CN117565386B - Cell or organoid chip and preparation method and application thereof - Google Patents

Cell or organoid chip and preparation method and application thereof Download PDF

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CN117565386B
CN117565386B CN202410067558.5A CN202410067558A CN117565386B CN 117565386 B CN117565386 B CN 117565386B CN 202410067558 A CN202410067558 A CN 202410067558A CN 117565386 B CN117565386 B CN 117565386B
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cell
hydrogel
substrate
precursor
organoid
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CN117565386A (en
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苏萌
谢岱希
陈炳达
宋延林
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Institute of Chemistry CAS
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Institute of Chemistry CAS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/307—Handling of material to be used in additive manufacturing
    • B29C64/314—Preparation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B33Y40/10—Pre-treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12N2533/54—Collagen; Gelatin
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    • C12N2533/72—Chitin, chitosan
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate
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    • C12N2533/78—Cellulose
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/80—Hyaluronan
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention provides a cell or organoid chip, a preparation method and application thereof, wherein the method comprises the following steps: s1: preparing a bioactive ink, the bioactive ink comprising a dispersion medium and a biological unit; s2: manufacturing a micro groove on the surface of a substrate to obtain a pretreated substrate; s3: carrying out surface modification on the pretreated substrate to obtain a surface modified substrate; s4: and printing the bioactive ink on a surface-modified substrate by a 3D printing method to obtain the cell or organoid chip. The invention prepares the drug screening chip with high adhesiveness by printing technology, and can realize drug screening by using a small amount of samples.

Description

Cell or organoid chip and preparation method and application thereof
Technical Field
The invention relates to the technical field of cell or tissue culture, in particular to a cell or organoid chip and a preparation method and application thereof.
Background
With the continuous development, application and popularization of 3D printing technology, more people use and experience, and the application field is wider and wider. How to apply 3D printing to cell culture has become another hotspot, for example, bone cell research of orthopedics medical science such as fracture, bone loss, bone tumor, etc., culture of dermal tissue cells, oral cells, cells of organs of viscera, etc., and more researchers are researching, reporting or marketing some products.
The conventional cell culture apparatus, device or scaffold belongs to a two-dimensional planar culture mode, and although a great part of industrial requirements can be met to a certain extent, the conventional cell culture apparatus, device or scaffold also has some defects or drawbacks, such as crowding of cells after proliferation in the culture process, partial non-adhesion of the cells to a implantation space, exposure to the surface of a culture solution, and the like. The appearance of the three-dimensional culture support well solves the defects and drawbacks existing in two-dimensional culture, and the rich network space structure of the three-dimensional support can provide larger specific surface area for proliferation and adhesion of cells, improve the yield and quality of the cells, reduce the contact and inhibition between the cells, simulate the three-dimensional structure growth environment of a human body or an animal body, facilitate interaction between the cells and cytoplasms and removal of metabolites, and facilitate differentiation of the cells and expression of original attribute functions.
Most of the three-dimensional culture scaffolds at present use polycaprolactone and the like as raw materials, and gel dispensing or printing forming and the like are adopted to obtain the three-dimensional cell culture scaffolds. Due to its hydrophobicity, adhesion of cells is not favored. Therefore, there is a need to develop a three-dimensional cell culture scaffold with good cell affinity and adhesion to meet the demands of production development.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a cell or organoid chip, a preparation method and application thereof, which solve the problem of poor adhesiveness of the cell or organoid chip existing in the prior art.
In one aspect, the invention provides a cell or organoid chip, in particular a highly adherent cell or organoid chip, having a micro-groove on its surface, and a highly adherent molecular layer. In another aspect, the invention provides a method for preparing a cell or organoid chip, in particular a method for preparing a highly adhesive cell or organoid chip.
Specifically, the invention provides the following technical scheme:
a method for preparing a cell or organoid chip, in particular to a method for preparing a high-adhesion cell or organoid chip, which comprises the following steps:
s1: preparing a bioactive ink, the bioactive ink comprising a dispersion medium and a biological unit;
s2: manufacturing a micro groove on the surface of a substrate to obtain a pretreated substrate;
s3: carrying out surface modification on the pretreated substrate to obtain a surface modified substrate;
s4: and printing the bioactive ink on a surface-modified substrate by a 3D printing method to obtain the cell or organoid chip.
In one embodiment of the invention, the dispersion medium comprises at least a hydrogel cross-linking precursor and a hydrogel adhesion enhancing agent.
In one embodiment of the present invention, the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancing agent in the dispersion medium is (1-10): 0.5; illustratively, 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 therebetween.
In one embodiment of the invention, the dispersion medium comprises at least a hydrogel cross-linking precursor, optionally with or without the addition of a cross-linking initiator. When a crosslinking initiator is added, the hydrogel crosslinking precursor is capable of reacting with the corresponding crosslinking initiator to form a hydrogel upon formation of the bio-ink.
