An organ-on-a-chip simulating acute myeloid leukemia and its applications

Abstract

This invention relates to the field of biochips, and particularly to an organ-on-a-chip simulating acute myeloid leukemia and its applications. The organ-on-a-chip provided by this invention includes a cell culture compartment, a culture medium exchange module, and a hydrogel injection module. The culture medium exchange module and the hydrogel injection module are respectively connected to the cell culture compartment. The cell culture compartment, from the center outwards, includes a biomimetic central sinus region, a biomimetic medullary cavity region, and a biomimetic endosteal region. The biomimetic medullary cavity region completely surrounds the biomimetic central sinus region, and the biomimetic endosteal region partially surrounds the biomimetic medullary cavity region. The organ-on-a-chip provided by this invention can serve as an in vitro model that can accurately simulate the physiological structure and multicellular microenvironment of human bone marrow. Combining the advantages of simple fabrication and high reproducibility of organ-on-a-chip, it can meet the high accuracy and high throughput requirements for drug screening and evaluation, drug resistance testing, and principle research.

Description

Technical Field

[0001] This invention relates to the field of biochips, and in particular to an organ-on-a-chip that simulates acute myeloid leukemia and its uses. Background Technology

[0002] Acute myeloid leukemia (AML) is a common hematologic malignancy, accounting for 60% of all leukemia cases. Currently, the main clinical treatment for AML is chemotherapy with cytarabine combined with anthracyclines, but the five-year survival rate is only 31.7%. Patients over 65 years of age often cannot tolerate highly toxic chemotherapy regimens, resulting in a worse prognosis compared to younger patients, with a five-year survival rate of less than 10%. Chemotherapy resistance is the main reason for the low survival rate of AML clinical treatment; the effectiveness of treatment often declines with the extension of the treatment cycle. The mechanisms of AML drug resistance are complex. Multiple studies have shown that AML drug resistance is not only related to the biological characteristics of AML cells themselves, but also to the important role played by the bone marrow microenvironment in which AML cells reside. The bone marrow microenvironment consists of the endosteum, medullary cavity, and central sinus anatomical regions, and can be divided into the endosteum microenvironment and the vascular microenvironment. The former is mainly composed of osteoblasts and osteoclasts, while the latter includes various cell types such as vascular endothelial cells and stromal cells. The microenvironment also contains various substances such as nutrients and metabolites, oxygen, extracellular matrix, and cytokines. AML cells alter the phenotype and function of cells in the microenvironment through direct contact and secretion of factors, reshaping the microenvironment into one that is more conducive to their own survival and proliferation.

[0003] Although the bone marrow microenvironment plays a crucial role in the challenge of AML drug resistance, existing preclinical models have limitations in simulating the AML bone marrow microenvironment: traditional cell models are simple to construct and cannot simulate the tissue structure of real bone marrow, and there is a lack of reports on the co-culture of AML cells with various microenvironment cells; while animal models can simulate the bone marrow microenvironment to some extent, their construction is time-consuming and complex, and species differences often limit the reliability of animal experimental results. These shortcomings lead to a high failure rate in the clinical trial phase of developing novel AML treatments or regimens. Therefore, there is an urgent need to develop preclinical models that mimic bone marrow anatomy, simulate the vascular and endosteal microenvironment, and are easy to construct, to explore the mechanisms of chemotherapy resistance induced by the AML bone marrow microenvironment, and to provide theoretical guidance and reliable data for novel AML clinical treatment regimens.

[0004] Organ-on-a-chip and other technologies can highly simulate the key structural and functional characteristics of tissues and have been widely used in disease modeling and drug development. However, existing AML organ-on-a-chips have certain shortcomings. For example, the classic three-channel structure used in existing AML organ-on-a-chips does not conform to the anatomical structure of bone marrow in vivo; and existing AML organ-on-a-chips only contain one or two types of microenvironment cells, failing to comprehensively simulate the vascular and endosteal microenvironment of bone marrow. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an organ-on-a-chip that simulates acute myeloid leukemia and its uses, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides an organ-on-a-chip that simulates acute myeloid leukemia. The organ-on-a-chip includes a cell culture compartment, a culture medium exchange module, and a hydrogel injection module. The culture medium exchange module and the hydrogel injection module are respectively connected to the cell culture compartment. The cell culture compartment includes, from the center outwards, a biomimetic central sinus region, a biomimetic medullary cavity region, and a biomimetic endosteal region. The biomimetic medullary cavity region completely surrounds the biomimetic central sinus region, and the biomimetic endosteal region partially surrounds the biomimetic medullary cavity region.

