CN114181883B - Hardness-adjustable hydrogel organoid ball containing matrigel, and preparation method and application thereof - Google Patents
Hardness-adjustable hydrogel organoid ball containing matrigel, and preparation method and application thereof Download PDFInfo
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- CN114181883B CN114181883B CN202111475196.6A CN202111475196A CN114181883B CN 114181883 B CN114181883 B CN 114181883B CN 202111475196 A CN202111475196 A CN 202111475196A CN 114181883 B CN114181883 B CN 114181883B
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 70
- 210000002220 organoid Anatomy 0.000 title claims abstract description 60
- 108010082117 matrigel Proteins 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229940072056 alginate Drugs 0.000 claims abstract description 37
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 37
- 229920000615 alginic acid Polymers 0.000 claims abstract description 37
- 238000004132 cross linking Methods 0.000 claims abstract description 17
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 13
- 108010035532 Collagen Proteins 0.000 claims abstract description 8
- 102000008186 Collagen Human genes 0.000 claims abstract description 8
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229920001436 collagen Polymers 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000002861 polymer material Substances 0.000 claims abstract description 5
- 229920005615 natural polymer Polymers 0.000 claims abstract description 4
- 238000007877 drug screening Methods 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 31
- 239000002131 composite material Substances 0.000 claims description 23
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 21
- 239000000499 gel Substances 0.000 claims description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 16
- 235000010413 sodium alginate Nutrition 0.000 claims description 16
- 239000000661 sodium alginate Substances 0.000 claims description 16
- 229940005550 sodium alginate Drugs 0.000 claims description 16
- 239000011259 mixed solution Substances 0.000 claims description 15
- 229920001223 polyethylene glycol Polymers 0.000 claims description 15
- 239000002202 Polyethylene glycol Substances 0.000 claims description 14
- 150000001768 cations Chemical class 0.000 claims description 12
- -1 salt ions Chemical class 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 8
- 239000012362 glacial acetic acid Substances 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 5
- 210000000056 organ Anatomy 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000011575 calcium Substances 0.000 claims description 4
- 159000000007 calcium salts Chemical class 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims description 3
- AUDYZXNUHIIGRB-UHFFFAOYSA-N 3-thiophen-2-ylpyrrole-2,5-dione Chemical compound O=C1NC(=O)C(C=2SC=CC=2)=C1 AUDYZXNUHIIGRB-UHFFFAOYSA-N 0.000 claims description 2
- 210000004556 brain Anatomy 0.000 claims description 2
- 210000000481 breast Anatomy 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- FNAQSUUGMSOBHW-UHFFFAOYSA-H calcium citrate Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FNAQSUUGMSOBHW-UHFFFAOYSA-H 0.000 claims description 2
- 239000001354 calcium citrate Substances 0.000 claims description 2
- QXDMQSPYEZFLGF-UHFFFAOYSA-L calcium oxalate Chemical compound [Ca+2].[O-]C(=O)C([O-])=O QXDMQSPYEZFLGF-UHFFFAOYSA-L 0.000 claims description 2
- 239000001506 calcium phosphate Substances 0.000 claims description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 2
- 235000011010 calcium phosphates Nutrition 0.000 claims description 2
- GUPPESBEIQALOS-UHFFFAOYSA-L calcium tartrate Chemical compound [Ca+2].[O-]C(=O)C(O)C(O)C([O-])=O GUPPESBEIQALOS-UHFFFAOYSA-L 0.000 claims description 2
- 235000011035 calcium tartrate Nutrition 0.000 claims description 2
- 239000001427 calcium tartrate Substances 0.000 claims description 2
- JHECKPXUCKQCSH-UHFFFAOYSA-J calcium;disodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate;hydrate Chemical compound O.[Na+].[Na+].[Ca+2].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O JHECKPXUCKQCSH-UHFFFAOYSA-J 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 210000003734 kidney Anatomy 0.000 claims description 2
- 210000004072 lung Anatomy 0.000 claims description 2
- 210000000496 pancreas Anatomy 0.000 claims description 2
- 235000010408 potassium alginate Nutrition 0.000 claims description 2
- 239000000737 potassium alginate Substances 0.000 claims description 2
- MZYRDLHIWXQJCQ-YZOKENDUSA-L potassium alginate Chemical compound [K+].[K+].O1[C@@H](C([O-])=O)[C@@H](OC)[C@H](O)[C@H](O)[C@@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@@H](O)[C@H]1O MZYRDLHIWXQJCQ-YZOKENDUSA-L 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 210000002784 stomach Anatomy 0.000 claims description 2