In an embodiment of the present invention, the crosslinking initiator is one or more selected from a photoinitiator and an ionic initiator.
In one embodiment of the present invention, the photoinitiator includes, but is not limited to, at least one of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (abbreviated as LAP), 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylbenzophenone (I2959), ethyl 2,4, 6-trimethylbenzoyl phenylphosphonate (TPO-L).
In one embodiment of the present invention, the ionic initiator includes, but is not limited to, at least one of a calcium chloride solution, a barium chloride solution.
In one embodiment of the present invention, the hydrogel crosslinked precursor is selected from one or more of photosensitive hydrogel precursor, ionic hydrogel precursor and thermosetting hydrogel precursor. Preferably, the hydrogel cross-linking precursor is a photosensitive hydrogel precursor and/or an ionic hydrogel precursor.
In one embodiment of the present invention, the thermoset hydrogel precursor includes, but is not limited to, at least one of a matrigel, polyether F127 diacrylate.
In one embodiment of the present invention, the hydrogel crosslinked precursor includes one or more of a photosensitive hydrogel precursor, an ionic hydrogel precursor, and a matrigel.
In one embodiment of the invention, the hydrogel cross-linked precursor comprises a matrigel, and optionally a photosensitive hydrogel precursor and/or an ionic hydrogel precursor.
In one embodiment of the invention, the hydrogel cross-linked precursor includes matrigel, photosensitive hydrogel precursor, and ionic hydrogel precursor.
According to an embodiment of the present invention, the content of the matrigel in the hydrogel crosslinked precursor is 50% by mass or less, preferably 40% by mass or less, further preferably 30% by mass or less, for example 35%, 25%, 20%, 18% by mass or less.
As an example, the content of the matrigel in the hydrogel crosslinked precursor is 20% by mass, and the content of the photosensitive hydrogel precursor and/or the ionic hydrogel precursor is 80%.
In an embodiment of the present invention, the Matrigel comprises one or more of Matrigel, cultrex Bme and Geltrex.
In an embodiment of the present invention, the hydrogel crosslinked precursor includes a photosensitive hydrogel precursor and an ionic hydrogel precursor, and the mass ratio of the photosensitive hydrogel precursor to the ionic hydrogel precursor is 1 (0-5), preferably the mass ratio of the photosensitive hydrogel precursor to the ionic hydrogel precursor is 1 (0-3), for example, 1:1.
In one embodiment of the invention, the photosensitive hydrogel precursors include, but are not limited to, methacryloylated sodium alginate (AlgMA), methacryloylated hyaluronic acid (HAMA), methacryloylated chitosan, methacryloylated carboxymethyl chitosan, methacryloylated Polylysine (PLMA), methacryloylated Gelatin (GM), methacryloylated silk fibroin (SilMA), methacryloylated dextran (DeXMA), methacryloylated chondroitin sulfate (ChSMA), polyether F127 diacrylate, polyethylene glycol diacrylate, tetra-arm polyethylene glycol acrylate, and other acrylated materials (e.g., acrylated RGD peptide, acrylated polyethylene glycol NHS ester).
In one embodiment of the invention, the ionic hydrogel precursors include, but are not limited to, carboxymethyl cellulose, sodium alginate, carboxymethyl chitosan.
In one embodiment of the invention, the hydrogel crosslinked precursor is a combination of a photosensitive hydrogel precursor and an ionic hydrogel precursor. It was found that when the composition is selected as a hydrogel cross-linking precursor, the photosensitive hydrogel precursor and the ionic hydrogel precursor can control the viscosity of the ink by controlling the photo-curing time, thereby controlling the growth state of cells; because the bonding strength of the ionic crosslinking is stronger than that of the photo-crosslinking, the addition of the ionic hydrogel precursor can further strengthen the space skeleton strength of the hydrogel, improve the stability of the hydrogel and widen the adjustable range of the cell growth environment. In addition, the methacryloylated sodium alginate, the methacryloylated gelatin and the methacryloylated hyaluronic acid in the photo-cured hydrogel precursor have photo-curing performance and ion curing performance, and when the photo-cured hydrogel precursor is used, even if the ion-type hydrogel precursor is not added, the photo-curing can be firstly performed to regulate and control the viscosity of the ink, and then the ion curing can be performed to strengthen the stability of the gel, so that the growth environment of cells can be effectively regulated and controlled, that is, when the methacryloylated sodium alginate, the methacryloylated gelatin and the methacryloylated hyaluronic acid are selected as the photosensitive hydrogel precursor, even if the photosensitive hydrogel precursor is only added, the same effect as the effect of adding the ion-type hydrogel precursor at the same time can be achieved.
In one embodiment of the invention, when the hydrogel cross-linking precursor comprises a photosensitive hydrogel precursor, the dispersion medium comprises a photoinitiator.
In one embodiment of the invention, the mass ratio of the photosensitive hydrogel precursor to the photoinitiator is (2-15) 1; preferably, the mass ratio of the photosensitive hydrogel precursor to the photoinitiator is (3-12) 1; for example 4:1, 7:1, 9:1, 10:1, 12:1.