[0007] Preferably, the biomimetic endometrial region includes a first biomimetic endometrial region and a second biomimetic endometrial region arranged symmetrically, and the first biomimetic endometrial region or the second biomimetic endometrial region partially surrounds the biomimetic medullary cavity region.

[0008] The present invention also provides the use of the aforementioned organ-on-a-chip in constructing an in vitro model of acute myeloid leukemia.

[0009] This invention also provides a method for preparing an in vitro acute myeloid leukemia model, the method comprising the following steps:

[0010] 1) Place the fibrin hydrogel mixed with vascular endothelial cells in the biomimetic central sinus region of the aforementioned organ-on-a-chip;

[0011] 2) Place the fibrin hydrogel mixed with AML cells, vascular endothelial cells and bone marrow stromal cells in the biomimetic medullary cavity region of the aforementioned organ-on-a-chip;

[0012] 3) Place the fibrin hydrogel mixed with AML cells and osteoblasts in the biomimetic endosteal region of the aforementioned organ-on-a-chip.

[0013] The present invention also provides an in vitro acute myeloid leukemia model prepared by the aforementioned preparation method.

[0014] The present invention also provides the use of the aforementioned in vitro acute myeloid leukemia model in drug screening.

[0015] As described above, the organ-on-a-chip simulating acute myeloid leukemia and its uses according to the present invention have the following beneficial effects:

[0016] The organ-on-a-chip provided by this invention can serve as an in vitro model that can accurately simulate the physiological structure of human bone marrow and the multicellular microenvironment. Combining the advantages of organ-on-a-chip fabrication, such as its simplicity and high reproducibility, it can meet the high accuracy and high throughput requirements of drug screening and evaluation, drug resistance testing, and principle research, and help accelerate the development of novel AML treatment options, including small molecule targeted drugs, antibody drugs, and cell therapy. Attached Figure Description

[0017] Figure 1 The diagram shown is a planar view of the organ-on-a-chip according to the present invention.

[0018] Figure 2 The image shown is a three-dimensional diagram of the organ-on-a-chip according to the present invention.

[0019] Figure 3 The image shown is a physical diagram of the micropillars in the organ-on-a-chip of this invention.

[0020] Figure 4 The image shown is a cell fluorescence pattern in the organ-on-a-chip of the present invention, in which Molm-13, OCI-AML3: acute myeloid leukemia cells; hFOB1.19: human osteoblasts; HS-5: human bone marrow stromal cells; HUVEC: human umbilical vein endothelial cells.

[0021] Figure 5 The results shown are the drug resistance test results of the in vitro acute myeloid leukemia model constructed based on organ-on-a-chip according to the present invention. Among them, Single-Molm13: the chip contains only AML cells; env-Molm13: the chip contains HUVEC, hFOB1.19 and HS-5 cells in addition to AML cells; Arac: cytarabine, concentration of 100 nM; DMSO: dimethyl sulfoxide.

[0022] Figure 6 The diagram shown is a structural concept diagram of an organ-on-a-chip according to the present invention. Detailed Implementation

[0023] This invention provides an organ-on-a-chip that simulates acute myeloid leukemia. The organ-on-a-chip includes a cell culture compartment, a culture medium exchange module 4, and a hydrogel injection module 5. The culture medium exchange module 4 and the hydrogel injection module 5 are respectively connected to the cell culture compartment. The cell culture compartment includes, from the center outwards, a biomimetic central sinus region 1, a biomimetic medullary cavity region 2, and a biomimetic endosteal region 3. The biomimetic medullary cavity region 2 completely surrounds the biomimetic central sinus region 1, and the biomimetic endosteal region 3 partially surrounds the biomimetic medullary cavity region 2.