- 210000001541 thymus gland Anatomy 0.000 claims description 2
- 210000001685 thyroid gland Anatomy 0.000 claims description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 2
- 235000013337 tricalcium citrate Nutrition 0.000 claims description 2
- DAWJJMYZJQJLPZ-UHFFFAOYSA-N 2-sulfanylprop-2-enoic acid Chemical group OC(=O)C(S)=C DAWJJMYZJQJLPZ-UHFFFAOYSA-N 0.000 claims 1
- 125000002897 diene group Chemical group 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 8
- 230000001105 regulatory effect Effects 0.000 abstract description 6
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- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 4
- 238000005698 Diels-Alder reaction Methods 0.000 description 3
- SHWNNYZBHZIQQV-UHFFFAOYSA-J EDTA monocalcium diisodium salt Chemical compound [Na+].[Na+].[Ca+2].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O SHWNNYZBHZIQQV-UHFFFAOYSA-J 0.000 description 3
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- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 125000004185 ester group Chemical group 0.000 description 2
- 125000002541 furyl group Chemical group 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000288906 Primates Species 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
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- 150000001993 dienes Chemical class 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 239000000416 hydrocolloid Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
Abstract
The invention discloses a hydrogel organoid ball with adjustable hardness containing matrigel, and a preparation method and application thereof, and belongs to the technical field of organoids. The organoid ball is formed by hydrogel formed by natural polymer materials such as matrigel, collagen, alginate and the like, cells are uniformly dispersed in the hydrogel solution to form a water phase, the water phase is prepared into uniform-particle-diameter monodisperse liquid drops by a microfluidic water-in-oil technology, and then the uniform-structure cell-carrying interpenetrating network hydrogel organoid ball is formed by crosslinking by a crosslinking agent. The method has the greatest advantages that on the premise of not changing the content of matrigel and/or collagen, the hydrogel organoid ball with the hardness accurately regulated and controlled within the range of 100Pa-800kPa can be prepared by regulating the type of alginate, the concentration and the type of the cross-linking agent in the water phase, is used for simulating the hardness of various soft tissues microenvironments of a human body, can be particularly used for various tumor organoids, and provides experimental basis for high-flux drug screening and clinical individuation medication.
Description
Technical Field
The invention belongs to the technical field of organoids, and particularly relates to a hydrogel organoid ball with adjustable hardness containing matrigel, and a preparation method and application thereof.
Background
Organoids are a three-dimensional cell culture system that mimics part of the structural and functional characteristics of a human or animal organ. Compared with the traditional two-dimensional cell culture or non-primate animal model, the organoid culture system overcomes many limitations of standard single-layer cancer cell culture, so that the organ-level biological model can be studied in vitro, and the culture is closer to the actual physiological environment of a human body. Importantly, organoid culture, especially tumor organoid culture, reproduces the morphology and biological properties of tumors, providing a valuable new tool for cancer research, drug development and precise medicine.
In the classical organoid technology, cells for culturing organoids are mixed with matrigel at low temperature, inoculated into a cell culture plate, placed in a culture environment at 37 ℃ and subjected to gelation reaction through temperature sensitivity of the matrigel, and the cells are embedded into the matrigel, so that the matrigel provides a three-dimensional culture environment for the cells. The biggest problem of the technology is that the large-scale preparation of organoids is difficult to realize due to the culture of the cell culture plates, and the requirement of high-throughput screening of medicines cannot be met.
For this reason, researchers have introduced microfluidic technology into organoid sphere culture, CN110004111a discloses a method for preparing organoid spheres, which comprises maintaining matrigel sol state under low temperature (4 ℃) condition, taking matrigel in sol state as water phase, forming water-in-oil droplet by means of pipeline microfluidic, transferring droplet pipeline to 37 ℃ until matrigel droplet is solidified into gel sphere, thus preparing organoid sphere with uniform shape and size and large scale.