In one embodiment of the invention, the dispersion medium further comprises at least a hydrogel adhesion enhancer for enhancing adhesion of the hydrogel to the substrate. Specifically, the hydrogel adhesion enhancing agent includes, but is not limited to, at least one of polydopamine, polymethacrylamide, polyethylene glycol, polyvinyl alcohol, and the like.
As an example, the dispersion medium includes at least carboxymethyl cellulose and polydopamine.
As an example, the dispersion medium includes at least alginate, carboxymethyl cellulose, and polydopamine.
As an example, the dispersion medium includes at least polydopamine, a photosensitive hydrogel precursor, and a photoinitiator.
As an example, the dispersion medium includes at least alginate, carboxymethyl cellulose, polydopamine, and chitosan.
In one embodiment of the present invention, in the bioactive ink, the dispersion medium includes at least alginate, carboxymethyl cellulose, polydopamine, a photosensitive hydrogel precursor, and an initiator.
In a specific embodiment of the invention, the mass ratio of the alginate to the carboxymethyl cellulose to the polydopamine to the photosensitive hydrogel precursor to the initiator is (1-5) 1:0.5 (1-5) 0.05-0.25.
Illustratively, in the mass ratio, the ratio of the alginate is any one point value of 1 to 5, specifically, for example, 1, 2, 3, 4 or 5.
Illustratively, the ratio of the photosensitive hydrogel precursor in the mass ratio is any one point value of 1 to 5, specifically, for example, 1, 2, 3, 4 or 5.
The ratio of the initiator in the mass ratio is any one of 0.05 to 0.25, for example, 0.05, 0.10, 0.15, 0.20 or 0.25.
In one embodiment of the invention, the photosensitive hydrogel precursor comprises methacryloylated sodium alginate.
In a specific embodiment of the invention, the initiator comprises lithium phenyl-2, 4, 6-trimethylbenzoyl phosphite (LAP).
In a specific embodiment of the present invention, the dispersion medium further comprises one or more of laminin, RGD polypeptide, cytokine, antibiotic, small molecule compound, cellulase, and culture medium.
In a specific embodiment of the present invention, the concentration of laminin and/or RGD polypeptide is 0.1-1mg/ml.
In a further embodiment of the invention, the cytokines include, but are 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.
In one embodiment of the invention, the concentration of the cytokine is 50-100 ng/mL.
In a further embodiment of the invention, the antibiotic includes, but is not limited to, primocin ™ primary cell antibiotic.
In a specific embodiment of the invention, the concentration of the antibiotic is 50-100. Mu.g/mL.
In a further embodiment of the invention, the small molecule compound includes, but is not limited to, at least one of SB202190, gastin I, A83-01, nicotinamide, prostaglandin E2, and N-acetyl-L-cysteine.
In one embodiment of the invention, the concentration of the small molecule compound is 10 nM to 10 mM.
In a further embodiment of the invention, the cellulases include, but are not limited to, endoglucanases, exoglucanases, beta-glucosidase.
In one embodiment of the invention, the concentration of the cellulase is 0.01-0.1%.
In a further embodiment of the invention, the medium includes, but is not limited to, DMEM/F12 medium.
In one embodiment of the invention, the biological unit comprises a cell, tissue or organoid.
In one embodiment of the invention, the concentration of cells in the biological unit is 10 4 -10 6 And each ml.
In a specific 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, and the like.
In a specific embodiment of the invention, the tissue comprises, but is not limited to, one or more of a cell mass, a small piece of biological tissue, a cytoplasmic matrix, and the like. In particular, the biological tissue includes, but is not limited to, one or more of epithelial tissue, connective tissue, muscle tissue, nerve tissue.
In a specific embodiment of the present invention, the organoids include, but are not limited to, one or more of the group consisting of small intestine organoids, stomach organoids, colon organoids, lung organoids, bladder organoids, brain organoids, liver organoids, pancreas organoids, kidney organoids, ovary organoids, esophagus organoids, heart organoids.
In an embodiment of the present invention, in step S2, the method for manufacturing the micro grooves (also called micro grooves) on the surface of the substrate includes photolithography, dicing, and laser etching.
In one embodiment of the invention, the width of the micro grooves is 10-200 microns, preferably 20-50 microns; the depth is 10-100 microns, preferably 50 microns.
In a specific embodiment of the present invention, the patterns formed by the micro grooves include parallel straight lines, waves and crisscross shapes, and preferably, the patterns are parallel straight lines. In the parallel straight line shape, the distance between adjacent micro grooves is 40-2000 micrometers, preferably 100-200 micrometers.
In one embodiment of the present invention, the cross-sectional shape of the micro-groove includes, but is not limited to, rectangular, u-shaped, v-shaped, trapezoidal.
In one embodiment of the present invention, in step S3, the surface modification includes dopamine modification of the surface of the substrate.