[0024] In some specific embodiments, the bionic endosteal region 3 includes a first bionic endosteal region 31 and a second bionic endosteal region 32 arranged symmetrically, and the first bionic endosteal region 31 or the second bionic endosteal region 32 partially surrounds the bionic medullary cavity region 2.

[0025] In some specific embodiments, the organ-on-a-chip further includes a culture medium exchange module 4, which is connected to the biomimetic endosteal region 3.

[0026] Furthermore, the culture medium exchange module 4 includes a culture medium reservoir 41 and a culture medium flow channel 42. The culture medium reservoir (41) is connected to the first bionic endosteal region (31) or the second bionic endosteal region (32) through the culture medium flow channel (42).

[0027] Furthermore, the culture medium exchange module 4 is provided in one or more forms. Specifically, the culture medium exchange module 4 is provided in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more forms. Preferably, the culture medium exchange module 4 is provided in four forms.

[0028] Furthermore, when multiple culture medium exchange modules 4 are provided, the multiple culture medium exchange modules 4 are symmetrically arranged with the cell culture compartment as the center.

[0029] In some specific embodiments, the organ-on-a-chip further includes a hydrogel injection module 5, which is connected to the biomimetic medullary cavity region 2.

[0030] Furthermore, the hydrogel injection module 5 includes a hydrogel injection port 51 and a hydrogel fluid channel 52, with the hydrogel injection port 51 connected to the biomimetic medullary cavity region 2 via the hydrogel fluid channel 52.

[0031] Furthermore, the end of the hydrogel fluid channel 52 near the hydrogel injection port 51 is wider than the other end near the biomimetic medullary cavity region 2. This narrowing at the end near the biomimetic medullary cavity region 2 allows the hydrogel fluid channel 52 to be easily separated from the culture medium channel 42 during chip photolithography, facilitating chip etching.

[0032] Furthermore, the hydrogel injection module 5 is provided in one or more forms. Specifically, the hydrogel injection module 5 is provided in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more forms. Preferably, the hydrogel injection module 5 is provided in two forms.

[0033] Furthermore, when multiple hydrogel injection modules 5 are provided, the multiple hydrogel injection modules 5 are symmetrically arranged with the cell culture compartment as the center.

[0034] In some specific embodiments, a plurality of micropillars 6 are provided at the junction of the bionic central sinus region 1 and the bionic medullary cavity region 2, or at the junction of the bionic medullary cavity region 2 and the bionic endosteal region 3 in the organ-on-a-chip. The micropillars are used to prevent hydrogel in the bionic central sinus region 1, the bionic medullary cavity region 2, or the bionic endosteal region 3 from flowing into adjacent areas.

[0035] In some specific embodiments, the cross-sectional shape of the micropillar 6 is trapezoidal, circular, triangular, parallelogram, irregular quadrilateral, pentagonal, hexagonal, or any other closed planar figure. Preferably, the micropillar 6 is trapezoidal.

[0036] Furthermore, when the cross-sectional shape of the micropillar 6 is trapezoidal, the top edge of the trapezoid is closer to the geometric center of the biomimetic central sinus region 1 than the bottom edge of the trapezoid.

[0037] Furthermore, when the cross-sectional shape of the micropillar 6 is trapezoidal, the ratio of the side length of the top side to the side length of the trapezoid is 1:2.5-1:3.5. Specifically, the side length ratio is 1:2-1:2.5, 1:2.5-1:3, 1:3-1:3.5, or 1:3.5-1:4. Preferably, the side length ratio is 1:3.

[0038] Furthermore, when the cross-sectional shape of the micropillar 6 is trapezoidal, the length of the top edge of the trapezoid is 50-150 μm. Specifically, the length of the top edge of the trapezoid is 50-70 μm, 70-90 μm, 90-100 μm, 100-110 μm, 110-130 μm, or 130-150 μm. Preferably, the length of the top edge of the trapezoid is 100 μm.