However, there are some problems in practical application of this technology:
① The matrigel is a temperature sensitive hydrogel, but the conversion from sol to gel is relatively long, and the gel state can be formed after the temperature is increased to 37 ℃ for at least 30 minutes. Therefore, after the microfluidic technology forms monodisperse water-in-oil droplets in a low temperature environment, the droplets need to be converted to a 37 ℃ environment, and the monodisperse state of the water-in-oil droplets needs to be maintained in the pipeline for at least 30 minutes, and the substrate gel can not leave the micro-channel pipeline to enter the collecting area until the substrate gel forms gel. Which would otherwise cause the aqueous phase to agglomerate into large particles. This results in a longer line for the formation of monodisperse droplets in a 37 ℃ environment, which does not affect the flow rate of the original line, but ensures a sufficiently long time to complete the gelation reaction of the matrigel.
② While the matrigel provides a three-dimensional culture environment for cells, and the matrigel contains more than 1000 protein components and provides a lot of nutrient substances for cell proliferation and differentiation, the matrigel has very low hardness (usually 20-450 Pa) which is far lower than that of normal soft tissue organs or tumor tissues (100 Pa-500 kPa) of a human body.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a hydrogel organoball with adjustable hardness containing matrigel, a preparation method and application thereof, wherein alginate and matrigel form a composite hydrogel, and as an alginate solution and divalent cations (such as Ca 2+、Cu2+、Fe2+、Sr2+、Zn2+ and Ba 2+) or trivalent cations (such as Fe 3+、Ga3+) can be instantaneously cross-linked to form the hydrogel, and the hydrogel is not limited by temperature (can instantaneously occur in the range of 1-37 ℃), thereby solving the problem that gelation time is long under the environment of 37 ℃ when the matrigel is simply used; meanwhile, by carrying out chemical modification on the alginate in advance and by means of an ion-covalent double-crosslinking technology, the hardness of the composite hydrogel can be accurately regulated and controlled within the range of 100Pa-800kPa by changing the concentration, molecular weight and distribution of the alginate material, GM ratio, substitution degree of covalent modification groups, ionic crosslinking agent types, covalent crosslinking agent types, crosslinking reaction time and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a hardness-adjustable hydrogel organoid ball containing matrigel is mainly composed of matrigel containing cells and natural high-molecular materials and is an ion-covalent double-crosslinked hydrogel ball.
Further, the organoid spheres are covalently cross-linked by click chemistry.
Further, the natural polymer material comprises alginate, alginate covalently modified by one group in the click chemistry reaction group pair, hyaluronic acid, and hyaluronic acid covalently modified by one group in the click chemistry reaction group pair.
Further, the natural polymer material comprises alginate and alginate covalently modified by one group in the click chemistry reaction group pair.
Further, alginate covalently modified by one of the click chemistry reactive group pair is covalently crosslinked by click chemistry reaction with polyethylene glycol molecule covalently modified by the other of the click chemistry reactive group pair.
Further, the organoid sphere is loaded with a cell load of 10-10 7 cells/organoid sphere.
Further, the organoid sphere has a particle size of 30-800 μm.
Further, the hardness of the organoid ball is adjustable and controllable within the range of 100Pa-800 kPa.
Further, the organoids include head and neck organoids, thyroid organoids, breast organoids, pancreas organoids, liver organoids, thymus organoids, lung organoids, kidney organoids, stomach organoids, intestinal organoids, brain organoids, and tumor organoids of the above-mentioned respective organs.
