human bone marrow microenvironment.

[0003] The existing methods in this technical field are often based on the principle of "seed cells + biological scaffold". Conventional seed cells are bone marrow mesenchymal stem cells (BMSCs) isolated from human bone marrow tissue. After the seed cells are passaged and expanded in vitro, they are inoculated on biological scaffolds to induce chondrogenesis or ossification in vitro or in vivo. This tissue construction method is based on the "cell" level. The humanized bone marrow organoids constructed in this way have the following main problems: (1) The extraction of BMSCs requires aspiration of human bone marrow by bone marrow puncture. This operation is very traumatic, and the volume of bone marrow obtained each time is very small. In addition, the aspirated patient bone marrow may contain malignant blood cells and is not suitable for subsequent experimental culture; (2) After repeated enzymatic hydrolysis, passage and expansion in vitro, the multidirectional differentiation ability of stem cells such as BMSCs will gradually be lost, that is, more and more cells lose the ability to differentiate into cartilage / bone, resulting in low efficiency in constructing bone marrow organoids; (3) The extraction of seed cells, passage and expansion in vitro, inoculation of biological scaffolds and other operations are time-consuming and labor-intensive. The experimental process is complicated and tedious, the experimental economic cost is high, and the operation process is prone to introduce exogenous contamination.

[0004] In the existing methods in this technical field, when using adipose tissue to directly construct a bone marrow niche model, the adipose tissue is directly induced to differentiate in vitro and developed into a bone marrow niche model in nude mice. However, the bone marrow niche model constructed by the existing method still has the following problems: (1) It lacks the complete structure of natural bone marrow tissue. The bone marrow includes three major structural parts: the endosteal niche, the vascular niche and the hematopoietic cell niche. The bone marrow niche model constructed by the existing method only has external bone tissue and some blood cells inside. It lacks osteoblasts, osteoclasts and blood / immune cells (such as hematopoietic stem / progenitor cells, myeloid cells, etc.), and the degree of vascularization is low, that is, the vascular structure is simple and the vascular density is sparse, which is far lower than the vascular density in normal bone marrow tissue; (2) It cannot simulate the physiological function of bone marrow. The main physiological function of bone marrow is to maintain the self-renewal and multidirectional differentiation ability of hematopoietic stem / progenitor cells. Due to the incomplete structure, the bone marrow niche model constructed by the existing method cannot recruit human hematopoietic stem cells in immunodeficient mice. That is, it only has a preliminary structure and does not have the physiological function of bone marrow, and cannot be truly used in subsequent research and clinical transformation; (3) The operation is cumbersome, time-consuming and labor-intensive. For example, in existing methods, after in vitro proliferation and culture, a biopsy punch is used to drill a small sample of fat tissue for further experiments. Manual drilling is time-consuming and labor-intensive, requiring numerous disposable drilling instruments, resulting in high average per-experiment costs. Furthermore, mechanical tissue cutting significantly damages stem cells within the tissue, reducing cell activity. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention aims to provide a human bone marrow organoid, a construction method, and an application thereof. The construction method utilizes adipose tissue to directly construct humanized bone marrow organoids with complete structure and function, avoiding the steps of extracting, amplifying, and inoculating stem cells in the current methods. The constructed human bone marrow organoids can be used for research in major medical fields such as normal physiological hematopoiesis, blood diseases, and bone metastasis of solid tumors.

[0006] A first aspect of the present invention provides a method for constructing human bone marrow organoids, comprising the following steps:

[0007] Step (1) extracting microvessel fragments from human adipose tissue by enzymatic digestion;

[0008] Step (2) Dynamic culture to prepare pre-vascularized cartilage tissue: human micro-adipocytes are prepared by mechanical method, human micro-adipocytes and microvessel fragments are added into a bioreactor according to a predetermined ratio, and culture medium is added to perform dynamic culture to obtain pre-vascularized self-aggregated adipose tissue;

[0009] Step (3) transplants the pre-vascularized cartilage tissue into the subcutaneous tissue of the target animal. After a predetermined period of time, the pre-vascularized cartilage tissue develops into humanized bone marrow organoids with specific functions in the target organism.

