A biological ink based on inkjet printing and its preparation method and application

Abstract

The present invention provides a bio-ink based on inkjet printing and its preparation method and application, wherein the bio-ink is obtained by mixing the components in the bio-ink composition, and the composition includes a dispersion medium and a biological unit; the dispersion medium includes a hydrogel cross-linking precursor and a hydrogel adhesion enhancer. The viscosity of the bio-ink of the present invention can be adjusted in a large range, which can ensure the printing performance of high-precision ink droplets (micrometer level) and trace amounts (nanoliter level); it is suitable for printing on the surface and inside of various materials (such as hydrogels, water, polymers, etc.); the ink composition is liquid at 0-40°C, suitable for printing at 0-40°C, and will not clog the needle when printing at 0-40°C, which can reduce printing costs; it can realize the printing of points, lines, surfaces, and bodies; it can realize the printing of various patterns.

Description

Technical Field

[0001] The present invention belongs to the field of cell or organoid printing, and in particular relates to a biological ink based on inkjet printing, and a preparation method and application thereof. Background Art

[0002] In recent years, my country has ranked first in the world in new cancer cases and deaths, and the incidence and mortality of malignant tumors have continued to rise. The annual medical expenses caused by malignant tumors exceed 220 billion. Traditional cancer drugs need to be tested on animals before they can be used as clinical drugs to determine the efficacy and develop corresponding treatment plans. Animal experiments are costly and have ethical issues, so alternatives are urgently needed.

[0003] Organoids are complex three-dimensional cell culture systems that not only contain some of the key characteristics of the organs they represent, but are also able to self-renew and reorganize in vitro, reproducing the behavior and function of the organs. Organoids can be directly generated from the patient's cancer tissue and are called "organs in a dish". They are widely used in biomedicine as biological models for drug screening and design, disease modeling, developmental biology, etc., especially for ultra-sensitive early diagnosis and treatment of trace samples.

[0004] At present, the cultivation of organoids is usually expensive and takes a long time (at least one month), and requires a high level of basic knowledge and proficiency from experimental operators. The large differences in manual operations make it difficult to manufacture related detection chips, which hinders their application in high-throughput screening, regenerative medicine and diagnosis.

[0005] Therefore, designing and preparing a fast and convenient cell and organoid drug screening chip is of great significance for clinical diagnosis and biomedical research.

[0006] There have been studies on cell printing. The formulation design of bio-ink for cell printing is one of the key technologies to achieve cell printing. The current bio-ink still has many shortcomings:

[0007] 1. Poor printability: The industry's commonly used temperature-sensitive Matrigel matrix glue is solid below 0°C, and its viscosity is similar to that of water at 0-4°C. It will solidify when the temperature is higher than 4°C. A trace amount of Matrigel matrix glue (a single ink droplet is less than 1 microliter) is easily solidified during the printing process due to an ambient temperature higher than 4°C, which can easily clog the nozzle. However, it is difficult and costly to maintain 0-4°C when printing microneedles.

[0008] 2. The printing process may cause damage to cells: cells need to have good dispersion in the ink to ensure the uniformity of printing. At the same time, it is also necessary to avoid damage to cells caused by shear force during the printing process. Taking the industry-common temperature-sensitive Matrigel matrix as an example, the viscosity of Matrigel matrix is ​​very low, and cells are prone to sedimentation during the printing process, resulting in uneven cell concentration in the printed ink droplets and low cell activity.

[0009] 3. Low cell activity in ink: The cell culture effect of artificially prepared bio-ink is worse than that of biological extracts (the thermosensitive Matrigel matrix glue commonly used in the industry); this is because artificially prepared bio-ink has poor ability to maintain the stability of the cell microenvironment and poor cell adhesion.

[0010] 4. Low precision: In order to ensure high precision of printed ink droplets, a micron-level printing needle is required. In order to avoid damage to cells caused by printing, the concentration of cells in the ink needs to be limited, resulting in low printing precision.

[0011] 5. Poor reproducibility. The composition of the thermosetting Matrigel matrix glue (biological extract) commonly used in the industry is not completely clear, and there are large differences between batches, resulting in poor reproducibility, which is not conducive to precise control experiments in cell research. Summary of the invention

[0012] In order to improve the deficiencies of the prior art, the present invention provides a bio-ink based on inkjet printing and a preparation method and application thereof. Specifically, the present invention provides the following technical solutions:

[0013] A bio-ink composition based on inkjet printing comprises a dispersion medium and a bio-unit; the dispersion medium comprises a hydrogel cross-linking precursor and a hydrogel adhesion enhancer.

[0014] According to the present invention, in the dispersion medium, the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancer is (1-10):0.5.

[0015] According to the present invention, the dispersion medium at least includes a hydrogel crosslinking precursor, with or without a crosslinking initiator, and the crosslinking initiator is selected from one or more of a photoinitiator and an ion initiator.

[0016] According to the present invention, the hydrogel cross-linking precursor is selected from one or more of a photocurable hydrogel precursor, an ionic hydrogel precursor, and a thermally curable hydrogel precursor.

[0017] According to the present invention, the dispersion medium further comprises a cell adhesion enhancer, and the cell adhesion enhancer is used to enhance the adhesion of cells to the hydrogel.

[0018] According to the present invention, the dispersion medium further comprises one or more of cytokines, antibiotics, small molecule compounds, cellulase, additives, and culture medium.

[0019] According to the present invention, the biological unit comprises a cell, a tissue or an organoid.

[0020] The present invention also provides a bio-ink, which is obtained by mixing the components in the above-mentioned bio-ink composition.

[0021] The present invention also provides a method for preparing the bio-ink, which comprises: mixing the biological units in the bio-ink composition with a dispersion medium to obtain the bio-ink.

[0022] The present invention also provides application of the biological ink in cell or organoid printing.

[0023] The solution of the present invention achieves the following beneficial effects:

[0024] 1) Printability: The viscosity of the ink composition of the present invention can be adjusted in a wide range, which can ensure the printing performance of high-precision ink droplets (micrometer level) and trace amounts (nanoliter level); it is suitable for printing on the surfaces and insides of various materials (such as hydrogels, water, polymers, etc.); the ink composition is liquid at 0-40°C, suitable for printing at 0-40°C, and will not clog the needle when printing at 0-40°C, which can reduce printing costs; it can realize the printing of points, lines, surfaces, and bodies; it can realize the printing of various patterns.

[0025] 2) Combination of multiple curing methods: By adjusting the proportion of ink components, photo-crosslinking, ion-crosslinking, and thermal-crosslinking can be combined to increase the controllable range of the ink's morphology and viscosity during printing.

[0026] 3) High adhesion to a variety of substrates: Adding cell adhesion enhancers such as polydopamine can enhance the adhesion of cell-containing bio-ink to the printed substrate, especially when the ink droplets are continuously immersed, shaken, and agitated in the culture medium for 7-14 days, and still do not fall off the substrate.

[0027] 4) High cell activity: The hydrogel protects cells from shear damage during the printing process, provides a spatial skeleton for cell dispersion, prevents cells from settling in the ink during the printing process, and ensures long-term uniform dispersion; the basal culture medium provides the life substances required by the cells during the printing and cell culture process; biological additives maintain the stability of the cell environment (such as antibiotics) and enhance the adhesion of cells to the hydrogel (such as polypeptide RGD and laminin); cytokines regulate the rate of cell proliferation and differentiation, and regulate the degree of gene expression of cells; small molecule compounds regulate the screening of cell types to ensure the singleness of the final tumor cell types.

