Vesicle and composition thereof

Abstract

The invention relates to the field of biotechnology and biological materials, in particular to a vesicle chip and application thereof. The vesicle is characterized in that a macromolecule hydrogel solution forms vesicle liquid drops with a certain diameter through a microchannel under shearing of gas, the vesicle liquid drops are atomized and crosslinked to form the solidified vesicle, and the diameter of the vesicle ranges from 50 micrometers to 800 micrometers; the vesicles are vesicles containing cells. The vesicles of different sizes and the highly-bionic vesicle fusion chip can be used for preparing homogenized tumor organs, the extracellular matrix environment in the body and the interaction among multiple cells can be effectively simulated, the reversible hydrogel curing technology facilitates cell separation and detection, and the application prospect is wide. Meanwhile, the processes of tumor metastasis, blood vessel invasion and the like can be simulated.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and biomaterials, and in particular to a vesicle and a composition thereof. Background Art

[0002] The traditional dish culture method cannot simulate the in vivo tumor microenvironment, which is the main problem leading to the failure of anti-tumor drug research and development. In the body, tumor tissue grows in a 3D environment, and the tumor microenvironment also includes immune cells, tumor-associated fibroblasts, endothelial cells, and pericytes. Therefore, the huge physiological differences between dish culture and the in vivo environment lead to the inability of traditional 2D culture models to truly reflect the tumor's response to drugs. In order to better simulate the tissue microenvironment, the in vitro culture model should have a three-dimensional culture environment that simulates the natural extracellular matrix, a multicellular ecology that simulates the in vivo tissue structure, and high-throughput and high-reproducibility conditions.

[0003] With the advancement of science and technology, various three-dimensional cell culture conditions that can provide the required three-dimensional structure for cell growth and good biocompatibility have emerged in recent years. The patient-derived tumor organoid (PDO) model developed in recent years has been widely used in preclinical tumor research and clinical drug screening because of its characteristics of maintaining tumor heterogeneity and high modeling success rate. Current research believes that tumor organoids have the potential to be used as an in vitro detection platform to predict the response to personalized immunotherapy. However, due to the lack of important physiological processes in the body and the lack of microenvironments such as immune cells and mesenchymal cells, the organoids constructed in vitro cannot completely replicate all the cell types and maturity of real organs, and there is no distribution of blood vessels, lymphatic vessels and nervous systems, which makes it difficult for organoid models to evaluate the true therapeutic response and drug mechanism of immunomodulators. In addition, the existing organoid technology simulates the in vivo tumor microenvironment by culturing primary cells in extracellular matrix proteins (such as matrix gel) and expanding them into cell spheres, but it has the problem of uncontrollable growth and different sizes of cell spheres, so the batch reproducibility of test results is poor.

[0004] Bio-3D printing technology is a new technology that uses a computer three-dimensional model as a "blueprint" and assembles special "bio-ink" to ultimately create a three-dimensional structure. It has good application prospects and huge social value in the field of tissue repair and regenerative medicine. Although 3D printing can achieve the printing of multi-cellular and multi-component bionic structures, its preparation time cycle is relatively long and it is impossible to achieve high-throughput organoid preparation. In addition, 3D printing cannot achieve micro-manufacturing, and sufficient ink and cells are required to achieve printing, which places certain requirements on the amount of patient-derived tumor tissue. Porous inks based on immiscible two-phases can achieve the formation of cell and organoid spheroids, but the pore size in the printed structure is difficult to control and cannot be uniform, so there are also challenges of uncontrollable growth and uneven sizes of organoids.

[0005] Microfluidics is a technology that manipulates two-phase or multi-phase fluids at the micrometer scale. Although the traditional oil-in-water droplet microfluidics method can prepare vesicles of uniform size, the microfluidics flux is extremely low and a secondary cleaning process is required to remove the oil phase, which increases the difficulty of the process and may damage cells, which is not conducive to the formation and batch manufacturing of organoids.

[0006] Cell microencapsulation is to encapsulate the target cells in one or more materials with good biocompatibility and semi-permeable membrane properties, so that the target cells can achieve immune isolation and avoid attacks by large molecular immune substances and immune cells, while allowing metabolites, small molecular nutrients and cell active substances to freely enter and exit the microcapsule, which has better advantages than traditional cell culture. Among them, vesicle-encapsulated cell technology can prepare organoids in a high-throughput one-step process without a secondary cleaning process, which is convenient for industrialization and standardization; the size of the vesicles is uniform and controllable, and the effect of the size of the cell spheres on vitality and function can be studied (improving the metabolic activity of hepatocytes); it can be used for the study of stem cell differentiation; but the culture operation is difficult, and the vesicles are suspended in the culture medium, which is not conducive to the replacement of the culture medium; the multi-organoid system mixed with multiple cell vesicles is difficult to operate.