[0010] In one embodiment of the present invention, the sequential extraction method for extracting microvessel fragments in step (1) specifically includes:

[0011] (a) Obtain human adipose tissue, rinse and collect, mix with equal volumes of collagenase solution, shake under predetermined conditions, and centrifuge;

[0012] (b) After centrifugation, the undigested adipose tissue in the upper layer was collected, and PBS buffer was added to resuspend the microvascular fragments in the lower layer;

[0013] (c) The collected upper layer of undigested adipose tissue was remixed with an equal volume of collagenase solution, shaken, and centrifuged;

[0014] (d) Repeat steps (b) and (c) multiple times;

[0015] (e) The microvessel fragment suspensions obtained multiple times are mixed, centrifuged, and the supernatant is removed to obtain microvessel fragments.

[0016] In one embodiment of the present invention, the sequential extraction method for extracting microvessel fragments further comprises: filtering the microvessel fragment suspension through a filter before centrifugation in step (e).

[0017] In one embodiment of the present invention, the pore size of the filter is 40-600 microns.

[0018] In one embodiment of the present invention, the shaking conditions are 30-37° C., 50-500 rpm, and constant shaking for 3-15 minutes.

[0019] In one embodiment of the present invention, the centrifugation conditions in steps (a) to (e) are all 50-1000 g for 1-5 minutes.

[0020] In one embodiment of the present invention, the bioreactor in step (2) is a rotary or rocking bioreactor.

[0021] In one embodiment of the present invention, step (2) dynamically cultures the self-aggregated micro-adipocytes, and the dynamic culture includes dynamic proliferation culture, dynamic vascularization culture, and dynamic differentiation culture.

[0022] In one embodiment of the present invention, the composition of the proliferation medium used in the dynamic proliferation culture is as follows: a-MEM + 2-20% FBS + 0-5% PSG + 0-5% HEPES + 0-5% glutamine + FGF-2 (1-20ng / mL) + PDGF (1-20 ng / mL) + dexamethasone (0.5-5) × 10-7 mol / L + ascorbic acid (0.5-5) × 10-5 mol / L; the proportions of the pro-vascularization medium used in the dynamic pro-vascularization culture are as follows: 1-10% PSG, 1-10% HEPES, (0.5-2)% ITS, ECGS (1-300 ng / mL).

[0023] In one embodiment of the present invention, the perfusion rate of the proliferation culture and the vascularization promotion culture is 0.1-2 mL / min.

[0024] In one embodiment of the present invention, the culture time of the dynamic proliferation culture and the dynamic angiogenesis promotion culture is 1-4 weeks and 1-4 weeks, respectively.

[0025] In one embodiment of the present invention, the composition of the differentiation medium used in the dynamic differentiation culture is as follows: DMEM + 2-20% HSA + 0-5% PSG + 0-5% HEPES + 0-5% glutamine + 0-5% ITS + 0-5% linoleic acid + dexamethasone (0.5-5) × 10-7 mol / L + ascorbic acid (0.5-5) × 10-5 mol / L + BMP-6 (1-20 ng / mL) + TGF-β3 (1-20 ng / mL) + SB431542 (1-20 μmol / L).

[0026] In one embodiment of the present invention, the perfusion rate of the differentiation medium is 0.1-2 mL / min.

[0027] In one embodiment of the present invention, the dynamic differentiation culture time is 2-10 weeks.

[0028] In one embodiment of the present invention, the particle size of the micro-adipocytes is 1-6 mm.

[0029] In one embodiment of the present invention, step (2) of preparing micro-adipocytes specifically comprises the following steps:

[0030] (1) Collect human adipose tissue, rinse 1-3 times, mince, centrifuge at 500-2000 g for 1-5 min, remove the upper fat layer and the lower swelling fluid, and collect the middle fat layer in a syringe;

[0031] (2) Prepare another syringe and connect it to the syringe that collects the middle fat layer through the fat cutter. Push the two syringes back and forth 10-50 times, and then centrifuge at 500-2000 g for 1-5 minutes to remove the upper fat layer. The lower layer is the micro-fat tissue.

[0032] A second aspect of the present invention provides a human bone marrow organoid constructed using the above-mentioned construction method.

[0033] The third aspect of the present invention provides the use of human bone marrow organoids in preparing animal models for studying the normal physiological hematopoietic system, blood system diseases, bone metastasis of solid tumors, and drug screening for blood diseases and tumor diseases.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. In the construction method provided by the present invention, functionalized human bone marrow organoids are directly constructed from adipose tissue. By adding micro-fat tissue and microvascular fragments obtained by sequential extraction into a bioreactor for dynamic culture, the differentiation efficiency and vascularization degree of adipose tissue are greatly improved. The resulting human bone marrow organoids have complete structural components of the endosteal niche, vascular niche, and hematopoietic cell niche, and maintain the physiological function of human hematopoietic stem cells.