[0028] 5) Compared with the traditional ink prepared directly with PBS or water, the direct use of basal culture medium as solvent can ensure the stability of cell permeate during printing and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the intestinal cancer cells cultured in the high-throughput cell chip in Example 1.

[0030] Figure 2 This is a morphological picture of colon cancer organoids cultured in the high-throughput organoid chip in Example 1.

[0031] Figure 3 These are the “dots” formed by different inkjet pressures and inkjet times during the printing process of Example 4.

[0032] Figure 4 Schematic diagram and actual picture of the high-throughput cell or organoid chip prepared by the printing method in Example 4.

[0033] Figure 5 This is a fluorescent staining image of living cells on the third day of culture of colon cancer organoids printed in hydrogels of different concentrations in Example 5.

[0034] Figure 6 Schematic diagram of the device for printing the heat-cured matrix glue in Example 8.

[0035] Figure 7 These are SEM images of cells and organoids at different growth times under different matrix gel contents in Example 10.

[0036] Figure 8 This is a cell viability test chart of cells and organoids at different growth times under different matrix gel contents in Example 10.

[0037] Fig. 9 The staining comparison and quantitative statistics of the cell survival rates in the bio-ink before and after printing in Test Example 2. DETAILED DESCRIPTION

[0038] The study found that the nano green printing technology based on inkjet printing has the characteristics of high pattern accuracy and easy regulation. The inventor can effectively regulate the molding process of the printed ink droplets on the surface of the substrate by adjusting the characteristics of the biological ink containing cells, regulating the spreading and de-wetting behavior of the ink droplets and the substrate during the printing process, etc., and can solve the coffee ring effect that affects the printing accuracy, and finally realize the rapid, simple, and large-area preparation of the designed patterned cell chip. The method of the present invention can avoid the use of micro-nano structural technologies such as flat-panel lithography, electron beam lithography, and holographic lithography commonly used in current research, breaking the requirements of these methods for expensive equipment, complex processes, and harsh environments; on the other hand, the printing technology is used to achieve the addition of trace drugs, reduce the interference of human factors, and provide a theoretical and technical basis for the development of efficient, accurate, and rapid biological detection technology.

[0039] [Bio-ink composition]

[0040] The present invention focuses on the bio-ink used for inkjet printing. Specifically, the present invention first proposes a bio-ink composition based on inkjet printing, especially the composition is used for cell or organoid printing, the composition includes a dispersion medium and a biological unit; the dispersion medium includes a hydrogel cross-linking precursor and a hydrogel adhesion enhancer.

[0041] According to an embodiment of the present invention, in the dispersed medium, the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancer is (1-10):0.5; illustratively, it is 1:0.5, 2:0.5, 3:0.5, 4:0.5, 5:0.5, 6:0.5, 7:0.5, 8:0.5, 9:0.5, 10:0.5 or a specific ratio value therebetween.

[0042] According to an embodiment of the present invention, the dispersion medium comprises at least a hydrogel crosslinking precursor, optionally with or without a crosslinking initiator. When a crosslinking initiator is added, the hydrogel crosslinking precursor can react with the corresponding crosslinking initiator to form a hydrogel when forming the bio-ink.

[0043] According to an embodiment of the present invention, the cross-linking initiator is selected from one or more of a photoinitiator and an ion initiator.

[0044] According to an embodiment of the present invention, the photoinitiator includes but is not limited to at least one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP for short), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPO-L).

[0045] According to an embodiment of the present invention, the ion initiator includes but is not limited to at least one of a calcium chloride solution and a barium chloride solution.

[0046] According to an embodiment of the present invention, the hydrogel crosslinking precursor is selected from one or more of a photocurable hydrogel precursor, an ionic hydrogel precursor, and a thermally curable hydrogel precursor. Preferably, the hydrogel crosslinking precursor is a photocurable hydrogel precursor and / or an ionic hydrogel precursor.

[0047] According to an embodiment of the present invention, the thermally curable hydrogel precursor includes but is not limited to at least one of matrix glue and polyether F127 diacrylate.

[0048] According to an embodiment of the present invention, the hydrogel cross-linking precursor includes a photocurable hydrogel precursor and an ionic hydrogel precursor, and the mass ratio of the photocurable hydrogel precursor to the ionic hydrogel precursor is 1:(0-5), preferably the mass ratio of the photocurable hydrogel precursor to the ionic hydrogel precursor is 1:(0-3), for example, 1:1.

[0049] According to an embodiment of the present invention, the photocurable hydrogel precursor includes but is not limited to methacrylated sodium alginate (AlgMA), methacrylated hyaluronic acid (HAMA), methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated polylysine (PLMA), methacrylated gelatin (GM), methacrylated silk fibroin (SilMA), methacrylated dextran (DeXMA), methacrylated chondroitin sulfate (ChSMA), polyether F127 diacrylate, polyethylene glycol diacrylate, four-arm polyethylene glycol acrylate, and other acrylated materials (such as acrylated RGD peptide, acrylated polyethylene glycol NHS ester).

[0050] According to an embodiment of the present invention, the ionic hydrogel precursor includes but is not limited to carboxymethyl cellulose, alginate (such as sodium alginate, potassium alginate, etc.), and carboxymethyl chitosan.

[0051] According to an embodiment of the present invention, the hydrogel cross-linking precursor is a composition of a photocurable hydrogel precursor and an ionic hydrogel precursor. The study found that when the composition is selected as a hydrogel cross-linking precursor, the photocurable hydrogel precursor and the ionic hydrogel precursor can adjust the viscosity of the ink by adjusting the photocuring time, thereby controlling the growth state of the cells; since the bonding strength of ionic crosslinking is stronger than that of photocrosslinking, the addition of the ionic hydrogel precursor can further strengthen the spatial skeleton strength of the hydrogel, improve the stability of the hydrogel, and broaden the adjustable range of the cell growth environment; in addition, the methacrylated sodium alginate, methacrylated gelatin and methacrylated hyaluronic acid in the photocurable hydrogel precursor have both photocuring properties and ionic curing properties. When used, even if the ionic hydrogel precursor is not added, the viscosity of the ink can be first photocured, and then the ionic curing can be performed to enhance the stability of the gel, thereby effectively regulating the growth environment of the cells. That is to say, when sodium methacrylated alginate, methacrylated gelatin and methacrylated hyaluronic acid are selected as photocurable hydrogel precursors, even if only the photocurable hydrogel precursor is added, the same effect as adding the ionic hydrogel precursor at the same time can be achieved.

[0052] According to an embodiment of the present invention, the hydrogel cross-linking precursor includes matrix glue and optionally one or more of a photocurable hydrogel precursor, an ionic hydrogel precursor, and other thermally curable hydrogel precursors.

[0053] According to an embodiment of the present invention, the hydrogel cross-linking precursor includes matrix glue and optionally one or more of a photocurable hydrogel precursor and an ionic hydrogel precursor.

[0054] According to an embodiment of the present invention, in the hydrogel cross-linking precursor, the content of the matrix glue is ≤50% by mass percentage, preferably the content of the matrix glue is ≤40%, and further preferably, the content of the matrix glue is ≤30%, for example, 35%, 25%, 20%, or 18%.

[0055] As an example, in terms of mass percentage, in the hydrogel cross-linking precursor, the content of the matrix glue is 20%, and the content of the photocurable hydrogel precursor and / or the ionic hydrogel precursor is 80%.

[0056] According to an embodiment of the present invention, when the hydrogel cross-linking precursor comprises a photocurable hydrogel precursor, the dispersion medium comprises a photoinitiator.