[0007] Fusion chip preparation technology is a synchronous technology of gas-cut water + positioning light curing. Through the diameter of the liquid flow, the size of the vesicles is precisely controlled to achieve quantitative curing of regional vesicles. Droplet microfluidics based on oil-in-water emulsion system can produce cell-loaded microspheres of uniform size, but the flux is insufficient, and the process of demulsification and washing increases the complexity of the process. Gas-jet microfluidics technology combined with rapid ion cross-linking can prepare organoid microcapsules in large quantities, but usually only large-sized microcapsules (greater than 500μm) can be prepared under low gas flow conditions. With the increase of gas flow rate, the morphology of microcapsules is unstable, and it is difficult to prepare small microcapsules of uniform size. More importantly, the organoids prepared by the above technologies can only grow in a suspended state in culture medium, and a series of tumor physiological phenomena such as vascular infiltration and metastasis cannot be reproduced, making it difficult to evaluate the role and effect of anti-angiogenic drugs in tumor treatment. 3D bioprinting can simulate complex tissue structures and reproduce the tumor microenvironment through multi-cell and multi-ink; however, with the increase of manufacturing complexity, how to achieve high-throughput screening is a huge challenge.

[0008] In response to the above research status and existing problems, the present patent proposes a highly bionic fusion organ-on-a-chip technology, improved air-jet microfluidic microcapsule organoid technology and reversible hydrogel solidification fusion process. The prepared homogenized tumor organoids can effectively simulate the extracellular matrix environment in the body and the interaction between multiple cells. The reversible hydrogel solidification technology facilitates the separation and detection of cells, and can also simulate processes such as tumor metastasis and vascular invasion, providing a new strategy for the high-throughput screening of related drugs. Summary of the invention

[0009] In a first aspect, the present invention provides a vesicle, wherein the vesicle is formed by a polymer hydrogel solution passing through a microchannel under gas shear to form vesicle droplets of a certain diameter, and solidified vesicles are formed by atomization cross-linking, and the vesicle diameter ranges from 50 to 800 μm; the vesicle is a vesicle containing cells.

[0010] Further, the diameter of the vesicle is preferably 100-800 μm, 100-700 μm, 100-600 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 100-150 μm; 200-800 μm, 200-700 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-300 μm; 250-750 μm m; 250-650μm, 250-550μm, 250-450μm, 250-350μm; 300-800μm, 300-700μm, 300-600μm; 300-500μm ; 300-400μm; 350-750μm, 350-650μm, 350-450μm; 400-800μm, 400-700μm, 450-600μm, 450-500μm.

[0011] Furthermore, the diameter of the vesicle can be controlled by the gas flow rate and the inner / outer diameter ratio of the microchannel.

[0012] Furthermore, the gas may be selected from CO 2 , O 2 or N 2 .

[0013] Furthermore, the ratio of the inner and outer diameters is in the range of 0.2-1.

[0014] Furthermore, the inner and outer diameter ratio is preferably in the range of 0.3-0.9, 0.4-0.8, or 0.5-0.7.

[0015] Furthermore, the cells include, but are not limited to, one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

[0016] Furthermore, the vesicles are prepared by using ion-crosslinked hydrogel materials, and the hydrogel materials include but are not limited to sodium alginate, gellan gum, gelatin, polyethylene glycol, polyvinyl alcohol, 2-hydroxyethyl methacrylate and polyacrylamide.

[0017] Further, the concentration of the hydrogel material is 0.1-10%, preferably 0.1-9%, 0.1-8%, 0.1-7%, 0.1-6%, 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.1-0.5%; 0.5%-10%, 0.5-9%, 0.5-8%, 0.5-7%, 0.5-6% , 0.5-5%, 0.5%-4%, 0.5-3%, 0.5-2%; 1-9%, 1-8%, 1-7%; 1%-6%, 1-5%, 1-4%, 1-3%; 1%-2%; 2-8%, 2-7%, 2-6%; 2-5%, 2-4%, 2-3%; 3-8%, 3-6%, 3-5%, 3-4%; 4-8%, 4-6%, 4-5%.

[0018] Furthermore, the ion-crosslinked hydrogel material may be further modified, and the modification includes but is not limited to modification of cell adhesion ligands, and the adhesion ligands include but are not limited to integrin ligands and cadherin ligands.

[0019] Furthermore, the adhesion ligand material includes but is not limited to fibronectin, collagen, laminin, and RGD peptide.

[0020] Furthermore, the vesicles are selected from wall-adherent vesicles and non-adherent vesicles.

[0021] Furthermore, the vesicles can achieve vesicle uniformity and standardization by controlling the vesicle diameter and quantity.

[0022] Furthermore, the vesicle fusion can achieve uniform tissue growth by controlling the number of cells.

[0023] Furthermore, the metal required for the atomization cross-linking is selected from Ca 2+ , Ba 2+ , Cu 2+ 、Cd 2+ or Sr 2+ .

[0024] In a second aspect, the present invention provides a vesicle fusion chip, wherein the vesicle fusion chip is obtained by fusing the vesicle described in the first aspect with a chip through solidification, and the diameter of the vesicle is 50-800 μm.