[0036] 2. In the construction method provided by the present invention, the sequential extraction of microvascular fragments yields more microvascular fragments from the same volume of adipose tissue than the traditional consistent extraction method, and the microvascular fragments have higher activity and higher content of vascular endothelial cells and adipose stem cells, which helps promote the vascularization of microfatty tissue (see Example 1 for details).

[0037] 3. In the construction method provided by the present invention, the micro-adipocytes are dynamically cultured in a bioreactor and the obtained microvessel fragments are extracted by sequential extraction, which promotes the uniform fusion of micro-adipocytes and MVF, reduces the necrosis of micro-adipocytes, improves the self-aggregation efficiency and differentiation efficiency of micro-adipocytes, and significantly shortens the culture time.

[0038] 4. In the present invention, dynamic culture is performed in a bioreactor at a specific perfusion rate and perfusion time, so that the mixture of micro-adipocytes and MVF can spontaneously aggregate into small round particles, reflecting the effect of promoting the self-aggregation of micro-adipocytes; in addition, by controlling the perfusion rate and the volume of the adipose tissue block, the particle size of the small round particles is controlled to obtain round cartilage particles with a diameter of about 1-6 mm, so that round cartilage particles of different particle sizes can meet the needs of different application scenarios; cartilage particles of different particle sizes recruit different numbers of cells, and cartilage particles of a specific particle size are selected according to the specific purpose and application scenario of the organoid.

[0039] 5. The model obtained by implanting the human bone marrow organoids constructed in the present invention into mice is such that, when used, human leukemia cells, tumor cells, etc. will preferentially enter the human bone marrow organoids in the mouse body, thereby surviving stably and long-term, rather than entering the mouse's own bone marrow. This provides a good model for the study of major medical issues such as normal physiological hematopoiesis, blood diseases, and bone metastasis of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0041] Figure 1 Flowcharts of sequential and consistent MVF extraction methods;

[0042] Figure 2 The following are the statistical graphs of fat content before and after sequential extraction and consistent extraction;

[0043] Figure 3 The photos of MVF obtained by sequential extraction method and consistent extraction method under inverted microscope;

[0044] Figure 4 Statistical diagram of the number of MVFs obtained by sequential extraction method and consistent extraction method;

[0045] Figure 5 Live / dead cell staining images of MVF obtained by sequential extraction method and consistent extraction method;

[0046] Figure 6 Statistical graphs of the ratio of live and dead cells in MVF obtained by sequential extraction method and consistent extraction method;

[0047] Figure 7 Representative images of flow cytometric grouping of MVF;

[0048] Figure 8 Statistical graphs of the proportions of various types of cells in MVF obtained by sequential extraction and consistent extraction methods;

[0049] Figure 9 To prepare immediate micro-adipocytes and micro-adipocyte particles formed by self-aggregation after culture in a bioreactor;

[0050] Figure 10 Safranin-fast green staining and type II collagen immunohistochemical staining of cartilage particles prepared from experimental and control groups;

[0051] Figure 11 Statistical graph showing the ratio of the maximum cross-sectional cartilage area to the total area of ​​the cartilage particles prepared for the experimental and control groups;

[0052] Figure 12 VEGF immunohistochemical staining of cartilage particles prepared for the experimental and control groups;

[0053] Figure 13 Statistical graph of VEGF immunohistochemical staining intensity of cartilage particles prepared for experimental and control groups;

[0054] Figure 14 HE staining images of bone marrow organoids obtained from the experimental group and the control group;

[0055] Figure 15 Statistical graph showing the ratio of the maximum cross-sectional bone marrow cavity area to the total area of ​​the bone marrow organoids obtained in the experimental and control groups;

[0056] Figure 16 Osterix immunohistochemical staining and TRAP staining of bone marrow organoids obtained from the experimental and control groups;

[0057] Figure 17 Immunofluorescence staining of CD31 and α-SMA in bone marrow organoids obtained from the experimental and control groups;

[0058] Figure 18 Statistical graph of the average number of blood vessels in a single field of view of bone marrow organoids obtained for the experimental and control groups;