[0057] According to an embodiment of the present invention, the mass ratio of the photocurable hydrogel precursor to the photoinitiator is (2-15):1; preferably, the mass ratio of the photocurable hydrogel precursor to the photoinitiator is (3-12):1; for example, 4:1, 7:1, 9:1, 10:1, 12:1.

[0058] According to an embodiment of the present invention, the dispersion medium further comprises at least a hydrogel adhesion enhancer, which is used to enhance the adhesion of the hydrogel to the substrate. Specifically, the hydrogel adhesion enhancer includes but is not limited to at least one of polydopamine, polymethacrylamide, polyethylene glycol, polyvinyl alcohol, etc.

[0059] As an example, the dispersion medium includes at least carboxymethyl cellulose and polydopamine.

[0060] As an example, the dispersion medium includes at least alginate, carboxymethyl cellulose and polydopamine.

[0061] As an example, the dispersion medium at least includes polydopamine, a photocurable hydrogel precursor and a photoinitiator.

[0062] As an example, the dispersion medium at least includes alginate, carboxymethyl cellulose, polydopamine and chitosan.

[0063] As an example, the dispersion medium at least includes alginate, carboxymethyl cellulose, polydopamine, a photocurable hydrogel precursor and a photoinitiator.

[0064] According to an embodiment of the present invention, the mass ratio of alginate, carboxymethyl cellulose, polydopamine, photocurable hydrogel precursor and photoinitiator is (1-5):1:0.5:(1-5):(0.05-0.25).

[0065] According to an embodiment of the present invention, the dispersion medium further includes a cell adhesion enhancer, which is used to enhance the adhesion of cells to the hydrogel. Specifically, the cell adhesion enhancer is selected from a bio-extracted polymer, such as at least one of poly-lysine, RGD polypeptide, laminin, etc.

[0066] According to an embodiment of the present invention, in the dispersion medium, the concentration of the cell adhesion enhancer is 0.1-1 mg / ml. For example, the concentration of the laminin and / or RGD polypeptide is 0.1-1 mg / ml.

[0067] According to an embodiment of the present invention, the dispersion medium further includes one or more of cytokines, antibiotics, small molecule compounds, cellulase, additives, and culture medium.

[0068] According to an embodiment of the present invention, the cytokine includes but is not limited to: at least one of R-Spondin cell growth factor, mNoggin cell growth factor, EGF cell growth factor, and Wnt3a cell growth factor.

[0069] According to an embodiment of the present invention, the concentration of the cytokine is 50-100 ng / mL.

[0070] According to an embodiment of the present invention, the antibiotics include but are not limited to Primocin TM Antibiotics for primary cells.

[0071] According to an embodiment of the present invention, the concentration of the antibiotic is 50-100 μg / mL.

[0072] According to an embodiment of the present invention, the small molecule compounds include but are not limited to SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridinyl)-1H-imidazole), Gastrin I gastrin, A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolyl)-1H-pyrazole-1-thiocarboxamide), nicotinamide, Prostaglandin E2 prostaglandin E2, and N-acetyl-L-cysteine, GlutaMAX TM At least one of .

[0073] According to an embodiment of the present invention, the concentration of the small molecule compound is 10 nM-10 mM.

[0074] According to an embodiment of the present invention, the cellulase includes but is not limited to endoglucanase, exoglucanase, β-glucosidase, and most of them are commercially available mixed cellulase (Cas No: 9012-54-8) (including all the above ingredients).

[0075] According to an embodiment of the present invention, the concentration of the cellulase is 0.01-0.1%.

[0076] According to an embodiment of the present invention, the additive includes B-27 TM additive.

[0077] According to an embodiment of the present invention, the concentration of the additive is 1%-2%.

[0078] According to an embodiment of the present invention, the culture medium includes but is not limited to DMEM / F12 culture medium.

[0079] According to an embodiment of the present invention, the biological unit comprises a cell, a tissue or an organoid.

[0080] According to an embodiment of the present invention, the cells include but are not limited to at least one of normal primary cells, tumor primary cells, cell lines, induced pluripotent stem cells, etc. Specifically, the normal primary cells include epithelial cells, neural cells, red blood cells, white blood cells, platelets, phagocytes (neutrophils, basophils, eosinophils, etc.), B lymphocytes, effector B cells, memory B cells, T lymphocytes, memory T cells, effector T cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, cardiomyocytes, osteoblasts, glial cells, hepatocytes, kidney cells, glandular cells, endocrine cells (thyroid cells, thymus cells, pancreatic B cells, pancreatic islet cells, etc.), hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, etc., and the tumor primary cells include prostate cancer cells, epithelial cells, mitochondrial stem ... Cancer cells, breast cancer cells, renal cancer cells, lung cancer cells, colon cancer cells, colorectal cancer cells, gastric cancer cells, brain cancer cells, glioblastoma cells, pancreatic cancer cells, myeloid leukemia cells, cervical cancer cells, medullary thyroid cancer cells, stromal ovarian cancer cells, astrocytoma cells, epithelial cancer cells, neuroendocrine cancer cells, gastrointestinal pancreatic tumor cells, non-Hodgkin's lymphoma cells, exocrine pancreatic cancer cells, Ewing's sarcoma cells, skin cancer cells, melanoma cells, the cell line includes at least one of Hela human cervical cancer cells, SW480 human colon adenocarcinoma cells, HCT116 human colon cancer cells, Caco-2 human colon cancer cells, LOVO human colorectal cancer cells, DLD1 human colon adenocarcinoma cells, HepG2 human liver cancer cells, etc.

[0081] According to an embodiment of the present invention, the tissue includes but is not limited to one or more of cell clusters, small pieces of biological tissue, cytoplasmic matrix, etc. Specifically, the biological tissue includes but is not limited to one or more of epithelial tissue, connective tissue, muscle tissue, and neural tissue.

[0082] According to an embodiment of the present invention, the organoids include but are not limited to one or more of small intestinal organoids, stomach organoids, colon organoids, lung organoids, bladder organoids, brain organoids, liver organoids, pancreatic organoids, kidney organoids, ovarian organoids, esophageal organoids, and heart organoids.

[0083] According to an embodiment of the present invention, the concentration of cells in the biological unit is 10 4 -10 6 Pieces / mL.

[0084] According to an embodiment of the present invention, the biological unit may further include a drug.

[0085] Specifically, the drug is a drug to be screened, including any particles or molecules that can be uniformly dispersed in a medium such as water, a buffer solution (PBS, HEPES, physiological saline, etc.), serum, culture medium, etc., and can be a single drug or a mixture of multiple drugs.

[0086] Taking the treatment of colon cancer as an example, the drugs may be oxaliplatin, LV, 5-FU, bevacizumab, cetuximab, capecitabine, irinotecan, regorafenib, furiquintinib, trifluridine tipiracil, trastuzumab, lapatinib, vemurafenib, dabrafenib, trametinib, raltitrexed or pembrolizumab, etc.

[0087] According to an embodiment of the present invention, the mixed medicine can be printed by mixing multiple medicines in the same ink tube in advance; or multiple medicines can be packaged in different ink tubes and the medicine mixing can be completed by performing multiple rounds of printing at the same site; the latter is preferred to reduce manual operation steps.

[0088] According to an embodiment of the present invention, the source of the biological unit includes one or more of patient tissue sections, tissue isolates, primary cells (tissues), passage cells (tissues), laboratory cells, and the like.