[0025] Further, the vesicle diameter is preferably 100-800 μm, 100-700 μm, 100-600 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 100-150 μm; 200-800 μm, 200-700 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-300 μm; 250-750 μm m; 250-650μm, 250-550μm, 250-450μm, 250-350μm; 300-800μm, 300-700μm, 300-600μm; 300-500μm ; 300-400μm; 350-750μm, 350-650μm, 350-450μm; 400-800μm, 400-700μm, 450-600μm, 450-500μm.

[0026] Furthermore, the solidification fusion is to place solidified vesicles of a certain size into a chip containing a hydrogel matrix, and fuse the vesicles and the chip together by solidification and cross-linking.

[0027] Furthermore, the gel matrix includes but is not limited to gelatin methacrylate, gelatin, hyaluronic acid, fibrin material and derivatives thereof.

[0028] Furthermore, the curing crosslinking includes but is not limited to enzymatic crosslinking, photocrosslinking, and physical crosslinking.

[0029] Furthermore, the chip may also contain one or more cells, including but not limited to one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

[0030] Furthermore, the vesicle fusion chip can release tissue by breaking the cross-linking of microcapsules through microcapsule lysis solution.

[0031] Furthermore, the vesicle fusion chip is a plastic vesicle fusion chip.

[0032] Furthermore, the plastic vesicle fusion chip means that the vesicle and the chip can be separated by enzyme cleavage, and the separated vesicles can be fixed in a new chip by re-solidification.

[0033] In a third aspect, the present invention provides a vesicle composition, which contains one or more different vesicles; the vesicles contain vesicles of the same or different diameters and / or vesicles containing the same or different types of cells, and the vesicle diameter ranges from 50 to 800 μm.

[0034] Further, the vesicle diameter is preferably 100-800 μm, 100-700 μm, 100-600 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 100-150 μm; 200-800 μm, 200-700 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-300 μm; 250-750 μm m; 250-650μm, 250-550μm, 250-450μm, 250-350μm; 300-800μm, 300-700μm, 300-600μm; 300-500μm ; 300-400μm; 350-750μm, 350-650μm, 350-450μm; 400-800μm, 400-700μm, 450-600μm and 450-500μm.

[0035] Furthermore, the cells include, but are not limited to, one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

[0036] In a fourth aspect, the present invention provides an organoid, comprising vesicle organoids, vesicle composition organoids and organoids formed by vesicle fusion chips; the vesicles, vesicle fusion chips and vesicle compositions are consistent with the vesicles, vesicle fusion chips and vesicle compositions described in the first, second and third aspects of the present invention; the vesicle diameter ranges from 50 to 800 μm.

[0037] Furthermore, the cells include, but are not limited to, one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

[0038] In a fifth aspect, the present invention provides the use of the aforementioned vesicles, vesicle fusion chips and vesicle compositions in drug screening.

[0039] Furthermore, the drug may be a tumor prodrug, an immune preparation, an anti-vascular drug or other active ingredients that can inhibit tumor cells.

[0040] Furthermore, the drugs include but are not limited to cyclophosphamide and cisplatin.

[0041] In one embodiment, after vascular endothelial cells are prepared into vascular vesicles, a vascular-tumor vesicle fusion chip is prepared, and whether the anti-vascular drug works is determined by observing the sprouting of vascular vesicles in the fusion chip.

[0042] In another embodiment, after the vascular endothelial cells are prepared into vascular vesicles, a vascular-tumor vesicle fusion chip is prepared, and drugs are screened by determining whether the tumor cells in the vascular-tumor vesicle fusion chip break through the vesicle boundary and invade the surrounding matrix.

[0043] In a sixth aspect, the present invention provides a method for sorting cells or organoids, the method comprising sedimenting the aforementioned different vesicles, vesicle fusion chips, vesicle compositions, vesicle organoids, vesicle composition organoids, and organoids formed by vesicle fusion chips, and achieving sorting of various types of cells or organoids by differences in sedimentation speeds.

[0044] Furthermore, the vesicles may be vesicles of different sizes, and the diameter of the vesicles ranges from 50 to 800 μm.

[0045] Further, the vesicle diameter is preferably 100-800 μm, 100-700 μm, 100-600 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 100-150 μm; 200-800 μm, 200-700 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-300 μm; 250-750 μm m; 250-650μm, 250-550μm, 250-450μm, 250-350μm; 300-800μm, 300-700μm, 300-600μm; 300-500μm ; 300-400μm; 350-750μm, 350-650μm, 350-450μm; 400-800μm, 400-700μm, 450-600μm and 450-500μm.

[0046] Furthermore, the cells include, but are not limited to, one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

[0047] In one embodiment, endothelial cells are packed into vesicles with a diameter of 200 μm, and tumor cells are packed into vesicles with a diameter of 300 μm, so that two different cells, endothelial cells and tumor cells, can be clearly sorted.

[0048] In another embodiment, vesicles of different sizes will have different sedimentation speeds after collection due to gravity differences, and small vesicles in the supernatant and large vesicles that settle to the bottom are directly collected to achieve vesicle typing, that is, cell typing.