[0039] In some specific embodiments, gaps exist between multiple micropillars 6. When the cross-sectional shape of the micropillar 6 is trapezoidal, the top edge gap distance a between adjacent micropillars 6 between the bionic central sinus region 1 and the bionic medullary cavity region 2 is 220-230 μm, the bottom edge gap distance b is 90-100 μm, and the included angle β between the geometric centers of two adjacent micropillars 6 is 20°-25°; and / or, the top edge gap distance a between adjacent micropillars 6 between the bionic medullary cavity region 2 and the protected bionic endosteal region 3 is 250-255 μm, the bottom edge gap distance b is 90-100 μm, and the included angle β between the geometric centers of two adjacent micropillars 6 is 12-15°. Preferably, the top edge gap distance a between adjacent micropillars 6 in the bionic central sinus region 1 and the bionic medullary cavity region 2 is 224 μm, the bottom edge gap distance b is 96 μm, and the included angle β between the geometric centers of two adjacent micropillars 6 is 22.5°; and / or, the top edge gap distance a between adjacent micropillars 6 in the bionic medullary cavity region 2 and the protected bionic endosteal region 3 is 253 μm, the bottom edge gap distance b is 94 μm, and the included angle β between the geometric centers of two adjacent micropillars 6 is 13.3°. Wherein, the included angle β is the angle formed by the normals of adjacent top and bottom edges perpendicular to the cross-section of the micropillar 6 and passing through the geometric center of the trapezoid; the geometric center is the intersection of the diagonals of the trapezoid.

[0040] In some specific embodiments, 14-18 micropillars 6 are arranged in a circular pattern between the bionic central sinus region 1 and the bionic medullary cavity region 2. Specifically, there are 14, 15, 16, 17, or 18 micropillars. Preferably, there are 16 micropillars 6.

[0041] In some specific embodiments, 22-30 micropillars 6 are provided between the biomimetic medullary cavity region 2 and the biomimetic endosteal region 3 (11-15 on each side), arranged in a circular pattern. Specifically, there are 22, 23, 24, 25, 26, 27, 28, 29, or 30 micropillars 6. Preferably, there are 26 micropillars 6.

[0042] In some specific embodiments, the cross-sectional shape of the biomimetic central sinus region 1 is a circle, rectangle, triangle, trapezoid, parallelogram, irregular quadrilateral, pentagon, hexagon, or any other closed planar figure. Preferably, the biomimetic central sinus 1 is circular.

[0043] Furthermore, when the cross-sectional shape of the bionic central sinus 1 is circular, the cross-sectional shape of the bionic medullary cavity region 2 is annular.

[0044] Furthermore, when the cross-sectional shape of the bionic central sinus 1 is circular and the cross-sectional shape of the bionic medullary cavity region 2 is annular, the cross-sectional area ratio of the bionic central sinus 1 to the bionic medullary cavity region 2 is 16:30-36. Specifically, the area ratio is 16:30-32, 16:32-33, 16:33-34, or 16:34-36. Preferably, the area ratio is 16:33.

[0045] Furthermore, when the cross-sectional shape of the biomedullary cavity region 2 is annular, the cross-sectional shape of the biomimetic endosteal region 3 is semi-annular.

[0046] Furthermore, when the cross-sectional shape of the biomedullary cavity region 2 is annular and the cross-sectional shape of the biomedullary endosteal region 3 is semi-annular, the cross-sectional area ratio of the biomedullary cavity region 2 to the biomedullary endosteal region 3 is 13:5-9. Specifically, the area ratio is 13:5-6, 13:6-7, 13:7-8, or 13:8-9. Preferably, the area ratio is 13:7.

[0047] The present invention also provides the use of the aforementioned organ-on-a-chip in constructing an in vitro model of acute myeloid leukemia.