[0089] According to an embodiment of the present invention, the viscosity of the bio-ink composition is 1.5 mPa·s-100000 mPa·s (37° C.).

[0090] According to an embodiment of the present invention, the bio-ink composition is in a liquid state at a temperature of 0-40°C.

[0091] [Bio-ink]

[0092] As mentioned above, the present invention also provides a bio-ink, which is obtained by mixing the components of the above-mentioned bio-ink composition.

[0093] According to an embodiment of the present invention, the bio-ink includes a hydrogel formed by the hydrogel cross-linking precursor and a cross-linking initiator. Studies have found that the hydrogel has a spatial skeleton, and the biological units can be dispersed and supported in the spatial skeleton, thereby preventing the biological units from settling in the ink and ensuring that the biological units can be evenly dispersed in the bio-ink for a long time.

[0094] [Method for preparing bio-ink]

[0095] As mentioned above, the present invention also provides a method for preparing the above-mentioned bio-ink, which comprises: mixing the biological units in the bio-ink composition with a dispersion medium to obtain the bio-ink.

[0096] According to an embodiment of the present invention, the preparation method includes: mixing a hydrogel adhesion enhancer, an ionic hydrogel precursor and / or a photocurable hydrogel precursor with other components to obtain the dispersion medium, mixing the dispersion medium with a biological unit to obtain a biological ink, wherein the other components are at least one of the components listed in the above composition except the hydrogel adhesion enhancer, the ionic hydrogel precursor and / or the photocurable hydrogel precursor.

[0097] According to an embodiment of the present invention, the preparation method comprises the following steps: mixing and reacting a hydrogel adhesion enhancer with a solvent, and then mixing it with an ionic hydrogel precursor and / or a photocurable hydrogel precursor, other components and a biological unit to obtain a bio-ink, wherein the other components are at least one of the components listed in the above composition except the hydrogel adhesion enhancer, the ionic hydrogel precursor and / or the photocurable hydrogel precursor.

[0098] According to an embodiment of the present invention, the preparation method comprises the following steps: mixing a hydrogel adhesion enhancer with a solvent, mixing with an ionic hydrogel precursor and / or a photocurable hydrogel precursor, other components and a biological unit, and obtaining a bio-ink after reaction, wherein the other components are at least one of the components listed in the above composition except the hydrogel adhesion enhancer, the ionic hydrogel precursor and / or the photocurable hydrogel precursor.

[0099] According to an embodiment of the present invention, the reaction temperature is 0-80°C, preferably the reaction temperature is 0-60°C, for example 4°C.

[0100] According to an embodiment of the present invention, the reaction time is 10 to 30 hours, preferably the reaction time is 15 to 25 hours, for example 24 hours.

[0101] According to an embodiment of the present invention, the solvent is selected from at least one of DMEM culture medium, F12 culture medium, PBS solution, and Tris-HCl solution, preferably PBS solution, and the pH value of the PBS solution is 8-12, for example, 11.

[0102] As an example, the dispersion medium is prepared by the following method: alginate, hydroxymethyl cellulose, polydopamine, a photocurable hydrogel precursor and an initiator are mixed with other components to obtain the dispersion medium; the other components include at least one of laminin / RGD polypeptide, cytokines, antibiotics, small molecule compounds, cellulase, and culture medium.

[0103] According to an embodiment of the present invention, the mixing is performed by conventional methods in the art, such as one or more of stirring, ultrasound, blowing, centrifugation, shaking, and the like.

[0104] According to an embodiment of the present invention, the dispersion medium needs to be sterilized before use. Specifically, the sterilization method includes cobalt 60 irradiation sterilization, high pressure steam sterilization, ultraviolet light irradiation sterilization or bacterial filtration by a bacterial filter; specifically, the performance of the substance cannot be changed during sterilization, and preferably, the bacterial filter is used to filter bacteria.

[0105] [Application of bio-ink in cell or organoid printing]

[0106] As mentioned above, the present invention also provides the use of the biological ink in cell or organoid printing.

[0107] According to an embodiment of the present invention, it is used for cell printing, comprising the following steps: printing the biological ink composition onto a substrate by 3D printing, inkjet printing, microcontact printing, or the like.

[0108] According to an embodiment of the present invention, the printing comprises the following steps: filling the bio-ink composition into an ink tube and photocuring the bio-ink composition, and then printing the photocured bio-ink composition, ie, the bio-ink, onto a substrate.

[0109] The photocuring includes irradiating the bio-ink composition with light.

[0110] According to an embodiment of the present invention, the wavelength of light used in the light irradiation is 365-430 nm, for example, 405 nm blue light.

[0111] According to an embodiment of the present invention, the light curing time is 1 to 100 s, preferably the light curing time is 1 to 50 s, for example, 1 s, 5 s, 10 s, 30 s, or 60 s.

[0112] According to an embodiment of the present invention, when the bio-ink composition contains an ionic hydrogel precursor, after printing the photocurable bio-ink composition onto a substrate, the following step is further included: immersing the printed substrate in a solution containing an ionic initiator for curing.

[0113] According to an embodiment of the present invention, the ionic initiator has the definition as described above.

[0114] According to an embodiment of the present invention, in the solution containing the ion initiator, the mass concentration of the ion initiator is 1-3%, for example, 1.5%.

[0115] According to an embodiment of the present invention, the substrate is selected from a flexible material or a hard material.

[0116] According to an embodiment of the present invention, the flexible material comprises one or more materials such as a polymer film and a hydrogel. The polymer film is, for example, a polydimethylsiloxane film (PDMS), a polyethylene terephthalate film (PET), an ABS plastic film, or a polytetrafluoroethylene film. The hydrogel is, for example, a hyaluronic acid hydrogel, an alginate hydrogel, a cellulose hydrogel, a chitosan hydrogel, a collagen hydrogel, a gelatin hydrogel, a silk hydrogel, an agar hydrogel, a DNA hydrogel, a decellularized tissue hydrogel, a polyvinyl alcohol hydrogel, a polyacrylic acid hydrogel, a polyethylene glycol hydrogel, a polydopamine (PDA) hydrogel, or a polyacrylate hydrogel, etc.; preferably, a polyethylene terephthalate film or a polydopamine hydrogel.

[0117] According to an embodiment of the present invention, the hard material includes one or more materials such as plastics, silicon, metals, etc. The plastics are, for example, common culture dishes and cell culture well plates (the materials may be polystyrene, polyethylene, polypropylene, polyurethane, polycarbonate, polysilazane, polytetrafluoroethylene), the silicon is, for example, glass sheets, single crystal silicon sheets, silicon dioxide sheets, quartz sheets or polydimethylsiloxane films, etc., the metals are, for example, aluminum sheets, copper sheets, nickel sheets or aluminum oxide sheets, etc.; preferably, aluminum sheets or glass sheets.

[0118] According to an embodiment of the present invention, the substrate (base) needs to be sterilized before use. Specifically, the sterilization method includes 75% ethanol immersion sterilization, cobalt 60 irradiation sterilization, high-pressure steam sterilization or ultraviolet light irradiation sterilization; from the perspective of cost-effectiveness, ultraviolet light irradiation for a certain period of time (such as 30 minutes) is preferred.

[0119] According to an embodiment of the present invention, the parameter settings of the 3D printing include: at least one of the printing pattern, the height of the printing needle from the substrate, the inkjet pressure, the unit point inkjet time, the substrate contact angle, and the ink drop printing accuracy.