In one embodiment of the present invention, the surface modification comprises the steps of:
(1) Dissolving dopamine salt in a buffer solution to obtain a dopamine aqueous solution;
(2) Immersing the pretreated substrate into aqueous solution of dopamine, heating and standing to obtain the modified substrate.
In a further embodiment of the invention, the surface modification comprises the steps of:
a. dissolving dopamine hydrochloride in Tris-HCl buffer (pH=8.8) to obtain 90mg/mL aqueous dopamine solution;
b. immersing the substrate into the aqueous solution of dopamine, heating at 60 ℃ for 6 hours, standing at room temperature overnight, removing the residual solution, washing with PBS, and drying with nitrogen to obtain the polydopamine grafted high-adhesion substrate.
In some embodiments, the substrate comprises a flexible material such as Polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), ABS plastic, polytetrafluoroethylene, hyaluronic acid hydrogel, alginic acid hydrogel, cellulose hydrogel, chitosan hydrogel, collagen hydrogel, gelatin hydrogel, silk fibroin hydrogel, agar hydrogel, DNA hydrogel, decellularized tissue hydrogel, polyvinyl alcohol hydrogel, polyacrylic acid hydrogel, polyethylene glycol hydrogel, polydopamine (PDA) hydrogel, or polyacrylate hydrogel; preferably a polyethylene terephthalate film or a polydopamine hydrogel.
In some embodiments, the substrate comprises one or more rigid materials selected from plastics, silicon, metal, and the like.
In some embodiments, in step S4, the parameter settings for 3D printing include: the printing pattern is set to be a dot matrix, a line or a 2D/3D pattern; the height of the printing needle head from the substrate is 20-100 micrometers; the single-point ink-jet time is 0-10s; the ink jet air pressure is 0-60 psi, and the precision is 0.1 psi; the contact angle of the substrate is 0-100 degrees, preferably 50+/-10 degrees; the drop printing accuracy is 100-1500 microns, preferably 300-500 microns.
The invention also provides a cell or organoid chip, in particular a highly adherent cell or organoid chip, prepared by any of the methods of preparation described above.
The invention also provides application of the cell or organoid chip in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, the micro grooves are formed on the substrate through pretreatment, so that on one hand, the roughness of the surface of the substrate can be increased, and on the other hand, the ink drops and the substrate can form rivet action, and the adhesiveness of the ink drops on the surface of the substrate is improved.
(2) According to the invention, the surface of the substrate is subjected to chemical molecular modification, so that molecular bonds are formed between ink drops and the substrate, and the adhesion between a printed matter and the substrate can be obviously improved through molecular acting force.
(3) Compared with the traditional manual sample application technology, the method has the advantages of less cell quantity, high adhesiveness and high automation degree, greatly reduces the cell culture cost, the culture time and the labor cost, and reduces the labor error.
(4) The chip of the invention may be a drug screening chip with an organoid microarray that can be used to achieve drug screening with a small sample.
Drawings
FIG. 1 is a pictorial view of ink droplets in different types of array-type highly adherent cells or organoid chips.
Fig. 2 is a scanning electron microscope image of the surface of the substrate after polydopamine treatment.
FIG. 3 is a graph showing the results of ATP assay for normal cell growth activity.
FIG. 4 is an SEM image of cells and organoids at different matrigel contents for different growth times.
FIG. 5 is a graph showing cell viability assays of cells and organoids at different matrigel levels for different growth times.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
Example 1 cell culture
1. Cell separation
The tissue of the isolated intestinal cancer is soaked in a 1:2 iodophor/PBS mixed solution for five minutes, and washed with PBS containing streptomycin and penicillin for five times. The washed tissue is put into 15 mL digestion liquid, sheared and digested in a 37 ℃ water bath for 40 minutes. After adding 15 mL of DMEM/F12 medium, undigested tissue pieces were removed by filtration, and the filtrate was centrifuged at 1200 rpm for 5 minutes to remove the supernatant. 10 mL red blood cell lysate was added for 10 minutes, centrifuged at 1200 rpm for 5 minutes, and the supernatant was removed. After washing the cells with 5 mL isolate, an appropriate amount of Matrigel was added, mixed by blowing and sucking, transferred to a 24-well plate (50. Mu.L per well), solidified at 37℃for 30 minutes, and placed in an incubator after adding medium (500. Mu.L per well).
The digestion solution is a separating solution containing 10 mu M Y-27632 and 100 mu g/mL Primocin ™ primary cell antibiotic and 2 mg/mL Collagenase Type enzyme. The isolation was DMEM/F12 medium supplemented with 2mM Glutamax ™, 25 mM HEPES, 1% streptomycin and penicillin.