[0059] Figure 19 Representative images of flow cytometric analysis of cells in bone marrow organoids obtained from the experimental and control groups;

[0060] Figure 20 Statistical graph of the human chimerism rate of cells in bone marrow organoids obtained from the experimental and control groups;

[0061] Figure 21 This is a statistical chart of the number of colony formation of cells in bone marrow organoids obtained from the experimental group and the control group. DETAILED DESCRIPTION

[0062] The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several changes and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0063] The relevant terms in the following embodiments are explained as follows:

[0064] MVF: Microvascular fragments are functional fragments of arterioles, capillaries and venules, composed of smooth muscle cells and endothelial cells. They can be isolated in large quantities from adipose tissue and retain the characteristics of natural microvessels, such as the ability to secrete angiogenic factors.

[0065] PBS: The most widely used buffer in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve and protect reagents.

[0066] α-MEM: MEM is a minimum essential culture medium containing 13 essential amino acids and 8 vitamins. Its composition is simple and can be widely used in animal cell lines and most adherent mammalian cells. α-MEM contains more amino acids, pyruvate, and vitamins than MEM and is often used to culture difficult-to-maintain cells or mesenchymal stem cells.

[0067] DMEM: A commonly used, nutrient-rich culture medium suitable for the in vitro culture of a variety of mammalian cells. It contains a variety of essential amino acids, vitamins, and inorganic salts, providing the nutrients and environment required for cell growth, promoting cell proliferation and differentiation.

[0068] FBS: Fetal bovine serum

[0069] PSG: Penicillin-Streptomycin-Glutamine

[0070] HEPES: 4-Hydroxyethylpiperazineethanesulfonic acid

[0071] ITS: Insulin-Transferrin-Zinc

[0072] HSA: human serum albumin

[0073] PDGF: Platelet-derived growth factor

[0074] FGF-2: Fibroblast Growth Factor 2

[0075] TGF-β3: transforming growth factor beta 3

[0076] BMP-6: bone morphogenetic protein 6

[0077] ECGS: Endothelial cell growth supplement, an indispensable supplement for the in vitro culture of endothelial cells, can optimize the in vitro growth environment of cells and promote the normal proliferation and growth of endothelial cells.

[0078] HE staining: A common staining method used in paraffin sectioning. Hematoxylin staining is alkaline, staining the chromatin in the cell nucleus and the nucleic acids in the cytoplasm purple-blue; eosin, an acidic dye, stains components of the cytoplasm and extracellular matrix red.

[0079] Type II collagen: A high-molecular-weight protein. Filamentous collagen fibers interweave with elastin and polysaccharides to form a network structure, providing mechanical strength. Produced primarily by chondrocytes, it is found in tissues such as bones, joints, and tendons.

[0080] Safranin-Fast Green staining: A commonly used staining method that can visually reflect the structure of articular cartilage, subchondral bone, and bone tissue. Cartilage appears red, while fibrous tissue and bone tissue appear green.

[0081] VEGF: Vascular endothelial growth factor, promotes vascular endothelial growth, increases vascular permeability, and promotes vascular endothelial migration, proliferation and angiogenesis.

[0082] Osterix: A newly discovered transcription factor related to osteoblast differentiation and bone formation, which is specifically expressed only in developing bone tissue.

[0083] TRAP: Tartrate-resistant acid phosphatase, is a specific stain for detecting osteoclasts in bone tissue, making osteoclasts appear red.

[0084] CD31: Platelet-endothelial cell adhesion molecule, mainly located in the tight junctions between vascular endothelial cells.

[0085] α-SMA: α-smooth muscle actin

[0086] Immunohistochemistry: It is a research method that applies the basic principle of immunology, namely the principle of specific binding between antigen and antibody, to determine the antigen in tissue cells through chemical reaction to make the color developer of the labeled antibody develop color, and to locate, characterize and relatively quantify it.

[0087] Flow cytometry: Used to count and sort tiny particles, such as cells, suspended in a fluid. It can be used to continuously analyze multiple parameters of individual cells as they flow past optical or electronic detectors.

[0088] Colony-forming unit (CFU) assay: An effective method for measuring the proliferation capacity of cultured cells. The principle is that when a single cell proliferates for more than six generations in vitro, its offspring form a cell population called a colony.