[0120] Preferably, the printing pattern is set as a dot matrix, a line or a 2D / 3D pattern;

[0121] Preferably, the height of the printing needle from the substrate is 20-100 microns;

[0122] Preferably, the unit point inkjet time is 0-10s;

[0123] Preferably, the inkjet air pressure is 0-60psi with an accuracy of 0.1psi;

[0124] The inkjet pressure is related to the viscosity of the ink. The inkjet pressure corresponding to the low viscosity liquid (such as water, temperature 5°C, viscosity 1.5 mPa·s) is 0.5-5.0 psi, preferably 1.0 psi; the inkjet pressure corresponding to the high viscosity liquid (such as a mixture of 2% carboxymethyl cellulose and 2% sodium alginate, temperature 4°C, shear rate 0.1 / s, shear stress 26.554 Pa, viscosity 250000±50000 mPa·s) is 10-55 psi, preferably 30 psi.

[0125] Preferably, the substrate contact angle is 0-100 degrees, preferably 50±10 degrees;

[0126] Preferably, the ink droplet printing accuracy is 100-1500 microns, preferably 300-500 microns. The ink droplet printing accuracy is defined as the ink dot size formed on the substrate by the minimum inkjet amount, such as the diameter of a single dot, the width of a line, etc. The design of the bioactive ink of the present invention can obtain ink droplets with significantly improved printing accuracy, achieving nanoliter-level printing.

[0127] [Substrate pretreatment and surface modification]

[0128] According to an embodiment of the present invention, before printing the bio-ink onto a substrate, the method further includes pre-treating and surface modifying the substrate.

[0129] According to an embodiment of the present invention, the pretreatment comprises subjecting the substrate to acetone treatment to obtain a pretreated substrate.

[0130] According to an embodiment of the present invention, the pretreatment time is 10-20 minutes.

[0131] According to an embodiment of the present invention, the surface modification includes modifying the surface of the substrate with dopamine.

[0132] According to an embodiment of the present invention, the concentration of dopamine is 10-90 mg / mL.

[0133] According to an embodiment of the present invention, the surface modification comprises the following steps: (1) dissolving a dopamine salt in a buffer solution to obtain a dopamine aqueous solution; (2) immersing the pretreated substrate in the dopamine aqueous solution, heating it, and allowing it to stand to obtain a modified substrate.

[0134] According to an embodiment of the present invention, the surface modification comprises the following steps: (1) dissolving dopamine hydrochloride in Tris-HCl buffer (pH=8.8) to obtain a 90 mg / mL dopamine aqueous solution; (2) immersing a substrate in the above dopamine aqueous solution, heating at 60°C for 6 hours and then standing at room temperature overnight, removing the remaining solution, washing with PBS, and drying with nitrogen to obtain a polydopamine-grafted high-adhesion substrate.

[0135] In the present invention, the adhesion between the printed object (ie, the hydrogel pattern) and the substrate can be significantly improved through pretreatment and surface modification.

[0136] [Cell or organoid chips and their applications]

[0137] The present invention also provides a cell or organoid chip, specifically, a high-throughput cell or organoid chip, which is prepared by any of the above preparation methods.

[0138] According to an embodiment of the present invention, the chip is composed of a substrate and a hydrogel (containing cells) pattern.

[0139] According to an embodiment of the present invention, the bioactive ink is patterned on the surface of the substrate by printing, and a high-throughput cell or organoid chip is obtained after curing.

[0140] The present invention also provides the use of the above-mentioned cell or organoid chip in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.

[0141] Specifically, the cell or organoid chip can realize high-throughput drug screening, drug toxicity and efficacy testing.

[0142] Technical terms

[0143] The "printing" mentioned in the present invention is to squeeze out the ink containing biological units pre-filled in the ink tube by air pressure, and the nozzle moves according to the pre-programmed printing path to write the ink on the substrate and regulate the formation and distribution of ink droplets. It can be divided into multi-nozzle printing mode and single-nozzle printing mode; on the one hand, multi-nozzle printing equipment (dispensing machine) can be used to achieve simultaneous printing of cell or organoid ink and drug dripping, that is, chip preparation and drug screening application can be carried out simultaneously; on the other hand, the chip preparation and drug screening links can be completed in batches.

[0144] The "printing" described in the present invention includes two ink droplet forming modes, namely "dot" and "line"; intermittent inkjet to form "dots", continuous inkjet to form "lines" (dots move to form lines), long-distance inkjet to form "surfaces" (lines move to form surfaces), and different height printing combinations to form "bodies" (surfaces move to form bodies). The printing effect of the "dots" described above is determined by the inkjet pressure, needle model, unit point inkjet time, substrate (substrate) contact angle, and ink viscosity; the greater the inkjet pressure, the larger the needle, the longer the unit point inkjet time, the smaller the substrate (substrate) contact angle, and the smaller the ink viscosity, the larger the final "dot" diameter, and vice versa. The printing effect of the "line" described above is determined by the inkjet pressure, needle model, substrate (substrate) contact angle, and ink viscosity; the greater the inkjet pressure, the larger the needle, the slower the needle movement speed, the smaller the substrate (substrate) contact angle, and the smaller the ink viscosity, the larger the final "line" width, and vice versa.

[0145] The "printing equipment (dispensing machine for short)" described in the present invention mainly includes a program host, a micron-level precision mobile platform, a temperature controller, an air source, a dispensing machine (inkjet air pressure controller), an ink tube, and a micron dispensing needle; the purchased brands include Nordson, Japan Musashi, and preferably Nordson; the printing equipment and its related consumables need to be sterilized before use, and the methods include 75% ethanol immersion sterilization, cobalt 60 irradiation sterilization, high-pressure steam sterilization, ultraviolet light irradiation sterilization, and bacterial filter filtration of bacteria; from the perspective of cost-effectiveness, it is preferably high-pressure steam sterilization (120°C, 40 minutes) or ultraviolet light irradiation for 30 minutes. The dispensing needle has a cross-sectional size of 100-1500 microns, preferably 100 or 200 microns; the purchased brands include Nordson, Japan Musashi, and preferably Nordson.

[0146] The "programming" mentioned in the present invention includes the printing path and inkjet pressure of zero-dimensional, one-dimensional, two-dimensional, or three-dimensional patterns, and printing is performed after being programmed by software. The printing path of the zero-dimensional, one-dimensional, two-dimensional, or three-dimensional pattern includes but is not limited to dots, dot matrix, line segment, grid, cylinder, etc.

[0147] The "printing ink droplet forming" described in the present invention can be one-round printing forming or multiple-round printing composite forming; the printing path of each round in the multiple-round printing can be the same or different.

[0148] The "software" mentioned in the present invention includes but is not limited to 3D MAX, CAD, and programming software that comes with the printing device.

[0149] The "temperature controller" mentioned in the present invention includes temperature control of the ink tube, needle, and printing platform (base or substrate) to prevent the evaporation of water in the ink droplets during the printing process, maintain the biological activity of the cell chip, and ensure the performance quality of the drug; the control range includes -10°C to room temperature, preferably (3±2)°C.

[0150] The "ink tube" described in the present invention has volume specifications of 3 ml and 5 ml, and can be filled with 50 μL to 4 ml of ink. For rare or expensive cells or drugs, 100-200 μL is preferred. After the ink tube is filled with ink, especially high-viscosity substances, some bubbles will be generated, which need to be removed by centrifugation to ensure the stability of the printing process and prevent bubbles from affecting cell activity.