[0049] Beneficial effects:

[0050] The vesicles of different sizes and the highly biomimetic vesicle fusion chip proposed in the present invention can be used to prepare homogenized tumor organoids, which can effectively simulate the extracellular matrix environment in the body and the interaction between multiple cells. The reversible hydrogel solidification technology facilitates the separation and detection of cells, and can also simulate processes such as tumor metastasis and vascular invasion. Specifically, the advantages of the vesicle fusion chip of the present invention are: (1) the vesicle size can be controlled to achieve homogenization of the vesicles; (2) quantitative solidification of vesicles + quantification of the size of organoids in the vesicles can be achieved; (3) the vesicle system is solidified in different regions to achieve the partitioning of the immune system, vascular system, tumor cells, etc., to form a highly biomimetic tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the growth pattern of cells in adherent vesicles.

[0052] Figure 2 Schematic diagram of the growth pattern of cells in non-adherent vesicles.

[0053] Figure 3 The effects of vesicles of different sizes on organoid formation: A. Bright field photograph of organoids B. Quantification of organoid size D. Statistics of the filling rate of organoids in vesicles.

[0054] Figure 4 Metabolic activity and quantitative analysis of hepatic vesicles of different sizes.

[0055] Figure 5 Quantification of cell number after multiple medium changes in non-confluent and confluent chips.

[0056] Figure 6 Bright field and live-dead fluorescent staining of uniform cell-laden vesicles.

[0057] Figure 7 This is the skeleton fluorescence staining of cell clusters in vesicles on days 3 / 5 / 7.

[0058] Figure 8 Schematic diagram of vesicle sorting implemented for the Fusion Chip.

[0059] Fig. 9 Fluorescence distribution image of the tumor / vascular organoid fusion vesicle chip.

[0060] Fig.10 Bright field microscopic images of cell-laden vesicles before and after lysis.

[0061] Fig.11 Cytotoxicity of anti-tumor drugs on tumor vesicles and tumor-liver fusion chips. A. CCK8 proliferation statistics of tumor vesicles and tumor-liver fusion chips after cyclophosphamide treatment. B. Proliferation inhibition rate of cells in tumor vesicles and tumor-liver fusion chips.

[0062] Fig.12 Fluorescence photos and diffusion quantitative statistics of nanodrugs diffusing in the fusion chip.

[0063] Fig.13 The drug sensitivity of 2D primary tumors, vesicle organoids, and Matrigel organoids to cisplatin.

[0064] Fig.14 Tumor organoid-vascular organoid fusion vesicle chip to observe endothelial sprouting. A. Schematic diagram of tumor organoid vesicles inducing endothelial organoid sprouting. B. Bright field micrograph of vascular sprouting. C. Quantitative statistical results of vascular sprout length and number.

[0065] Fig.15 Observation of tumor infiltration in a tumor organoid-vascular organoid fusion vesicle chip.

[0066] Fig.16 To prepare and test the integrated fusion chip prototype.

[0067] Fig.17 Schematic diagram of the lysis of the fusion vesicle chip, vesicle typing and secondary fusion chip formation. DETAILED DESCRIPTION

[0068] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0069] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0070] Example 1

[0071] 1. Preparation of Microencapsulated Organoids

[0072] Microcapsules were prepared using ion-crosslinked hydrogel materials. Cells were resuspended in a 2% solution of a polymer hydrogel prepolymer sodium alginate and introduced into a coaxial microfluidic channel. Primary tumor cells were selected. A microinjection pump was used to control the flow rate of the solution, while CO was introduced into the external phase. 2 The polymer solution carrying the cells forms droplets under the shear of the gas. The size of the droplets is mainly controlled by changing the gas flow rate and the inner / outer diameter ratio of the microchannel. In order to improve the stability of crosslinking, the atomization crosslinking method is used instead of the traditional crosslinking bath. The dispersed droplets fall vertically through the atomization crosslinking compartment, which is filled with metal ions Ca 2+The solution atomization spray can gently cross-link the droplets to solidify them, and the inner / outer diameter ratio of the microchannel is 0.5.

[0073] 2. Drug screening based on regional curing

[0074] The 96-well plate is filled with a hydrogel prepolymer solution, and gelatin methacrylate is selected as the hydrogel matrix. The tumor organoids formed in the microcapsules are added to the 96-well plate by photocrosslinking and curing, mixed and cured, and finally supplemented with culture medium. For the screening of anti-tumor drugs, the drug is added for 24 hours and then replaced with growth medium. CCK8 solution is added for incubation at a fixed time point, and the absorbance at 450nm is measured using an ELISA reader to examine the inhibitory effect of the drug on tumor cells.

[0075] 3. Preparation of Fusion Chip

[0076] The 96-well plate is filled with a hydrogel prepolymer solution. To simulate a multicellular ecology, the prepolymer solution in the 96-well plate is mixed with one or more other cells. In this example, tumor cell lines and endothelial cells are selected. The microcapsule organoids are sprayed into the 96-well plate using a mobile nozzle to mix, and then the hydrogel matrix is ​​solidified and supplemented with culture medium.