[0089] Example 1 Comparison of consistent and sequential MVF extraction

[0090] 1. Consistent extraction method:

[0091] (a) Human adipose tissue obtained from liposuction surgery was rinsed three times with saline, and the upper layer of adipose tissue was collected.

[0092] (b) Washed fat was mixed with an equal volume of 0.15% collagenase solution (final collagenase concentration) and shaken at 37°C, 150 rpm, for 15 minutes. The suspension was centrifuged (300 g, 3 minutes), the supernatant removed, and the MVF pellet resuspended in PBS. The pellet was passed through a sterile 500-μm filter and centrifuged at 300 g for 3 minutes. The supernatant was removed and the pellet resuspended in PBS to obtain the consistently extracted MVF.

[0093] 2. Sequential extraction method:

[0094] (a) Human adipose tissue obtained from liposuction surgery was rinsed three times with saline, and the upper layer of adipose tissue was collected.

[0095] (b) First digestion: Mix the washed fat with an equal volume of 0.15% collagenase solution (final collagenase concentration) and incubate at 37°C, shaking at 150 rpm for 5 minutes. Centrifuge at 300 g for 3 minutes. Collect the undigested fat in the upper layer into a new centrifuge tube for the second digestion. Resuspend the MVF pellet in PBS.

[0096] (c) Second digestion: The undigested fat layer collected after centrifugation in step (b) was mixed with an equal volume of 0.15% collagenase solution, and the mixture was shaken at 37°C and 150 rpm for 5 minutes. The mixture was centrifuged at 300 g for 3 minutes, and the undigested fat layer was collected. PBS buffer was added to resuspend the MVF pellet at the bottom.

[0097] (d) The undigested fat collected from the upper layer after centrifugation in step (c) was mixed with an equal volume of 0.15% collagenase solution, and the mixture was shaken at 150 rpm at 37°C for 5 minutes. The mixture was centrifuged at 300 g for 3 minutes, the supernatant was removed, and PBS buffer was added to resuspend the MVF pellet at the bottom.

[0098] (e) The microvessel fragment suspensions obtained from the digestion in steps (b), (c), and (d) were mixed evenly, filtered through a sterile 500-μm filter to remove larger tissue fragments, and centrifuged at 300 g for 3 minutes. The supernatant was removed and the pellet was resuspended in PBS buffer to obtain sequentially extracted MVFs. Figure 1Flowchart of traditional consistent extraction method and sequential extraction method.

[0099] 3. Experimental testing

[0100] 1. Calculate the fat volume before and after digestion using the two methods. Statistical analysis shows that when the total digestion time is 15 minutes, sequential extraction consumes more fat than consistent extraction, and has a significantly higher fat utilization efficiency ( Figure 2 ).

[0101] 2. Observe the morphology of MVF extracted by the two methods under an inverted microscope ( Figure 3 , white arrows indicate MVF) and counted them. Statistical analysis showed that when the total digestion time was 15 minutes, the number of MVF obtained by sequential extraction was significantly higher than that by consistent extraction ( Figure 4 ).

[0102] 3. Live / dead cell staining of MVF extracted by two methods ( Figure 5 , live cells are green, dead cells are red), and the ratio of live cells to dead cells was statistically analyzed. The results showed that the ratio of live cells in MVF obtained by sequential extraction method was significantly higher than that by consistent extraction method ( Figure 6 ).

[0103] 4. Flow cytometry analysis of MVFs extracted by the two methods was performed, as follows: Figure 7 As shown in Figure 2, MVF contains endothelial cells, mesenchymal stem cells, and pericytes. Statistical analysis showed that the proportions of endothelial cells, mesenchymal stem cells, and pericytes in MVF obtained by the sequential extraction method were significantly higher than those obtained by the consistent extraction method ( Figure 8 ).

[0104] Example 2 In vitro culture of self-aggregated microfat tissue

[0105] 1. Preparation of the experimental group:

[0106] 1. Preparation of Micro-micronized Adipose Tissue

[0107] (a) Human adipose tissue obtained from liposuction surgery was rinsed three times with saline, and the upper layer of adipose tissue was collected.

[0108] (b) The upper layer of fat tissue was minced with scissors and centrifuged at 1600 g for 3 min to remove the upper fat layer and the lower tumescent fluid. The middle fat layer was collected into a 20 mL syringe.