[0151] The "printability" mentioned in the present invention includes stable uniformity or dispersion of ink, stability of ink droplet formation, uniformity of continuous printing, and high resolution of printed patterns; stable uniformity or dispersion of ink means that there will be no aggregation, phase separation, solidification, volatilization, or denaturation during the printing process; stability of ink droplet formation means that the shape on the substrate is stable after printing, and it will not spread, evaporate, shrink, or collapse, for example, the printed dots are uniformly circular with smooth boundaries; uniformity of continuous printing means that the size and shape of multiple prints are not much different, for example, the diameter of each dot in the printed dot matrix is ​​not much different, the width of the printed lines is not much different at different places, and there will be no line breakage or fusion of multiple ink droplets; the resolution of the printed pattern refers to the distribution of ink droplets after printing and the size and boundary of the pattern involved.

[0152] The "ink droplet forming" mentioned in the present invention includes the shape and size of the ink droplets, which are affected by the inkjet pressure, substrate characteristics, and ink properties; the greater the inkjet pressure, the larger the ink droplets; the smaller the substrate contact angle, the more obvious the spread of the printed ink droplets, and the larger the final ink droplets; the smaller the viscosity of the ink, the worse the forming effect (forming: the difference between the shape of the ink droplet at the moment of printing and the shape after printing, the higher the degree of forming, the smaller the difference).

[0153] The "ink droplet distribution" described in the present invention is the same as the printing path, that is, the air pressure continuously or intermittently squeezes out the ink, and at the same time controls the movement of the needle to leave the squeezed ink on the substrate surface during the movement. Different ink droplet distributions are formed by designing the printing path, that is, patterning of ink droplets.

[0154] The composition, bio-ink, preparation method and application of the present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection of the present invention.

[0155] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0156] Example 1

[0157] Cell / Organoid Culture

[0158] 1. Cell separation

[0159] Soak the ex vivo colorectal cancer tissue in a 1:2 iodine / PBS mixture for five minutes and wash it five times with PBS containing streptomycin and penicillin. After washing, put the tissue into 15mL digestion solution, cut it into pieces, and digest it in a 37℃ water bath for 40 minutes. After adding 15mL of culture medium, filter to remove indigestible tissue pieces, centrifuge the filtrate at 1200 rpm for 5 minutes, and remove the supernatant. Add 10mL of red blood cell lysis solution to lyse for 10 minutes, centrifuge at 1200 rpm for 5 minutes, and remove the supernatant. After washing the cells with 5mL of separation solution, add an appropriate amount of Matrigel matrix glue, pipette and mix, and move it into a 24-well plate (50μL per well), solidify it at 37℃ for 30 minutes, add culture medium (500μL per well) and place it in the incubator.

[0160] The above digestion solution is supplemented with 10μM Y-27632 and 100μg / mL Primocin TM Primary cell antibiotics, 2mg / mL collagenase type 2 enzyme separation solution. Separation solution is supplemented with 2mM GlutaMAX TM The culture medium was DMEM / F12 medium containing 25 mM HEPES, 1% streptomycin and penicillin.

[0161] 2. Cell culture and passaging

[0162] The cells were cultured at 37°C and 5% CO2. The culture medium was changed every two days and the cells were passaged every seven days. The passage steps were as follows: 500 μL of separation solution was added to each well, and the matrix gel was broken by aspiration with a pipette and then all the liquid was transferred to a centrifuge tube. 1 mL of separation solution was taken to rinse each well, and all the liquid was transferred to the above centrifuge tube and centrifuged at 2000 rpm for five minutes. The supernatant was removed, and TrypLEexpress enzyme (250 μL per well) and 10 μM Y-27632 were added, and digested in a water bath at 37°C for 5 minutes. After the digestion, the liquid was aspirated several times with a pipette to further break up the large pieces of matrix gel, and then centrifuged at 2000 rpm for five minutes. The supernatant was removed, 2 mL of separation solution was added to resuspend, and then centrifuged at 2000 rpm for five minutes again. The supernatant was removed, an appropriate amount of Matrigel matrix gel was added, and the mixture was mixed by aspiration and then transferred to a 24-well plate (50 μL per well), solidified at 37°C for 30 minutes, and the culture medium (500 μL per well) was added and placed in an incubator.

[0163] The separation solution in this step is 2 mM GlutaMAX TM , DMEM / F12 medium containing 25 mM HEPES, 1% streptomycin and penicillin.

[0164] The culture medium in this step is supplemented with 2 mM GlutaMAX TM , 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Additives, 100ng / mL Wnt3a cell growth factor, 1.25mM N-acetyl-L-cysteine, 500ng / mL R-Spondin, 100ng / mL mNoggin cell growth factor, 50ng / mL EGF cell growth factor, 10nM gastrin Ihuman, 0.5μM A83-01, 3μM SB202190, 10nM prostaglandin E2, 10mM nicotinamide, 100μg / mL Primocin TM Primary cells were cultured in antibiotic-free DMEM / F12 medium.

[0165] Cells that have been stably passaged for 4 times were extracted for printing and drug screening. Figure 1 These are the intestinal cancer cells cultured in this example.

[0166] 3. Organoid Culture

[0167] Organoid culture medium is supplemented with 2 mM GlutaMAX TM , 25mM HEPES, 1% streptomycin and penicillin, 2% B-27 TM Additives, 100ng / mL Wnt3a cell growth factor, 1.25mM N-acetyl-L-cysteine, 500ng / mL R-Spondin, 100ng / mL mNoggin cell growth factor, 50ng / mL EGF cell growth factor, 10nM gastrin Ihuman, 0.5μM A83-01, 3μM SB202190, 10nM prostaglandin E2, 10mM nicotinamide, 100μg / mL Primocin TM Primary cells were cultured in DMEM / F12 medium with antibiotics. Figure 2 shown. Figure 2 Morphology of colon cancer organoids cultured from patient tissue sections.

[0168] Example 2

[0169] Formulating the Dispersing Medium in Bioactive Inks

[0170] The formulation includes 100ng / mL Wnt3a cell growth factor, 1.25mM N-acetyl-L-cysteine, 500ng / mL R-Spondin, 100ng / mL mNoggin cell growth factor, 50ng / mL EGF cell growth factor, 10nM gastrin Ihuman, 0.5μM A83-01, 3μM SB202190, 10nM prostaglandin E2, 10mM nicotinamide, 100μg / mL Primocin TM DMEM / F12 culture medium with antibiotics for primary cells. Prepare the culture medium and sterilize it with a bacterial filter.

[0171] Preparation of the dispersion medium of the bioactive ink: Sodium alginate powder and carboxymethyl cellulose powder were spread flat and sterilized under ultraviolet light for 3 hours. 20 mg, 50 mg, 75 mg, 100 mg of sodium alginate (NaA) powder, 20 mg of carboxymethyl cellulose (CMC) powder (2% NaA-2% CMC, 5% NaA-2% CMC, 7.5% NaA-2% CMC, 10% NaA-2% CMC), 10 mg of polydopamine powder, 20 mg of methacryloyl sodium alginate, and 1 mg of LAP were dissolved uniformly by stirring and ultrasonication to prepare four dispersion media.

[0172] Table 1 lists the contents and types of sodium alginate and carboxymethyl cellulose in the four dispersion media.

[0173] 1500 mg of dopamine hydrochloride was dissolved in 30 mL of PBS (pH=11) buffer solution, filtered and sterilized with a cell filter, and printed after mixing with other components, finally ensuring that polydopamine accounted for 0.1% in the bio-ink.