[0077] 4. Cell Isolation and Extraction

[0078] The fusion chip technology proposed in the present invention can selectively separate microcapsule organoids or surrounding stromal cells. For the separation of microcapsule organoids, EDTA is added to the well plate to chelate metal ions to decompose the microcapsules without affecting the stability of the surrounding matrix. For the separation of surrounding stromal cells, collagenase or hyaluronidase is added to the well plate to selectively degrade the surrounding matrix.

[0079] 5. Organoid Fusion Chip for Detection of Anti-angiogenic Drugs

[0080] Microcapsules (100 μm) loaded with endothelial cells and fibroblasts were prepared and cultured for 7 days until vascular organoids were formed in the microcapsules. The vascular organoid vesicles and tumor organoid vesicles were added to a gelatin methacrylate (Gelatinmethacryloyl, GelMA) solution, the GelMA matrix was cross-linked under 405nm blue light, and then the culture medium was supplemented. Under the induction of tumor organoids, vascular organoids showed a tendency to sprout and infiltrate into the tumor. Angiogenesis inhibitors were added to the drug group, and the number and average length of sprouts were photographed using a laser confocal microscope to investigate the effect of anti-vascular drugs on vascular infiltration of transplanted tumors.

[0081] Example 2 Experimental results

[0082] 1. Microcapsule preparation process and microcapsule size uniformity results

[0083] (1) Preparation of wall-adherent vesicles

[0084] Additional modification of cell adhesion ligands (e.g. Figure 1 ). The modified adhesion ligand is RGD (Arg-Gly-Asp) peptide. Cells can adhere and spread in the adherent vesicles, which can be used to simulate processes such as tumor infiltration and vascular sprouting.

[0085] (2) Preparation of non-adherent vesicles

[0086] Cells were dispersed and solidified using an ionically cross-linked hydrogel solution that did not contain cell adhesion sites (e.g. Figure 2 As shown in Figure 2 ). Since cells cannot adhere to the wall in the vesicles, they grow in aggregates and clusters, which can be used for the preparation and culture of organoids.

[0087] 2. Comparison of vesicle size and time required to fill the cell

[0088] The shorter the microcapsule filling time, the shorter the time it takes for organoids to be cultured until they can be detected and analyzed, which is conducive to tumor drug screening and other detection and analysis in a short period of time.

[0089] Analysis of primary tumor organoid culture time: Human colorectal cancer cell lines were injected intraperitoneally into mice to establish a colorectal cancer PDX model. Tumor tissue was formed 60 days after cell injection, and tumor tissue was isolated from PDX mice; the tumor tissue was washed three times with PBS and cut into pieces with ophthalmic scissors. The tumor tissue was then digested with 0.25% type I collagenase. Digestion was performed in an incubator for 4 hours, and then complete medium was added to terminate the digestion. The tissue digestion solution was filtered through a 70 μm cell strainer to remove undigested tissue clumps. The tissue filtrate was centrifuged at 800 rpm for 5 minutes to obtain a cell pellet, and the supernatant was removed. 1.5% sodium alginate solution was added to resuspend the cell pellet and gently blown until uniform. Microcapsules with different diameters (400, 280, 215, 150 and 100 μm) were prepared by changing the speed of the airflow and the inner diameter of the needle. Tumor organoid microcapsules were cultured for one week, and the size of the organoid clumps was photographed and measured.

[0090] The results are as follows Figure 3 A shows that larger microcapsule sizes (400, 280 μm) are not conducive to the formation of organoids. Microcapsules of 215, 150, and 100 μm all grew well, and organoids were seen breaking through the microcapsule shell in the 100 μm microcapsule. Figure 3 As shown in B, it can be seen that the vesicles contain a variety of cell types and have the characteristics of tumor organoids. The size analysis results of the organoids are shown in Figure 3As shown in Figure C, the average diameters of the organoids were 85, 98, 147, 107, and 81 μm, respectively. It can be seen that the tumor organoids formed in the microcapsules with a size of 215 μm had the largest volume. The filling rate of the organoids was quantified by calculating the ratio of the organoid mass and the microcapsule size. The results are shown in Figure 5. Figure 3 D shows that the organoid filling rates are 21, 35, 68, 71, 75 and 80%, respectively. This indicates that microcapsules with sizes of 215, 150 and 100 μm have better effects.

[0091] 3. Relationship between vesicle size and cell activity

[0092] The effect of vesicle size on the metabolic function of hepatocytes was investigated using the hepatocyte cell line Hep G2 / C3A. CYP3A4 is an important regulator that is present in the human liver, lungs, and kidneys and is an important drug-metabolizing enzyme. C3A cells were mixed into the vesicles and the production process was changed to prepare hepatic vesicles with an average size of approximately 400 (large), 300 (medium), and 200 μm (small). After 3 days of culturing the hepatic vesicles, proteins were extracted using RIPA lysis buffer, quantified using a BCA assay kit, separated by SDS-PAGE gel, and transferred to a PVDF membrane. After blocking with 5% skim milk powder, the PVDF membrane was incubated with diluted CYP3A4 and GAPDH primary antibodies at 4°C overnight. After washing, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 hour and then developed. Protein bands were analyzed using Image J software.