In another aspect, the present invention provides a method for preparing the hydrogel organoid sphere containing matrigel and having adjustable hardness, the method mainly comprising the following steps:
(1) Covalently modifying one group of the click chemistry reaction group pair on the carboxyl group of the alginate molecule;
(2) Mixing the modified alginate obtained in the step (1) with unmodified alginate according to the mass ratio of 1:0-1:20, and preparing an alginate mixed solution with the final concentration of 3-100mg/mL by using a slightly soluble salt solution of an ion crosslinking agent of the alginate with the concentration of 2-200 mmol/L;
(3) Preparing matrigel and collagen solution, wherein the concentration of matrigel protein in the solution is 10-20mg/mL, the concentration of collagen protein in the solution is 0-20mg/mL, and the pH of the solution is 6.8-7.4;
(4) Mixing the solution prepared in the step (3) and the mixed solution of the alginate prepared in the step (2) into a uniform solution in a low-temperature environment (1-4 ℃), wherein the content of the alginate in the solution is 3-50mg/mL, the concentration of the insoluble salt in the solution is 2-100mmol/L, the content of the matrigel is 5-20mg/mL, and the content of the collagen is 0-10mg/mL;
(5) Mixing the mixed solution prepared in the step (4) with cells, wherein the cell density in the mixed solution is 1X 10 3-5×106/mL; through a microfluidic pipeline, firstly forming uniform and monodisperse water-in-oil droplets in a low-temperature environment (1-4 ℃), then introducing an oil-soluble acid solution into the pipeline to trigger release of insoluble salt ions in the water phase, forming ionic crosslinking hydrogel spheres by the droplets, and collecting the hydrogel spheres through water-oil layering;
(6) Incubating the hydrogel ball prepared in the step (5) for 10-120 minutes at the temperature of 30-38 ℃ to form a composite hydrogel ball;
(7) The other group in the click chemistry reaction group is covalently modified with polyethylene glycol to form 2-arm, 4-arm and 8-arm polyethylene glycol respectively;
(8) Mixing the hydrogel spheres collected in the step (5) or (6) with the modified polyethylene glycol solution (1-20 g/L) prepared in the step (7) to perform covalent crosslinking reaction to form ion-covalent double-crosslinked hydrogel spheres;
(9) Placing the double-crosslinked hydrogel spheres prepared in the step (8) into a bioreactor for culture to form the spheres with the cell load of 10-10 7 cells/organoids.
Further, the alginate in the step (1) comprises sodium alginate and potassium alginate.
Further, the click chemistry reaction group pair described in step (1) includes any one of azide-alkyne groups, thiol-olefins, thiol-acrylates, conjugated dienes-yoke-philic dienes; the thiol-olefin is preferably thiol-maleimide and the conjugated dienophile-yoke-philic dienome is preferably furyl-maleimide.
Further, the ionic crosslinking agent in the step (2) is a divalent cation or a trivalent cation, the divalent cation includes Ca 2+、Cu2+、Fe2+、Sr2+、Zn2+ and Ba 2+, and the trivalent cation includes Fe 3+、Ga3+.
Further, the insoluble salt in the step (2) is insoluble calcium salt, and the insoluble calcium salt is one or more of EDTA calcium, calcium carbonate, calcium citrate, calcium oxalate, calcium tartrate and calcium phosphate.
Further, the cells described in step (5) include primary cells and immortalized cell lines.
Further, the oil-soluble acid in step (5) comprises glacial acetic acid.
Further, the microfluidic pipeline in the step (5) includes all microfluidic pipelines suitable for preparing sodium alginate droplets by internal gelation reaction, including a T-shaped pipeline (Monodisperse Alginate Hydrogel Microbeads for Cell Encapsulation,Adv.Mater.2007,19,2696–2701;Alginate gelation in microfluidic channels,Food Hydrocolloids 22(2008)97–104)、", a return pipeline (Microfluidic Encapsulation of Single Cells by Alginate Microgels Using a Trigger-Gellified Strategy,Frontiers in Bioengineering and Biotechnology,2020,8,583065)、", a ten-shaped pipeline (Using a microfluidic chip and internal gelation reaction for monodisperse calcium alginate microparticles generation,Frontiers in Bioscience 2007,12,3061-3067)、"Y", a multi-Y-shaped combined pipeline (Monodisperse hybrid microcapsules with an ultrathin shell of submicron thickness for rapid enzyme reactions,J.Mater.Chem.B,2015,3,796–803), and combined pipelines with different shapes.
Further, the hardness of the hydrogel spheres is regulated by regulating the concentration, the type and the reaction time (1-30 minutes) of the ionic crosslinking agent and the covalent crosslinking agent, the molecular weight of the crosslinked alginate, the distribution thereof, the G content, the concentration, the substitution degree of the covalent modification groups and the like.
The invention also provides application of the hydrogel organoid ball with the matrix gel and adjustable hardness in high-throughput screening of medicines.