[0174] Table 1 Content of sodium alginate and carboxymethyl cellulose

[0175] Sodium alginate Carboxymethylcellulose, 20 mg Hydrogel A 20mg 2%NaA-2%CMC Hydrogel B 50mg 5%NaA-2%CMC Hydrogel C 75mg 7.5%NaA-2%CMC Hydrogel D 100mg 10%NaA-2%CMC

[0176] Example 3

[0177] Preparation of substrate

[0178] 1. Pretreatment of substrate

[0179] Soak the polystyrene substrate in acetone for 10-20 minutes, then take it out and dry it.

[0180] 2. Modification of the base

[0181] Dopamine hydrochloride was dissolved in Tris-HCl buffer (pH=8.8) to obtain a 90 mg / mL dopamine aqueous solution. The pretreated substrate was immersed in the above dopamine aqueous solution, heated at 60°C for 6 hours and then allowed to stand at room temperature overnight, the remaining solution was removed, washed with PBS, and dried with nitrogen to obtain a polydopamine-grafted high-adhesion substrate.

[0182] Example 4

[0183] Preparation of high-throughput cell chips

[0184] 1. Preparation of bioactive ink

[0185] The intestinal cancer cell suspension after enzyme digestion in Example 1 was centrifuged, the supernatant was removed, and the four dispersion media prepared in Example 2 were added respectively and mixed evenly to obtain four bioactive inks with a cell concentration of 10 5 -10 6 Pieces / mL.

[0186] 2. Printing bioactive ink

[0187] 1) Place the ink tube, needle, ink tube pressure plug and other consumables in a high-pressure steam sterilizer for sterilization (130°C, 40 minutes), and irradiate the printing substrate (PET film, well plate) with ultraviolet light for 30 minutes.

[0188] 2) Using the built-in program of the printing device, the printing process is programmed, including setting the printing pattern coordinates to a dot matrix, the height of the printing needle from the substrate to 20-100 microns, the inkjet pressure to 0-60psi, the unit point inkjet time to 0-10s, the substrate contact angle to 0-100 degrees, and the ink droplet printing accuracy to 100-1500 microns.

[0189] In one example, the height of the printing needle from the substrate is 50 microns, the inkjet pressure is 10psi and 50psi, the unit point inkjet time is 0.1s, 0.4s, and 0.8s, the substrate contact angle is 50 degrees, and the ink drop printing accuracy is 350 microns.

[0190] 3) Fix the printed substrate flat on the printing platform after UV irradiation; fill 300 microliters of bioactive ink into the ink tube, remove bubbles in the dispersion medium by centrifugation, and irradiate with a blue light lamp (405nm) for 30-60 seconds to complete the cross-linking of the methacrylated sodium alginate. Connect the ink tube to the dispensing machine (inkjet air pressure controller) and different types of needles with cross-sectional sizes ranging from 100 to 1500 microns, place it on the mobile platform, use a temperature controller to control the temperature at 4°C, and run the program to print. After printing is completed, use 1.5% Ca 2+(PBS is the dispersion liquid, pH = 7.0) The ink droplets were soaked for 10 minutes to complete the solidification, and a high-throughput cell chip was prepared. The printed chip was placed under a microscope for observation. The printing results of the ink prepared by hydrogel A are shown in Figure 2. Figure 3 As shown in the results, it can be seen that the ink droplet forming effect is good; the greater the inkjet pressure, the larger the ink dot diameter; the longer the unit point inkjet time, the larger the ink dot diameter.

[0191] Example 5

[0192] Detecting cell viability in a chip

[0193] 1) The chip printed in Example 4 was subjected to a live-dead cell immunostaining test to examine the survival rate of cells in the ink spots on the chip.

[0194] The specific method is: take out the sample (i.e., the chip printed in Example 4), wash it with PBS 1-2 times, and wash away the residual culture medium solution. Use a cell live and dead staining kit for staining, first add enough prepared propidium iodide working solution to ensure that the cells are covered, and incubate at room temperature for 10 minutes. Remove the propidium iodide working solution, then gently wash it with enough PBS to remove the supernatant. Add enough prepared calcein working solution to ensure that the cells are covered, and incubate at room temperature for 20-45 minutes. Remove the calcein working solution, then gently wash it with enough PBS to remove the supernatant. Add PBS or anti-fluorescence quencher, and finally observe the live and dead labeling of the cells under a fluorescence microscope. The proportion of live cells in the ink dots is statistically 80-99%.

[0195] 2) The chip printed in Example 4 was placed in an organoid culture medium for culture, and after 14 days, live-dead cell immunostaining test was performed to check the survival rate of cells in the ink dots. After 14 days of culture, the proportion of live cells in the ink dots was 80-99%.

[0196] 3) Comparison of the proliferation and differentiation efficiency of human colon cancer cells (organoids) after printing with four hydrogel inks: 2% NaA-2% CMC, 5% NaA-2% CMC, 7.5% NaA-2% CMC, and 10% NaA-2% CMC. Figure 5 As shown, the cell growth corresponding to the hydrogel inks of 2% NaA-2% CMC and 5% NaA-2% CMC is better than that of the inks of 7.5% NaA-2% CMC and 10% NaA-2% CMC, indicating that the cell growth state can be regulated by adjusting the ink composition.

[0197] Example 6

[0198] Bio-inks with other dispersion media combinations

[0199] The dispersion medium of this embodiment is substantially the same as that of embodiment 2, except that the hydrogel cross-linking precursor in embodiment 2 is replaced with a hydrogel cross-linking precursor as shown in Table 2.

[0200] Table 2 Combinations of different hydrogel cross-linking precursors

[0201]

[0202] Example 7

[0203] Mixing method of hydrogel adhesion enhancer

[0204] The hydrogel was prepared according to Example 2, except for the mixing method of polydopamine (including solvent and addition method). The ink droplets were tested after being immersed in F12 culture medium for 7 days after printing, as shown in Table 3:

[0205] Table 3 Effects of different polydopamine mixing methods

[0206]

[0207]

[0208] In addition, changing the proportion of polydopamine relative to the bio-ink (0.1, 0.2, 0.5, 1%) had no significant difference on the above results.

[0209] Example 8

[0210] Ink mixing heat curing ink for printability

[0211] The dispersion medium was prepared according to the method of Example 2, and 5% NaA-2% CMC hydrogel mixed matrix glue was used for printing. The printing conditions were as follows. Figure 6 As shown in Table 3 (a simple low-temperature method: the ink tube is set in an environment of 1°C, the needle is at room temperature, and the temperature of the printing platform is 1°C), the printing conditions of the ink droplets are shown, and under the condition that the matrix gel content is less than 90%, the printed ink dots can reach the nanoliter level.

[0212] Table 4 Printing conditions after mixing different ratios of matrix gel and hydrogel

[0213]

[0214]

[0215] Example 9

[0216] Light-curing and ion-curing hybrid ink with adjustable ink viscosity

[0217] A photocurable and ion-curable mixed hydrogel was prepared according to the method of Example 2. Taking 2% methyl cellulose-3% sodium alginate-2% methacrylated sodium alginate as an example, printing was performed according to the method of Example 4, except that: a 405nm blue light lamp was used for irradiation for 1, 5, 10, 30, and 60 seconds before printing, and the viscosity of the ink was regulated by the degree of photocuring to regulate the growth state of the cells. The 3-day proliferation rate and survival rate of the cells in the ink were measured according to the method of Example 5, as shown in Table 5. It can be seen that the proliferation rate and survival rate of the cells can be controlled by controlling the photocuring time and the age of the ink.