[0093] The results are as follows Figure 4 As shown, the size of vesicles greatly affects the metabolic capacity of hepatocytes, with hepatic vesicles of 200 μm in size showing the highest metabolic activity.

[0094] 4. Comparison of homogeneity and standardization between vesicle fusion chip and simple vesicle samples

[0095] Maintaining the stability of the vesicle number by solidifying the vesicles is one of the important advantages of the fusion chip.

[0096] For the non-fusion pure vesicle group, the cell-laden vesicles were directly resuspended in complete medium and added to 96-well plates. The number of cells in each well was counted at the time of plating, after the first medium change, after the second medium change, and after the third medium change. For the non-fusion pure vesicle group, 0.1mM sodium citrate and 0.1mM EDTA were added to lyse the vesicles, and the cell suspension was collected and used the cell counting technique.

[0097] For the vesicle fusion chip, the cell-loaded vesicles were resuspended in the GelMA hydrogel prepolymer solution, added to a 96-well plate and crosslinked under 405nm blue light for 10 seconds, and then supplemented with complete medium. The number of cells in each well was counted at the time of plating, after the first medium change, after the second medium change, and after the third medium change. For the fusion chip group, 0.5% type I collagenase was added and the GelMA matrix was lysed at 37°C, and then sodium citrate and EDTA were used to lyse the vesicles and collect the cells and count them.

[0098] The results are as follows Figure 5 As shown in the figure, the non-fusion pure vesicle group will experience a loss of cell number every time the medium is changed. This is because the suspended vesicles and the medium will inevitably be sucked out during the medium change process. In the fusion chip group, GelMA is used to fix the vesicles, and the cell number remains unchanged after multiple medium changes.

[0099] 5. Drug Screening

[0100] Cell-loaded vesicles with a size of about 250 μm were prepared, and the esophageal cancer cell line KYSE 150 was used as a cell model. The viability of cells in the vesicles was examined using a live / dead cell staining kit. The results showed that most cells remained alive, and very few dead cells were observed (e.g. Figure 6 ).

[0101] Prepare cell-loaded vesicles with a size of about 200 μm, and use immunofluorescence staining to examine the skeletal morphology of cell spheres on the 3rd / 5th / 7th day of culture. The cell-loaded vesicles were fixed in 4% paraformaldehyde for half an hour, washed with PBST, and permeabilized with 0.1% Triton X-100, and then incubated with FITC-labeled phalloidin at room temperature for half an hour. After washing, add DAPI for counterstaining for 10 minutes and observe under a confocal fluorescence microscope.

[0102] Skeleton staining results showed that esophageal cancer cells grew in clusters in the microcapsules, which well simulated the growth pattern of tumors in vivo (such as Figure 7 ).

[0103] Based on the size of vesicles, vesicle classification and dynamic observation in the fusion chip are achieved (vesicles of different sizes in tumors and blood vessels, light microscopy + staining verification). Cell vesicles of different sizes are prepared, and cells can be directly distinguished by vesicle size in the fusion chip. (e.g. Figure 8 )

[0104] The GFP-labeled endothelial cells HUVEC and the RFP-labeled tumor cells KYSE 150 were prepared into vesicles. The size of the endothelial vesicles was about 200 μm, and the size of the tumor vesicles was about 300 μm. The endothelial vesicles and tumor vesicles were mixed in the GelMA prepolymer solution and cross-linked. The distribution of vesicles in the fusion chip was observed using a confocal fluorescence microscope (e.g. Fig. 9 shown).

[0105] 6. Reversible realization of vesicle structure

[0106] 0.1 mM sodium citrate and 0.1 mM EDTA were used to chelate the cross-linked metal ions in the microcapsules and break the cross-linked network of the microcapsules. It can be seen that the microcapsules are lysed and the cell clusters in the microcapsules are released (such as Fig.10 That is, removing the vesicle structure can form connections between different cells.

[0107] 7. Vesicle fusion chip for tumor drug screening and new drug development

[0108] (1) Add normal liver metabolic vesicles

[0109] Hepatocyte C3A vesicles and esophageal cancer cell KYSE 150 vesicles were prepared. C3A vesicles and KYSE 150 microcapsules were suspended in GelMA prepolymer solution and then added to a 96-well plate. 50 μL of GelMA prepolymer solution was added to each well. For the control group, the chip was composed entirely of KYSE 150 cell microcapsules, wherein the GelMA prepolymer solution in each replicate well contained 5000 KYSE 150 cells. For the fusion chip group, the chip was composed of KYSE 150 cell microcapsules and C3A cell microcapsules, wherein the GelMA prepolymer solution in each replicate well contained 2500 KYSE 150 cells and 2500 C3A cells. The well plate was irradiated with 405 nm blue light for 10 seconds to crosslink and solidify GelMA. Cyclophosphamide was used as a model drug. Cyclophosphamide has no toxicity to tumor cells in vitro. Only after being metabolized by the liver and converted into metabolites such as cyclophosphamide can it have a killing effect on tumor cells. 20 μg / ml cyclophosphamide was prepared in the culture medium and added to the chip for co-culture for 24 hours. CCK8 reagent was added and the absorbance at 450nm was measured using an ELISA reader to examine the cell proliferation in the chip. Proliferation inhibition rate was calculated = (absorbance of control well - absorbance of drug-added well) / (absorbance of control well - absorbance of blank well) to quantitatively measure the toxicity of metabolic drugs to tumor cells. The control wells were cells without drug treatment, and the blank wells contained no cells but only complete culture medium.