[0048] This invention also provides a method for preparing an in vitro acute myeloid leukemia model, the method comprising the following steps:

[0049] 1) Place the fibrin hydrogel mixed with vascular endothelial cells into the biomimetic central sinus region 1 of the aforementioned organ-on-a-chip;

[0050] 2) Place the fibrin hydrogel containing AML cells, vascular endothelial cells and bone marrow stromal cells into the biomimetic medullary cavity region 2 of the aforementioned organ-on-a-chip;

[0051] 3) Place the fibrin hydrogel mixed with AML cells and osteoblasts in the biomimetic endosteal region 3 of the aforementioned organ-on-a-chip.

[0052] In some specific embodiments, based on the volume of the fibrin hydrogel in step 1), the density of the vascular endothelial cells is 0.8-1.2 × 10⁻⁶. 7 / mL. Specifically, the density is 0.8-0.9×10⁻⁶. 7 / mL, 0.9-1×10 7 / mL, 1-1.1×10 7 / mL or 1.1-1.2×10 7 / mL. Preferably, the density is 1×10⁻⁶. 7 / mL.

[0053] In some specific embodiments, based on the volume of the fibrin hydrogel in step 2), the ratio of the AML cells, the vascular endothelial cells, and the bone marrow stromal cells is (1.8-2.2):(0.9-1.1):(0.9-1.1). Preferably, the ratio is 2:1:1.

[0054] Furthermore, based on the volume of the fibrin hydrogel in step 2), the density of the AML cells is 4.6-5.4 × 10⁻⁶. 6 / mL. Specifically, the density is 4.6-4.8×10 6 / mL, 4.8-5×10 6 / mL, 5-5.2×10 6 / mL or 5.2-5.4×10 6 / mL. Preferably, the density is 5×10⁻⁶. 6 / mL.

[0055] In some specific embodiments, based on the volume of the fibrin hydrogel in step 3), the ratio of the AML cells to the osteoblasts is 1:0.8-1.2. Specifically, the ratio is 1:0.8-0.9, 1:0.9-1, 1:1-1.1, or 1:1.1-1.2. Preferably, the ratio is 1:1.

[0056] Furthermore, based on the volume of the fibrin hydrogel in step 3), the density of the AML cells is 4.6-5.4 × 10⁻⁶. 6 / mL. Specifically, the density is 4.6-4.8×10 6 / mL, 4.8-5×10 6 / mL, 5-5.2×10 6 / mL or 5.2-5.4×10 6 / mL. Preferably, the density is 5×10⁻⁶. 6 / mL.

[0057] In some specific embodiments, based on the volume of the fibrin hydrogel in steps 1), 2), or 3), the working concentration of fibrin in the fibrin hydrogel is 2.5-3.5 mg / ml. Specifically, the working concentration of fibrin is 2.5-2.8 mg / ml, 2.5-3 mg / ml, 3-3.2 mg / ml, or 3.2-3.5 mg / ml. Preferably, the working concentration of fibrin is 3 mg / ml.

[0058] The present invention also provides an in vitro acute myeloid leukemia model obtained by the aforementioned preparation method.

[0059] The present invention also provides the use of the aforementioned in vitro acute myeloid leukemia model in drug screening.

[0060] In some specific embodiments, the drug is a treatment for acute myeloid leukemia.

[0061] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0064] Example 1: Organ-on-a-Chip Fabrication

[0065] This embodiment is based on Figure 1 , Figure 2 or Figure 6 The structure shown is used to fabricate organ-on-a-chip. In AML organ-on-a-chip, micropillars are designed to separate adjacent regions (e.g., Figure 3As shown), a certain distance is left between the micropillars to ensure material diffusion and cell migration between regions. The AML organ-on-a-chip is manufactured using soft photolithography. A 200-micrometer thick SU-8 negative photoresist is spin-coated onto a silicon wafer using a spin coater. After UV exposure, baking, and rinsing, a master mold is prepared on the silicon wafer. The PDMS substrate and curing agent are mixed evenly at a weight ratio of 10:1 and poured onto the master mold. After removing air bubbles, it is placed in an 80°C oven for 1 hour. After peeling the PDMS off the master mold, holes with a diameter of 1 mm are punched in the biomimetic central sinus region (1) and the hydrogel injection port 51, and a hole with a diameter of 4 mm is punched in the culture medium reservoir 41. The punched PDMS is bonded to a 30 mm diameter circular quartz glass plate using a plasma cleaner and placed in an 80°C oven for overnight heating. The obtained AML organ-on-a-chip is sterilized using ultraviolet light for 20 minutes.