2. Cell culture and passage
Cells at 37℃with 5% CO 2 Culturing under the condition. The medium was changed every two days and passaged every seven days. The passaging steps are as follows: 500 mu L of separating liquid is added into each hole, and after the broken matrigel is blown and sucked by a pipetting gun, all the liquid is transferred to a centrifuge tube. And taking 1 mL separating liquid for rinsing each hole, transferring all the liquid to the centrifuge tube, and centrifuging at 2000 rpm for five minutes. The supernatant was removed, trypLE expression enzyme (250. Mu.L per well) was added and digested in a water bath at 37℃for 5 minutes at 10. Mu. M Y-27632. After digestion, the liquid was sucked several times with a pipette to further break up the bulk matrigel, and centrifuged at 2000 rpm for five minutes. The supernatant was removed, resuspended in 2 mL isolate and centrifuged again at 2000 rpm for five minutes. The supernatant was removed, an appropriate amount of Matrigel was added, and after mixing by blowing and suction, the mixture was transferred to a 24-well plate (50. Mu.L per well), solidified at 37℃for 30 minutes, and after adding the medium (500. Mu.L per well), placed in an incubator. Cells after stable passage 4 times were extracted for printing and drug screening.
The separation in this step was DMEM/F12 medium supplemented with 2mM Glutamax ™, 25 mM HEPES, 1% streptomycin and penicillin. The medium was DMEM/F12 medium supplemented with 2mM Glutamax ™, 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 ™ supplement, 100 ng/mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine, 500ng/mL R-Spondin, 100 ng/mL mNagin cell growth factor, 50 ng/mL EGF cell growth factor, 10 nM gastrin I human, 0.5. Mu. M A83-01, 3. Mu.M SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, 100. Mu.g/mL Primocin ™ primary cell antibiotics.
3. Organoid culture
The cell density is adjusted to be 2 to 3 multiplied by 10 6 Adding organoid culture solution, placing the culture plate at 37deg.C CO 2 Culturing in an incubator. The culture medium was changed every 2 days.
The organoid medium was DMEM/F12 medium supplemented with 2mM Glutamax ™, 25 mM HEPES, 1% streptomycin and penicillin, 2% B-27 ™ supplement, 100 ng/mL Wnt3a cell growth factor, 1.25 mM N-acetyl-L-cysteine, 500ng/mL R-Spondin, 100 ng/mL mNagin cell growth factor, 50 ng/mL EGF cell growth factor, 10 nM gastrin I human, 0.5 μ M A83-01, 3 μM SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, 100 μg/mL Primocin ™ primary cell antibiotic.
EXAMPLE 2 preparation of dispersion Medium in bioactive ink
The culture broth was prepared and then sterilized with a bacterial filter. The culture solution comprises RGD polypeptide 1g, R-Spondin cell growth factor 50mg, SB202190 10 mu M, cellulase 0.05% and DMEM/F12 culture medium 1000ml.
9000mg of dopamine hydrochloride is dissolved in 300mL of Tris-HCl (ph=8.8) buffer solution, sterilized by filtration with a cell filter, then heated at 60-70 ℃ for 6 hours, then left for 18 hours, filtered, evaporated to dryness and ground into polydopamine powder.
Preparing a dispersion medium of the bioactive ink: tiling sodium alginate powder and carboxymethyl cellulose powder, and sterilizing by ultraviolet irradiation for 3 hr. The above culture solution was used to dissolve and disperse 20mg of sodium alginate (NaA) powder, 20mg of carboxymethyl cellulose (CMC) powder (2% NaA-2% CMC), 10mg of polydopamine powder, 20mg of methacryloylated sodium alginate and 1mg of LAP, and the above materials were stirred and dissolved uniformly by ultrasound to prepare a dispersion medium.
Example 3 preparation of a substrate
1. Pretreatment of substrates
The PET substrate was prepared for the trench array using a dicing saw. A 23 micron wide diamond blade was used, 50 microns deep, line spacing of 100, 500, 1000 or 2000 microns, dicing angles of 0 ° and 90 °. Such as the pattern of various grooves and ink drops in the chip shown in fig. 1.
2. Modification of substrates
Dopamine hydrochloride was dissolved in Tris-HCl buffer (ph=8.8) to give 90mg/mL aqueous dopamine solution. Immersing the pretreated substrate into the aqueous solution of dopamine, heating at 60 ℃ for 6 hours, standing at room temperature overnight, removing the residual solution, cleaning with PBS, and drying with nitrogen to obtain the polydopamine grafted high-adhesion substrate. Fig. 2 shows a scanning electron microscope image of the surface of the substrate after polydopamine treatment.
EXAMPLE 4 preparation of highly adhesive cell chip
1. Preparation of bioactive ink
Centrifuging the cell suspension digested with the enzyme in example 1, removing supernatant, adding into the dispersion medium prepared in example 2, and mixing to obtain bioactive ink with cell concentration of 10 5 -10 6 /mL。
2. Printing bioactive ink
1) Consumable materials such as ink tube, needle head, ink tube pressure plug, etc. are placed in a high pressure steam sterilizing pot for sterilization (130 ℃ C., 40 minutes), and a printing substrate (PET film, orifice plate) is irradiated by an ultraviolet lamp for 30 minutes.