[0110] Fig.11 The results of cell proliferation measured by CCK8 method shown in A show that the cell proliferation of KYSE150 esophageal cancer cells was significantly reduced in the fusion chip co-cultured with C3A liver vesicles. Fig.11 As shown in B, in the fusion chip, cyclophosphamide produced a significant inhibitory effect on cell proliferation, while cyclophosphamide did not produce obvious cytotoxicity when acting on tumor cells alone, proving that the fusion chip can be used for in vitro research on tumor prodrugs.

[0111] (2) Adding nano drug vesicles to achieve drug infiltration and diffusion

[0112] In order to simulate the infiltration and diffusion behavior of nanomedicine, gold nanoparticles (RhoB@AuNPs) were modified with rhodamine B. 100 μL (0.1 g / mL) of AuNPs solution was weighed and added to a 2 mL brown bottle, and 400 μL of PBS solution was added. The AuNPs were dispersed in an ultrasonic water bath at room temperature for 20 min. 1 mg of RhoB was weighed and dissolved in 500 μL of PBS solution. After it was fully dissolved, it was slowly added drop by drop to the AuNPs solution. After the addition was completed, it was stirred continuously for 12 hours overnight at room temperature. After the reaction was completed, it was placed in a centrifuge tube, centrifuged at 15000 rpm, and then the unbound free RhoB in the supernatant was removed. After washing three times, it was stored away from light for later use.

[0113] The RhoB@Au NPs solution and the vesicles were incubated overnight in the dark to prepare fluorescent microcapsules loaded with RB@Au NPs. The microcapsules were allowed to settle for ten minutes, the supernatant was removed and washed with PBS. The washing was repeated three times to remove free nanoparticles. The fluorescent microcapsules were resuspended in the GelMA prepolymer solution and cross-linked for 10 seconds under 405nm blue light. The culture medium was supplemented, and the fluorescence images were taken at fixed time points. The diffusion rate of the nanoparticles was calculated by calculating the ratio of the fluorescence intensity of the surrounding area and the fluorescent microcapsules.

[0114] The results are as follows Fig.12 As shown, the nanoparticles can diffuse slowly in the fusion chip, and the diffusion of the fluorescent nanoparticles reaches a peak after 1 hour.

[0115] (3) Primary tumors form organoids, complete drug killing experiments, and test the parallelism of multiple wells

[0116] Primary cells were isolated from colorectal cancer Pdx mice. The primary cells were digested and resuspended in sodium alginate solution to prepare organoid microcapsules, and a plate control group and a matrigel control group were set up at the same time. Cells were cultured in 96-well plates, and 5000 cells were seeded in each well of all groups. For the matrigel group, the primary cells were resuspended in the matrigel solution, operated on ice to prevent the matrigel from solidifying, and then added to the well plate and incubated in the incubator for half an hour to solidify the matrigel. Then complete medium was added and cultured for 6 hours. After the cells in the 2D group adhered to the wall, the drug was added to the well plate. Cisplatin was used as a model drug at a concentration of 0.4μg / ml. After the cells were treated with drugs for 24 hours, they were replaced with complete medium and cultured for another day. The toxicity of anti-tumor drugs to cells was investigated using CCK8 reagent.

[0117] The results are as follows Fig.13As shown in Figure 2, compared with 2D dishes, organoids formed in vesicles and Matrigel showed greater drug sensitivity. Compared with organoids formed in traditional Matrigel, organoid vesicles have a smaller standard deviation, which is more conducive to the standardization of drug screening.

[0118] 8. Organoid vesicle fusion chip for tumor mechanism research

[0119] (1) Vascular invasion

[0120] Vascular organoid vesicles are immobilized together with tumor organoid vesicles in the fusion chip ( Fig.14 A) Observation of budding of vascular organoid vesicles in the fusion chip. After 5 days of culture in the solidified matrix, endothelial budding was observed by fluorescence staining. Fig.14 B shows that in the vascular-tumor organoid fusion chip, the vascular organoids showed significant sprouting and growth into the tumor organoids. Fig.14 The quantitative results of the number and average length of vascular sprouts shown in C showed that the average length of vascular sprouts in the fusion chip and pure vascular vesicle chip were 49 and 20 μm, respectively, and the average number of sprouts were 6 and 1, respectively, proving that the fusion chip technology can be used to study tumor-vascular interactions and the role of antivascular drugs in inhibiting tumor vascular growth and invasion.