[0066] Example 2: Construction of an in vitro acute myeloid leukemia model

[0067] Using a pipette, 1 μL of fibrin hydrogel mixed with vascular endothelial cells was drawn and injected into the biomimetic central sinus region (1) through the 1 mm diameter pore as described in Example 1. The hydrogel was then placed in an incubator at 37°C and 5% CO2 and heated for 1 minute to allow cross-linking and solidification. Using a pipette, 5 μL of fibrin hydrogel mixed with AML cells, vascular endothelial cells, and bone marrow stromal cells was drawn and injected into the biomimetic medullary cavity region (2) through the 1 mm diameter pore at the hydrogel injection port 51 as described in Example 1. The hydrogel was then placed in an incubator at 37°C and 5% CO2 and heated for 1 minute to allow cross-linking and solidification. Using a pipette, 5 μL of fibrin hydrogel mixed with AML cells and osteoblasts was drawn and injected into the biomimetic endosteal region (3) through the culture medium reservoir 41 as described in Example 1. The hydrogel was then placed in an incubator at 37°C and 5% CO2 and heated for 1 minute to allow cross-linking and solidification. RPMI-1640 medium (containing 20% ​​fetal bovine serum), DMEM / F12 medium (containing 10% fetal bovine serum), and DMEM medium (containing 10% fetal bovine serum) were mixed at a volume ratio of 1:1:1 to prepare the culture medium for AML organ-on-a-chip. 50 μL of the chip culture medium was added to each of the four culture medium reservoirs 41 using a pipette. The AML organ-on-a-chip loaded with cells and culture medium was placed in an incubator at 37°C and 5% CO2, and the culture medium in reservoir 41 was changed every 24 hours. The distribution of cells in the chip under co-culture conditions is shown in the figure below. Figure 4 As shown.

[0068] Example 3: Application of an in vitro acute myeloid leukemia model

[0069] A 100 μM cytarabine solution (in dimethyl sulfoxide, DMSO) was mixed with the AML organ-on-a-chip medium at a volume ratio of 1:999 to achieve a final concentration of 100 nM cytarabine. 50 μL of the cytarabine-containing medium was added to each of the four culture media reservoirs 41 using a pipette. The AML organ-on-a-chip was then incubated at 37°C with 5% CO2, and the medium in reservoir 41 was replaced every 24 hours. Furthermore, fluorescence images of AML cells in the organ-on-a-chip were captured using a fluorescence microscope at 0, 24, 48, and 72 hours of culture, and the fluorescence area of ​​the AML cells was analyzed using image processing software. The fluorescence area of ​​AML cells in the untreated (DMSO) culture at 0, 24, 48, and 72 hours was used as a normalization benchmark to calculate the cell viability of AML cells treated with 100 nM cytarabine at 0, 24, 48, and 72 hours. Example results are shown below. Figure 5 As shown in the figure, the AML cells exhibit greater tolerance to the clinical chemotherapy drug cytarabine when the chip contains bone marrow microenvironment cells (HUVEC, HS-5, and hFOB1.19), without any change in the properties of the AML cells themselves.

[0070] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An organ-on-a-chip simulating acute myeloid leukemia, characterized in that, The organ-on-a-chip includes a cell culture compartment, a culture medium exchange module (4), and a hydrogel injection module (5). The culture medium exchange module (4) and the hydrogel injection module (5) are respectively connected to the cell culture compartment. The cell culture compartment includes a biomimetic central sinus area (1), a biomimetic medullary cavity area (2), and a biomimetic endosteal area (3) from the center outwards. The biomimetic medullary cavity area (2) completely surrounds the biomimetic central sinus area (1), and the biomimetic endosteal area (3) partially surrounds the biomimetic medullary cavity area (2).