2) The printing process is programmed by using a program built in the printing equipment, wherein the printing pattern coordinates are set to be a dot matrix, the height of a printing needle head from a substrate is 50 microns, the ink jet pressure is 50 psi, the ink jet time of a unit dot is 0.4s, the contact angle of the substrate is 50 degrees, and the printing precision of ink drops is 350 microns.
3) Flatly fixing the printing substrate irradiated by the ultraviolet lamp on a printing platform; 300. Mu.l of the bioactive ink was filled in the ink tube, and air bubbles in the dispersion medium were removed by centrifugation. The ink tube is connected with a dispensing machine (an ink jet air pressure controller) and a needle head, and is arranged on a moving platform, the temperature is controlled to be in an environment of 4 ℃ by a temperature controller, and a program is run for printing. After printing was completed, 1.5% Ca was used 2+ (PBS is dispersion, ph=7.0) the ink droplets were soaked for 10 minutes to complete curing, and a highly adhesive cell chip was prepared. The chip was observed under a microscope. The results show that the ink drop forming effect is good.
Example 5 detection of cell Activity in chip
1) The chip printed in example 4 was subjected to a live dead cell immunostaining test to examine the viability of cells in the ink dots on the chip.
The specific method comprises the following steps: samples (i.e., the chips printed in example 4) were removed, washed 1-2 times with PBS, and the remaining medium solution was washed away. And (3) staining by using a cell living and dying staining kit, firstly adding enough prepared propidium iodide working solution, ensuring that cells are not passed, and incubating for 10min at room temperature. The propidium iodide working solution was removed and the supernatant was removed by gentle washing with sufficient PBS. And adding a sufficient amount of prepared calciferous working solution to ensure that cells are not used, and incubating for 20-45 minutes at room temperature. The calciferous working solution was removed and the supernatant was removed by a gentle wash with sufficient PBS. And (3) dropwise adding PBS or an anti-fluorescence quenching agent, and finally observing the living and dead cell marking condition under a fluorescence microscope. The proportion of living cells in ink spots on the chip obtained through statistical printing is 80-95%.
2) The chip printed in example 4 was placed in organoid medium for culture, and after 14 days, live dead cell immunostaining was performed to examine the viability of cells in the ink dots. After 14 days of culture, the proportion of viable cells in the ink dots was 85-98%.
3) The cell chip prepared in example 4 was cultured for 7 days, and the ATP amount of the cells/organoids in the chip was measured once on days 0, 1, 4, and 7, respectively, as shown in FIG. 3, to confirm that the growth activity of the cells was good and increased stepwise.
ATP test method: cell viability was tested using CellTiter-Glo cube 3D Cell Viability Assay. Cells cultured in 96-well plates were replaced with medium and 100uL organoid complete medium was added per well. 100uL CellTiter-Glo 3D Reagent is added to each well in equal volume, and the mixture is fully mixed and incubated for 30 min at room temperature. The chemiluminescent values were measured by a multifunctional microplate reader (TECAN index M1000 PRO, swiss).
EXAMPLE 6 Effect of matrigel in different proportions on cell and organ growth
A dispersion medium was prepared in the same manner as in example 2, and Matrigel was added to the dispersion medium in a mass percentage of 5, 20, 50, respectively, and no Matrigel (0%) was added, and only Matrigel (100%) was included as a control. A high throughput cell chip was prepared and placed in organoid medium for culture as in example 4.
Organoid growth was observed after initial printing (Day 0) and after 7 days of incubation (Day 7), and the results are shown in fig. 4. As shown in fig. 4, when the mixing ratio of matrigel is 50% or more, day0 printing cell density is not uniform, cell sedimentation may occur, and organoids grow fast but are not uniform after 7 days of culture; when the matrigel proportion is 20%, the density of Day0 printing cells is uniform, and after 7 days of culture, the organoid grows fast and has the best uniformity; when the matrigel proportion is less than 20%, for example, 5% or 0%, day0 print cell density is uniform, and organoids grow slowly and with poor uniformity after 7 days of culture. Meanwhile, cell viability is detected at different times in the cell culture process. As a result, as shown in fig. 5, D0 is 1 day, D4 is 4 days, and D7 is 7 days after the initial printing. From the results, it was found that the cell viability was the highest when the matrigel proportion was 50%, and the cell viability was decreased when the matrigel proportion was 20%. From this, it was found that the cell growth structure and the printing effect were both best when the matrigel proportion was 20%.
Comparative example 1
The printing method is the same as that of example 4, except that: without addition of photosensitizer
The proportion of viable cells in the ink dots after printing was detected as 50% in the method of example 5.
Comparative example 2
The printing method is the same as that of example 4, except that: using O 2 -treating the substrate with a plasma having a contact angle of 0-10 °. Results: the contact angle of the substrate surface treated by the method is uneven compared with the contact angle of the substrate surface treated by polydopamine, and the contact angle returns to 50-60 ℃ within half an hour; in the process of culturing cells, ink points gradually fall off along with the soaking of the culture solution.