[0121] (2) Tumor Infiltration

[0122] In the tumor-vascular tumor fusion vesicle chip, it can be observed that tumor cells break through the vesicle boundary and invade into the surrounding matrix, proving that the fusion chip technology can simulate the situation in vivo where tumors break through the basement membrane and infiltrate into the surrounding tissue. Fig.15 )9. Fusion chip preparation technology and equipment

[0123] In this embodiment, the fusion chip is prepared by using gas-cutting water + positioning light curing synchronous technology, and the size of the vesicles is accurately controlled by the liquid flow diameter to achieve regionalized vesicle quantitative curing.

[0124] The liquid flow rate of hydrogel precursor solutions such as sodium alginate is controlled by Liquid controller; Pinheadchanger is a microchannel converter that can freely switch the models of the inner and outer channels according to needs to change the size of the microcapsule. The inner channel size can be switched between 28G-34G, and the outer channel can be switched between 18G-25G. The hydrogel precursor solution passes through the central pipe and is sheared by the gas (nitrogen, oxygen, and carbon dioxide, etc.) introduced from the right to form hydrogel droplets. The gas flow rate can be controlled by Gascontroller. Atomizer is an atomizer that can spray the cross-linked hydrogel solution (CaCl 2When the valve is opened, the crosslinker mist mixes with the hydrogel droplets, crosslinks and solidifies the hydrogel droplets to form microcapsules; when the valve is closed, the crosslinker gas can be discharged through the waste tube. The solidified microcapsules fall in the central pipe, and the fused solidified ink flows into the Bioink channel, mixes with the microcapsules, and is collected together in the orifice plate on the platform. The holder that fixes the orifice plate is controlled by a motor and can move freely on the XY axis. Finally, UV light irradiates the orifice plate to crosslink and solidify the fused ink to obtain a vesicle fusion chip.

[0125] 10. Cleavable Fusion Chip for Secondary Fixation and Re-fusion of Vesicles

[0126] The fusion chip can release the vesicles after enzyme lysis. Due to the difference in gravity, the vesicles of different sizes will have different sedimentation speeds after collection. The small vesicles in the supernatant and the large vesicles at the bottom can be directly collected to achieve vesicle typing. In addition, the separated vesicles can be fixed again in a new fusion chip for other experiments ( Fig.17 ).

Claims

1. A vesicle, wherein the vesicle is formed by passing a polymer hydrogel solution through a microchannel under gas shear to form vesicle droplets of a certain diameter, and solidified vesicles are formed by atomization cross-linking, and the vesicle diameter ranges from 50 to 800 μm; the vesicle is a vesicle containing cells.

2. The vesicle according to claim 1, characterized in that The cells include, but are not limited to, one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells, and epidermal cells.

3. The vesicle according to claim 1, characterized in that The vesicle diameter also includes 100-800 μm, 100-700 μm, 100-600 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, 100-150 μm; 200-800 μm, 200-700 μm, 200-600 μm, 200-500 μm, 200-400 μm, 200-300 μm; 250-750 μm; 250-650μm, 250-550μm, 250-450μm, 250-350μm; 300-800μm, 300-700μm, 300-600μm; 300-500μm; 300-400μm; 350-750μm, 350-650μm, 350-450μm; 400-800μm, 400-700μm, 450-600μm and 450-500μm.

4. A vesicle fusion chip, which is obtained by fusing the vesicle described in the first aspect with a chip through solidification, and the diameter of the vesicle is 50-800 μm.

5. The vesicle fusion chip according to claim 4, characterized in that: The solidification fusion is to place solidified vesicles of a certain size into a chip containing a hydrogel matrix, and fuse the vesicles and the chip together by solidification and cross-linking.

6. The vesicle chip according to claim 3, characterized in that The vesicle fusion chip is a plastic vesicle fusion chip, which means that the vesicles and the chip can be separated by enzyme cleavage, and the separated vesicles can also be fixed in a new chip by re-solidification.

7. A vesicle composition, comprising one or more different vesicles; the vesicles contain vesicles of the same or different diameters and / or vesicles containing the same or different types of cells, and the vesicle diameter ranges from 50 to 800 μm; the cells include but are not limited to one or more of liver cell lines, kidney cell lines, fibroblasts, endothelial cells, pericytes, immune cells, mesenchymal stem cells, tumor cell lines, primary tumor cells, lymphoepithelial cells and epidermal cells.

8. An organoid, comprising vesicle organoids, vesicle composition organoids and organoids formed by vesicle fusion chips; the vesicles, vesicle fusion chips and vesicle compositions are consistent with the vesicles, vesicle fusion chips and vesicle compositions described in claims 1-7; the vesicle diameter ranges from 50 to 800 μm.

9. Use of the vesicles, vesicle fusion chips and vesicle compositions described in claims 1-7 in drug screening, wherein the application refers to the screening of tumor prodrugs, immune preparations, anti-vascular drugs or other active ingredients that can inhibit tumor cells.

10. A method for sorting cells or organoids, the method comprising: using the vesicle, vesicle fusion chip, The vesicle composition and organoids are sedimented, and the sorting of various types of cells or organoids is achieved by the difference in sedimentation speed. The diameter of the vesicles ranges from 50 to 800 μm.

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