[0109] (c) Use a sterile fat cutter to connect two 20 mL syringes and push the two syringes back and forth 35 times to obtain micronized adipose tissue;

[0110] (d) The microfat tissue was centrifuged at 1600 g for 3 min, and the upper lipid layer was removed, leaving the microfat tissue mixture at the lower layer.

[0111] 2. Sequential Extraction Method for Extracting MVF

[0112] Same steps as in Example 1.

[0113] 3. Dynamic Culture of Self-aggregated Micro-adipocytes

[0114] Under sterile conditions, 3 mL of prepared microfat tissue was transferred into the rotary bioreactor and the mixture was diluted with 2.0 × 10 5 MVFs were added at a ratio of 100 MVFs / mL of micro-adipocytes, and 10 mL of culture medium was added. Dynamic culture was performed by perfusing the medium at a rate of 0.47 mL / min. The medium was changed twice weekly. After two weeks of culture, the micro-adipocytes spontaneously aggregated into round particles approximately 4 mm in diameter as the culture medium was perfused.

[0115] The culture medium and the corresponding culture time were: dynamic proliferation medium for 2 weeks, dynamic angiogenesis culture for 1 week, and dynamic differentiation medium for 3 weeks. After the differentiation culture was completed, cartilage particles of the experimental group were formed.

[0116] The composition of the proliferation medium used in the dynamic proliferation culture is as follows: α-MEM + 10% FBS + 1% PSG + 1% HEPES + FGF-2 (10 ng / mL) + PDGF (10 ng / mL) + dexamethasone (10 -7 mol / L) + ascorbic acid (10 -5 mol / L), and the perfusion rate of the dynamic proliferation medium was 0.47 mL / min;

[0117] The composition of the angiogenic culture medium used in the dynamic angiogenic culture is as follows: a-MEM + 10% FBS + 1% PSG + 1% HEPES + 1% ITS + ECGS (10 ng / mL).

[0118] The composition of the differentiation medium used in the dynamic differentiation culture is as follows: DMEM + 1% HSA + 1% PSG + 1% HEPES + 1% ITS + 0.5% linoleic acid + dexamethasone (10 -7mol / L) + ascorbic acid (1 mol / L) + BMP-6 (10 ng / mL) + TGF-β3 (10 ng / mL) + SB431542 (10 μmol / L), and the perfusion rate of the dynamic differentiation medium was 0.47 mL / min.

[0119] 2. Preparation of the control group

[0120] (1) Preparation of micro-adipocytes: The preparation of micro-adipocytes was the same as that described in step 1 of the experimental group.

[0121] (2) Static culture of microfat tissue: 2 mL of microfat tissue and 3 mL of proliferation medium were added to each well of a sterile 6-well plate (the culture medium was changed twice a week). After static culture in a common cell culture incubator for 2 weeks, the microfat tissue was cut into round particles with a diameter of 4 mm using a sterile tissue ring drill. The particles were cultured in angiogenic culture medium for 1 week and statically cultured in differentiation medium for 3 weeks to form cartilage particles in the control group. No MVF was added to the control group throughout the process, and no dynamic culture was used.

[0122] 3. Experimental testing

[0123] 1. The cartilage particles obtained from the experimental group and the control group were stained with safranin-fast green and immunohistochemically stained for type II collagen.

[0124] The results are as follows Figure 10 and Figure 11 As shown by Figure 10 It can be seen that the cartilage particles in both the experimental and control groups contained safranin fast green staining which was red ( Figure 10 ) and collagen type II stained red ( Figure 10 ) of cartilage tissue components, but the proportion of cartilage area in the experimental group to the total tissue area was significantly greater than that in the control group ( Figure 10 、 Figure 11 ). This shows that the culture method of the experimental group can more effectively promote the differentiation of adipose tissue into cartilage tissue compared with the control group under the same culture time.

[0125] 2. VEGF is a highly specific vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis.

[0126] VEGF immunohistochemical staining results showed that the experimental group was VEGF positive, while the control group was negative ( Figure 12 ), and the VEGF staining intensity in the experimental group was significantly higher than that in the control group ( Figure 13This indicates that the experimental group's culture method can produce more VEGF protein than the control group under the same culture time, which plays a very important role in the formation of highly vascularized bone marrow organoids from cartilage particles in subsequent in vivo experiments.