Comparative example 3
The printing method is the same as that of example 4, except that: the substrate has no micro-groove structure.
According to the method of step 3 of example 4, the results show that the ink drop forming effect is poor, and the ink dots gradually fall off along with the soaking of the culture solution in the process of culturing cells.
Comparative example 4
The printing method is the same as that of example 4, except that: polydopamine modification was not performed.
The results of the procedure of step 3 of example 4 showed poor droplet formation and drop-off after curing.
Comparative example 5
The printing method is the same as that of example 4, except that: the substrate is treated with polyacrylamide. The specific method comprises the following steps: 1600 mg acrylamide, 160 mg ammonium persulfate, 8 mg N, N-methylenebisacryl were dissolved in PBS (ph=11), stirred for 5min, and the solution was added dropwise to a substrate (e.g., 300 μl per well of a 24-well plate). A layer of high viscosity polyacrylamide was formed on the substrate surface after adding 3-5 microliters of ion chelating agent (TMEDA) per well. And printing, curing ink drops after printing, and soaking in a culture solution, wherein the polyacrylamide swells after soaking for a plurality of hours.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Claims (9)

1. A method for preparing a cell or organoid chip, characterized by: the preparation method comprises the following steps:
s1: preparing a bioactive ink, the bioactive ink comprising a dispersion medium and a biological unit;
s2: manufacturing a micro groove on the surface of a substrate to obtain a pretreated substrate;
s3: carrying out surface modification on the pretreated substrate to obtain a surface modified substrate;
s4: printing the bioactive ink on a substrate with a modified surface by a 3D printing method to obtain the cell or organoid chip;
wherein the dispersion medium at least comprises a hydrogel cross-linking precursor and a hydrogel adhesion enhancer, and the mass ratio of the hydrogel cross-linking precursor to the hydrogel adhesion enhancer is (1-10) 0.5;
the hydrogel crosslinked precursor comprises a photosensitive hydrogel precursor and an ionic hydrogel precursor, wherein the mass ratio of the photosensitive hydrogel precursor to the ionic hydrogel precursor is 1 (0-5); or the hydrogel crosslinking precursor comprises one or more of photosensitive hydrogel precursor, ionic hydrogel precursor and matrigel, wherein the content of the matrigel in the hydrogel crosslinking precursor is less than or equal to 50% by mass;
the dispersion medium also comprises a crosslinking initiator.
2. The method of preparing a cell or organoid chip according to claim 1, wherein: the dispersion medium also comprises one or more of laminin, RGD polypeptide, cytokine, antibiotics, small molecule compound, cellulase and culture medium.
3. The method of preparing a cell or organoid chip according to claim 1, wherein: the biological unit comprises a cell, tissue or organoid.
4. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S2, the method for manufacturing the micro-grooves on the substrate surface includes photolithography, dicing or laser etching.
5. The method of preparing a cell or organoid chip according to claim 1, wherein: in the step S2, the width of the micro groove is 10-200 micrometers, the depth is 10-100 micrometers, and the pattern formed by the micro groove comprises parallel straight lines, waves and crisscross shapes.
6. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S3, the surface modification includes dopamine modification of the surface of the substrate; the concentration of the dopamine is 10-90mg/mL.
7. The method of preparing a cell or organoid chip according to claim 1, wherein: in step S4, the parameter settings of the 3D printing comprise setting the printing pattern as a dot matrix, lines or 2D/3D pattern; the height of the printing needle head from the substrate is 20-100 micrometers; the ink jet air pressure is 0-60 psi, and the precision is 0.1 psi; single-site inkjet time is 0-10s; the contact angle of the substrate is 0-100 degrees; the ink drop printing accuracy is 100-1500 microns.
8. A cell or organoid chip, characterized in that: the chip is prepared by the preparation method of any one of claims 1-7.
9. Use of the cell or organoid chip of claim 8 in drug screening, drug toxicity and efficacy testing, organ model construction or tissue engineering.