2. The organ-on-a-chip according to claim 1, characterized in that, The bionic endometrial region (3) includes a first bionic endometrial region (31) and a second bionic endometrial region (32) arranged symmetrically, and the first bionic endometrial region (31) or the second bionic endometrial region (32) partially surrounds the bionic medullary cavity region (2).

3. The organ-on-a-chip according to claim 2, characterized in that, The culture medium exchange module (4) includes a culture medium reservoir (41) and a culture medium flow channel (42). The culture medium reservoir (41) is connected to the first bionic endosteal region (31) or the second bionic endosteal region (32) through the culture medium flow channel (42).

4. The organ-on-a-chip according to claim 1, characterized in that, The hydrogel injection module (5) includes a hydrogel injection port (51) and a hydrogel fluid channel (52), and the hydrogel injection port (51) is connected to the biomimetic medullary cavity region (2) through the hydrogel fluid channel (52).

5. The organ-on-a-chip according to claim 1, characterized in that, Multiple micropillars (6) are provided at the junction of the bionic central sinus region (1) and the bionic medullary cavity region (2) in the organ chip, or at the junction of the bionic medullary cavity region (2) and the bionic endosteal region (3), and each micropillar (6) is arranged in the same direction.

6. The organ-on-a-chip according to claim 5, characterized in that, There are gaps between multiple micropillars (6). When the cross-sectional shape of the micropillar (6) is trapezoidal, the top edge gap distance a between each adjacent micropillar (6) between the bionic central sinus region (1) and the bionic medullary cavity region (2) is 220-230 μm, the bottom edge gap distance b is 90-100 μm, and the included angle β between the geometric centers of two adjacent micropillars (6) is 20°-25°; and / or, the top edge gap distance a between each adjacent micropillar (6) between the bionic medullary cavity region (2) and the protected bionic endosteal region (3) is 250-255 μm, the bottom edge gap distance b is 90-100 μm, and the included angle β between the geometric centers of two adjacent micropillars (6) is 12-15°.

7. Use of the organ-on-a-chip as described in any one of claims 1-6 in constructing an in vitro model of acute myeloid leukemia.

8. A method for preparing an in vitro acute myeloid leukemia model, characterized in that, The preparation method includes the following steps: 1) Place the fibrin hydrogel mixed with vascular endothelial cells in the biomimetic central sinus region (1) of the organ-on-a-chip as described in any one of claims 1-6; 2) Place the fibrin hydrogel containing AML cells, vascular endothelial cells and bone marrow stromal cells into the biomimetic medullary cavity region (2) of the organ-on-a-chip as described in any one of claims 1-7; 3) Place the fibrin hydrogel containing AML cells and osteoblasts in the biomimetic endosteal region (3) of the organ-on-a-chip as described in any one of claims 1-6.

9. The preparation method according to claim 8, characterized in that, The preparation method includes one or more of the following features: I) Based on the volume of the fibrin hydrogel in step 1), the density of the vascular endothelial cells is 0.8-1.2 × 10⁻⁶. 7 / mL; II) Based on the volume of the fibrin hydrogel in step 2), the ratio of the AML cells, the vascular endothelial cells, and the bone marrow stromal cells is (1.8-2.2):(0.9-1.1):(0.9-1.1); preferably, based on the volume of the fibrin hydrogel in step 2), the density of the AML cells is 4.6-5.4 × 10⁻⁶. 6 / mL; III) Based on the volume of the fibrin hydrogel in step 3), the ratio of AML cells to osteoblasts is 1:0.8-1.2; preferably, based on the volume of the fibrin hydrogel in step 3), the AML cell density is 4.6-5.4 × 10⁻⁶. 6 / mL.

10. An in vitro acute myeloid leukemia model obtained by the preparation method according to claim 8 or 9.

11. Use of the in vitro acute myeloid leukemia model of claim 10 in drug screening.

Images