[0127] Example 3 Structural Identification of Bone Marrow Organoids

[0128] Under sterile conditions, the experimental and control cartilage particles obtained in Example 2 were implanted subcutaneously into the backs of 4-week-old female severely immunodeficient mice. After 8 weeks, the mice were killed and the implants removed. The experimental and control cartilage particles developed into bone marrow organoids in the body, respectively. The bone marrow organoids in the experimental and control groups were stained with hematoxylin and eosin (HE), and the location of the bone marrow cavity was marked with a yellow dotted line ( Figure 14 ), the results showed that the proportion of bone marrow cavity area in the bone marrow organoids in the experimental group was significantly greater than that in the control group ( Figure 14 、 Figure 15 The experimental group's bone marrow organoids contained abundant bone, bone marrow cells, and a small amount of adipose tissue, while the control group's bone marrow organoids were mostly adipose tissue vacuoles with a very small bone marrow cavity area ( Figure 14 ).

[0129] Osteoblasts and osteoclasts are important components of the endosteal niche in bone marrow organoids. Osterix and TRAP staining were used to label osteoblasts and osteoclasts in bone marrow organoids, respectively. The results showed that a large number of Osterix and TRAP staining positive cells were present in the bone marrow organoids of the experimental group ( Figure 16 The black arrows in the middle of the image indicate that the bone marrow organoids in the experimental group had a complete endosteal niche structure, while the endosteal niche structure in the bone marrow organoids in the control group was less complete.

[0130] The vascular niche is an important structural component of bone marrow organoids. The function of bone marrow organoids is to recruit hematopoietic stem cells and maintain their function. The density and diameter of blood vessels determine whether bone marrow organoids can function. α-SMA and CD31 were used to mark vascular smooth muscle cells and endothelial cells in bone marrow organoids, respectively. Figure 17 As shown, red is α-SMA, green is CD31, and blue is cell nucleus). The results showed that the blood vessel density in the bone marrow organoids of the experimental group was significantly higher than that of the control group ( Figure 17 、 Figure 18 This indicates that the bone marrow organoids in the experimental group have abundant blood vessels and complete vascular niche structure, while the bone marrow organoids in the control group have low blood vessel density and incomplete vascular niche structure.

[0131] Example 4 Functional Characterization of Bone Marrow Organoids

[0132] Under sterile conditions, the experimental group cartilage particles and the control group cartilage particles obtained in Example 2 were implanted subcutaneously on the backs of 6-week-old female severely immunodeficient mice. After 10 weeks, 4×10 5 human CD34 + After 12 weeks, the cell components of the bone marrow organoids in the experimental group and the control group were analyzed by flow cytometry ( Figure 19 ) and colony formation assays.

[0133] Definition of human cell chimerism = (human CD45 + cells) / (human CD45 + cells+mouse CD45 + Flow cytometry results showed that the chimerism rate of human cells in the bone marrow organoids of the experimental group was significantly higher than that in the bone marrow organoids of the control group ( Figure 19 、 Figure 20 ).

[0134] Cells were extracted from bone marrow organoids of the experimental and control groups, and human CD34 + For colony formation assay, count the number of cells per 2 × 10 4 The total number of cells that could form colonies after 14 days showed that human CD34 + Human CD34 in cells and control bone marrow organoids + Compared with cells, under the same starting cell number, culture conditions and culture time, the total number of knockdowns that can be formed is significantly increased ( Figure 21 The above results show that compared with the control group, the bone marrow organoids in the experimental group can recruit more human hematopoietic cells in mice and better maintain the recruited human CD34 + Functions of hematopoietic stem and progenitor cells.

[0135] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. The embodiments of this application and the features in the embodiments may be combined with each other in any manner unless there is a conflict.