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Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238735A (en) * 2015-11-13 2016-01-13 广州洁特生物过滤股份有限公司 Three-dimensional cell culture stent and preparation method thereof
CN107308505A (en) * 2017-05-11 2017-11-03 华南理工大学 A kind of sodium alginate-modified gelatin modified dopamine compound rest and preparation method thereof
CN107400661A (en) * 2017-06-29 2017-11-28 武汉枫霖科技有限公司 A kind of method of the three-dimensional liver cancer tissue structure based on biological 3D printing hydrogel
KR20180035162A (en) * 2016-09-28 2018-04-05 포항공과대학교 산학협력단 Novel Bioink for 3D Bioprinting and Uses Thereof
CN107998451A (en) * 2018-01-30 2018-05-08 扬州大学 A kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent
CN108187145A (en) * 2017-12-19 2018-06-22 中国科学院深圳先进技术研究院 Gelatin-alginate composite mortar, gelatin-alginate compound rest and preparation method thereof
CN109999226A (en) * 2018-11-27 2019-07-12 王翀 A kind of printed material, the bone tissue engineering scaffold and preparation method for loading stem cell
CN110804193A (en) * 2019-10-31 2020-02-18 南方医科大学南方医院 3D printing method of hydrogel support
CN111803706A (en) * 2020-07-04 2020-10-23 四川大学 Preparation method of bone-like bioactive polycaprolactone porous scaffold and porous scaffold
CN112451746A (en) * 2020-11-25 2021-03-09 广东省科学院生物工程研究所 Preparation method of light-curable sodium alginate hydrogel repair stent
CN113101419A (en) * 2021-03-19 2021-07-13 华南理工大学 Hydrogel stent with polydopamine coating and preparation method thereof
CN114773628A (en) * 2022-05-18 2022-07-22 中山大学 Self-curable 3D printing hydrogel ink, preparation method and tissue engineering scaffold
CN115154663A (en) * 2022-07-05 2022-10-11 中国科学院化学研究所 Hydrogel type tissue engineering scaffold with two-phase drug slow release function, photocuring 3D printing preparation method thereof and photosensitive resin
CN115382020A (en) * 2022-09-21 2022-11-25 银丰低温医学科技有限公司 Biological ink based on human acellular matrix and preparation method and application thereof
CN115887772A (en) * 2022-11-17 2023-04-04 南京工业大学 Gelatin/sodium alginate hydrogel-based 3D printing biological ink and application thereof
CN116218760A (en) * 2023-02-07 2023-06-06 北京理工大学 Arterial organ chip based on multi-material suspended organism 3D printing and preparation method

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8828203B2 (en) * 2004-05-20 2014-09-09 Sanofi-Aventis Deutschland Gmbh Printable hydrogels for biosensors
DE102019132214B3 (en) * 2019-11-27 2021-04-29 Cellbricks Gmbh 3D scaffold made of biocompatible polymer and its production, colonized with biological cells
US11918703B2 (en) * 2020-08-13 2024-03-05 Universidad De Los Andes Extrudable photocrosslinkable hydrogel and method for its preparation

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238735A (en) * 2015-11-13 2016-01-13 广州洁特生物过滤股份有限公司 Three-dimensional cell culture stent and preparation method thereof
KR20180035162A (en) * 2016-09-28 2018-04-05 포항공과대학교 산학협력단 Novel Bioink for 3D Bioprinting and Uses Thereof
CN107308505A (en) * 2017-05-11 2017-11-03 华南理工大学 A kind of sodium alginate-modified gelatin modified dopamine compound rest and preparation method thereof
CN107400661A (en) * 2017-06-29 2017-11-28 武汉枫霖科技有限公司 A kind of method of the three-dimensional liver cancer tissue structure based on biological 3D printing hydrogel
CN108187145A (en) * 2017-12-19 2018-06-22 中国科学院深圳先进技术研究院 Gelatin-alginate composite mortar, gelatin-alginate compound rest and preparation method thereof
CN107998451A (en) * 2018-01-30 2018-05-08 扬州大学 A kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent
CN109999226A (en) * 2018-11-27 2019-07-12 王翀 A kind of printed material, the bone tissue engineering scaffold and preparation method for loading stem cell
CN110804193A (en) * 2019-10-31 2020-02-18 南方医科大学南方医院 3D printing method of hydrogel support
CN111803706A (en) * 2020-07-04 2020-10-23 四川大学 Preparation method of bone-like bioactive polycaprolactone porous scaffold and porous scaffold
CN112451746A (en) * 2020-11-25 2021-03-09 广东省科学院生物工程研究所 Preparation method of light-curable sodium alginate hydrogel repair stent
CN113101419A (en) * 2021-03-19 2021-07-13 华南理工大学 Hydrogel stent with polydopamine coating and preparation method thereof
CN114773628A (en) * 2022-05-18 2022-07-22 中山大学 Self-curable 3D printing hydrogel ink, preparation method and tissue engineering scaffold
CN115154663A (en) * 2022-07-05 2022-10-11 中国科学院化学研究所 Hydrogel type tissue engineering scaffold with two-phase drug slow release function, photocuring 3D printing preparation method thereof and photosensitive resin
CN115382020A (en) * 2022-09-21 2022-11-25 银丰低温医学科技有限公司 Biological ink based on human acellular matrix and preparation method and application thereof
CN115887772A (en) * 2022-11-17 2023-04-04 南京工业大学 Gelatin/sodium alginate hydrogel-based 3D printing biological ink and application thereof
CN116218760A (en) * 2023-02-07 2023-06-06 北京理工大学 Arterial organ chip based on multi-material suspended organism 3D printing and preparation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
《可见光交联的生物墨水的制备及其生物相容性研究》;《郑子卓》;《工程科技Ⅰ辑》;20200715;1-85 *

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