[0218] Table 5 Printing conditions after different irradiation conditions

[0219] Light curing time(s) Value-added rate (%) Survival rate (%) 1 314 95 5 281 90 10 157 82 30 119 75 60 108 73

[0220] Example 10

[0221] Effects of inks with different matrigel ratios on cell and organ growth

[0222] The dispersion medium was prepared according to the method of Example 8, and the colorectal cancer cell suspension after enzyme digestion in Example 1 was centrifuged, the supernatant was removed, and the suspension was added to the dispersion medium prepared in Example 8, and the corresponding bioactive ink was obtained after mixing evenly. The cell concentration was 10 5 -10 6 The chip printed according to Example 4 was then cultured in an organoid culture medium.

[0223] See also Figure 7 As shown, the growth of organoids after initial printing (Day0) and after 7 days of culture. It can be seen from the figure that when the mixing ratio of matrix gel is greater than or equal to 50%, the cell density printed on Day0 is uneven, and cell sedimentation may occur. After 7 days of culture, the organoid grows fast but unevenly; when the matrix gel ratio is 20%, the cell density printed on Day0 is uniform, and after 7 days of culture, the organoid grows fast and has the best uniformity; when the matrix gel ratio is less than 20%, for example 5% or 0%, the cell density printed on Day0 is uniform, and after 7 days of culture, the organoid grows slowly and has poor uniformity.

[0224] See also Figure 8 As shown, it is a cell viability detection diagram at different times during the cell culture process. It can be seen from the figure that when the matrix gel ratio is 50%, the cell viability is the highest, and when the base gel ratio is 20%, the cell viability is reduced.

[0225] It can be seen that when the proportion of matrix gel is 20%, the cell growth structure and printing effect are the best.

[0226] Comparative Example 1

[0227] The printing method is the same as that of Example 4, except that no photosensitizer is added.

[0228] According to the method of Example 5, the ink dots after printing showed slight defects, the number of cells in the ink dots decreased, and the proportion of live cells was 50%. It can be seen that the photosensitizer is a necessary component to promote the formation of the hydrogel skeleton and provide physical support for cell growth, proliferation, differentiation and other processes.

[0229] Comparative Example 2

[0230] The printing method is the same as that of Example 4, except that no acetone pretreatment is performed.

[0231] According to the method of step 3 of Example 4, the results show that the ink droplet forming effect is poor and is easy to fall off.

[0232] Comparative Example 3

[0233] The printing method is the same as in Example 4, except that no polydopamine modification is performed.

[0234] According to the method of step 3 of Example 4, the results show that the ink droplet forming effect is poor.

[0235] Comparative Example 4

[0236] The printing method of this embodiment is the same as that of Example 8, except that: water or DMEM F12 culture medium (representing a general cell suspension) is used for mixing and then printing, resulting in a smaller printable range and a higher air pressure being required to ensure that the printing needle is not blocked by thermal curing of the matrix glue during the printing process, and the ink dot volume can only achieve a microliter level.

[0237] Table 6 Printing conditions after mixing different proportions of Matrigel with water or DMEM F12

[0238]

[0239]

[0240] Test Example 1

[0241] Determination of the volume of the printed ink droplets by printing method: The volume of the printed ink droplets is controlled by adjusting the printing air pressure to control the size of the printed ink droplets formed on the substrate. Test method: The printing method is the same as in Example 4, 5% NaA-2% CMC hydrogel is selected, 100 ink dots are printed on the substrate, and the volume of each ink dot is calculated according to the density (the main component of the hydrogel is water, and the density is 1g / mL) after weighing the mass, as shown in Table 7. By adjusting the printing air pressure, nanoliter-level ink dot printing can be achieved.

[0242] Table 7 Effect of printing air pressure on ink droplets

[0243] Printing air pressure (psi) Ink dot diameter (μm) Dot volume (nL) 50 1347.53 457 30 703.91 63 10 390.12 12

[0244] Test Example 2

[0245] The printing method is the same as in Example 4. The cell viability in the bio-ink before and after printing is tested. The testing method is the same as in Example 5. The results are as follows: Fig. 9 As shown, the results show that the cell survival rate in the bio-ink remains almost unchanged before and after printing, which shows that the printing process will not cause harm to the cells.

[0246] Test Example 3

[0247] Cell printing was performed according to the ratio of Example 6, and the method was the same as that of Example 4. Activity testing was then performed, and the method was the same as that of Example 5. Under the condition of using PBS (pH=11) as the solvent, as shown in Table 8, the cell survival rate and proliferation rate obtained by printing with different hydrogel precursors were shown.

[0248] Table 8 Cell survival rate and proliferation rate after printing with different hydrogel cross-linking precursors

[0249]

[0250]

[0251] The above examples are used to illustrate the specific implementation of the present invention. However, the protection scope of the present invention is not limited to the above exemplary implementation. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A bio-ink composition based on inkjet printing, characterized in that: The composition comprises a dispersion medium and a biological unit; the dispersion medium comprises a hydrogel cross-linking precursor, a cross-linking initiator, a hydrogel adhesion enhancer and a cell adhesion enhancer; the biological unit comprises a cell, a tissue or an organoid; Wherein, the hydrogel cross-linking precursor comprises a photocurable hydrogel precursor, a thermally curable hydrogel precursor and an ionic hydrogel precursor, the mass ratio of the photocurable hydrogel precursor to the ionic hydrogel precursor is 1:(0-5), and the content of the ionic hydrogel precursor is not 0; The heat-curable hydrogel precursor includes matrix glue, and in the hydrogel cross-linked precursor, the content of the matrix glue is ≤50% and greater than 0 by mass percentage; The hydrogel adhesion enhancer is selected from polydopamine, and the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancer is (1-10):0.5; The cell adhesion enhancer is selected from at least one of poly-lysine, RGD polypeptide, and laminin, and the concentration of the cell adhesion enhancer in the dispersion medium is 0.1-1 mg / ml; The bio-ink composition is in liquid state at a temperature of 0-40°C.

2. The bio-ink composition according to claim 1, characterized in that: The cross-linking initiator is selected from one or more of a photoinitiator and an ion initiator.

3. The bio-ink composition according to claim 1, characterized in that: The dispersion medium also includes one or more of cytokines, antibiotics, small molecule compounds, cellulase, additives, and culture medium.

4. The bio-ink composition according to any one of claims 1 to 3, characterized in that: The photocurable hydrogel precursor comprises at least one of the following substances: methacrylated sodium alginate, methacrylated hyaluronic acid, methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated polylysine, methacrylated gelatin, methacrylated silk fibroin, methacrylated dextran, methacrylated chondroitin sulfate, polyether F127 diacrylate, polyethylene glycol diacrylate, four-arm polyethylene glycol acrylate, and other acrylated materials, wherein the other acrylated materials are selected from acrylated RGD peptide and acrylated polyethylene glycol NHS ester; And / or, the ionic hydrogel precursor comprises at least carboxymethyl cellulose.

5. The bio-ink composition according to claim 4, characterized in that: The ionic hydrogel precursor further comprises at least one of alginate and carboxymethyl chitosan.

6. The bio-ink composition according to claim 5, characterized in that: The alginate is selected from at least one of sodium alginate and potassium alginate.

7. A biological ink, characterized in that: The bio-ink is obtained by mixing the components in the bio-ink composition according to any one of claims 1 to 6.

8. A method for preparing the biological ink according to claim 7, characterized in that: The method comprises: mixing the biological units in the biological ink composition with a dispersion medium to obtain the biological ink.

9. Use of the bio-ink described in claim 7 in cell or organoid printing.

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