Claims

1. A method for constructing human bone marrow organoids, characterized in that: The following steps are involved: Step (1) extracting microvessel fragments from human adipose tissue by enzymatic digestion; Step (2) Mechanically prepare human microfat tissue, add human microfat tissue and 2.0×10 5 The microvessel fragments were added at a ratio of 10 microvessel fragments / mL of microfatty tissue, and culture medium was added to perform dynamic culture to obtain cartilage particles; Step (3) transplanting the cartilage particles into the subcutaneous tissue of a severely immunodeficient mouse, wherein after a predetermined period of time, the cartilage particles develop into humanized bone marrow organoids with specific functions in the severely immunodeficient mouse; wherein the specific functions include having a complete endosteal niche, a vascular niche, and a hematopoietic cell niche and maintaining the physiological function of human hematopoietic stem cells; Wherein, step (2) dynamically cultures cartilage particles, wherein the dynamic culture includes dynamic proliferation culture, dynamic vascularization culture and dynamic differentiation culture; The composition of the proliferation medium used in the dynamic proliferation culture is as follows: a-MEM, 2-20% FBS, 0-5% PSG, 0-5% HEPES, 0-5% glutamine, 1-20 ng / mL FGF-2, 1-20 ng / mL PDGF, 0.5×10 -7 -5×10 -7 mol / L dexamethasone, 0.5×10 -5 -5×10 -5 mol / L ascorbic acid; The composition of the angiogenesis medium used in the dynamic angiogenesis culture is as follows: a-MEM, 10% FBS, 1% PSG, 1% HEPES, 1% ITS, 10 ng / mL ECGS; The composition of the differentiation medium used in the dynamic differentiation culture is as follows: DMEM, 2-20% HSA, 0-5% PSG, 0-5% HEPES, 0-5% glutamine, 0-5% ITS, 0-5% linoleic acid, 0.5×10 -7 -5×10 -7 mol / L dexamethasone, 0.5×10 -5 -5×10 -5 mol / L ascorbic acid, 1-20 ng / mL BMP-6, 1-20 ng / mL TGF-β3, and 1-20 μmol / L SB431542.

2. The method for constructing human bone marrow organoids according to claim 1, characterized in that: Extracting microvessel fragments in step (1) specifically includes: (a) Obtain human adipose tissue, rinse and collect, mix with equal volumes of collagenase solution, shake under predetermined conditions, and centrifuge; (b) After centrifugation, the undigested adipose tissue in the upper layer was collected, and PBS buffer was added to resuspend the microvascular fragments in the lower layer; (c) The collected upper layer of undigested adipose tissue was remixed with an equal volume of collagenase solution, shaken, and centrifuged; (d) Repeat steps (b) and (c) multiple times; (e) The microvessel fragment suspensions obtained multiple times are mixed, centrifuged, and the supernatant is removed to obtain microvessel fragments.

3. The method for constructing human bone marrow organoids according to claim 2, characterized in that: Extracting the microvessel fragments in step (1) specifically further includes: filtering the microvessel fragment suspension through a filter before centrifugation in step (e).

4. The method for constructing human bone marrow organoids according to claim 3, characterized in that: The pore size of the filter is 40-600 microns.

5. The method for constructing human bone marrow organoids according to claim 2, wherein: The shaking conditions are 30-37°C, 50-500 rpm, and constant shaking for 3-15 minutes.

6. The method for constructing human bone marrow organoids according to claim 2, characterized in that: The centrifugation conditions in steps (a) to (e) are all 50-1000 g for 1-5 minutes.

7. The method for constructing human bone marrow organoids according to claim 1, wherein: The bioreactor in step (2) is a rotary or rocking bioreactor.

8. The method for constructing human bone marrow organoids according to claim 1, wherein: The perfusion rate of the proliferation culture and the vascularization promotion culture is 0.1-2 mL / min.

9. The method for constructing human bone marrow organoids according to claim 1, wherein: The culture time of the dynamic proliferation culture and the dynamic angiogenesis promotion culture is 1-4 weeks and 1-4 weeks respectively.

10. The method for constructing human bone marrow organoids according to claim 1, wherein: The perfusion rate of the differentiation medium is 0.1-2 mL / min.

11. The method for constructing human bone marrow organoids according to claim 1, wherein: The dynamic differentiation culture time is 2-10 weeks.

12. The method for constructing human bone marrow organoids according to any one of claims 1 to 11, characterized in that: The particle size of the micro-fat tissue is 1-6 mm.

13. The method for constructing human bone marrow organoids according to claim 1, wherein: Step (2) of preparing micro-fat tissue specifically includes the following steps: (1) Collect human adipose tissue, rinse 1-3 times, mince, centrifuge at 500-2000 g for 1-5 min, remove the upper fat layer and the lower swelling fluid, and collect the middle fat layer in a syringe; (2) Prepare another syringe and connect it to the syringe that collects the middle fat layer through the fat cutter. Push the two syringes back and forth 10-50 times, and then centrifuge at 500-2000 g for 1-5 minutes to remove the upper fat layer. The lower layer is the micro-fat tissue.

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