The invention has the beneficial effects that:
(1) According to the invention, the alginate and matrigel form the composite hydrogel, and as the alginate solution and divalent cations (such as Ca 2+、Cu2+、Fe2+、Sr2+、Zn2+ and Ba 2+) or trivalent cations (such as Fe 3+、Ga3+) can be instantaneously subjected to ionic crosslinking to form the hydrogel, the ionic crosslinking reaction is not affected by temperature, and can instantaneously occur within the range of 1-37 ℃, the gel can enter a collecting area, and organoid balls can be collected by oil-water layering. The matrigel in the gel is still in a liquid state due to short time, but is still uniformly distributed in the alginate ion crosslinked gel balls due to the constraint of the alginate ion crosslinked gel network bracket, and the gelation reaction can be continuously completed in a 37 ℃ incubator. Thus, the present invention does not require a long pipeline to maintain a monodisperse droplet state.
(2) The reaction process of the alginate for ion-covalent double cross-linking can be completed under physiological conditions, and the biological activity of cells is not affected, so that the composite high molecular organoid sphere can realize in-situ cell preparation.
(3) According to the invention, the hardness of the composite hydrogel is accurately regulated and controlled within the range of 100Pa-800kPa by carrying out chemical covalent modification on the alginate in advance and by means of an ion-covalent double-crosslinking technology, and by changing the concentration, molecular weight and distribution of the alginate, GM ratio, substitution degree of covalent modification groups, ionic crosslinking agent types, covalent crosslinking agent types, crosslinking reaction time and the like of the alginate material, so that the composite hydrogel is suitable for the mechanical environment requirements of different organs.
(4) According to the invention, covalent bonds are introduced through click chemical reaction, covalent modification groups are modified on materials in advance, the modified materials are firstly subjected to ionic crosslinking to prepare gel with a certain shape, and finally the gel is subjected to mild click chemical reaction to form covalent crosslinking gel, wherein the ionic crosslinking and the click chemical reaction are reactions instantaneously occurring at normal temperature and normal pressure, so that the preparation process of the gel can carry cell operation, and the overall structural stability of the composite scaffold is improved without influencing cell activity.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
Fig. 1 is a schematic diagram of a microfluidic device in a pipeline according to embodiment 1 of the present invention.
FIG. 2 is a schematic representation of the preparation of 6 experimental group samples in example 1 of the present invention.
FIG. 3 shows the morphology (B) of the breast cancer organoids prepared in example 2 of the present invention after the initial culture (A) and 14 days of culture.
FIG. 4 shows a schematic diagram (A) of the preparation of the hydroxyl modified sodium alginate material Alg (OH) -g-BAT prepared in example 3 and an infrared spectrogram (B) of a novel synthesized material.
FIG. 5 shows morphology (B) of liver organoids prepared in example 3 of the present invention after initiation of culture (A) and 14 days of culture and results of live and dead staining of cells (C).
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
Example 1
Preparing a hardness-adjustable hydrogel organoid sphere, which comprises the following steps:
(1) And (3) grafting the furyl furfuryl furylamine onto the carboxyl of the sodium alginate glycogen through amidation reaction of the sodium alginate molecules to obtain sodium alginate (Alg-furan) containing furanyl, wherein the grafting rate is 60% through nuclear magnetic mass spectrometry.
(2) And (3) mixing the modified sodium alginate prepared in the step (1) with unmodified sodium alginate according to the following formula 1:2, mixing the materials in proportion, and preparing a sodium alginate mixed solution with the final concentration of 15mg/mL by using a calcium EDTA solution with the concentration of 0.1 mol/L.
(3) And (3) uniformly mixing the sodium alginate mixed solution prepared in the step (2) with a commercial matrigel solution according to a volume ratio of 1:1 at the temperature of 4 ℃.
(4) And (3) taking the mixed solution prepared in the step (3) as a disperse phase, taking liquid paraffin as a mobile phase, preparing water-in-oil liquid drops according to a pipeline microfluidic device of the attached figure 1, then introducing liquid paraffin containing glacial acetic acid (the volume ratio of the glacial acetic acid to the liquid paraffin is 1:100) into a subsequent pipeline as the mobile phase, and preparing and collecting the alginate-matrigel composite hydrogel microsphere with the particle size of 500 microns.
(5) And (3) placing the microspheres prepared in the step (4) in a 37 ℃ incubator for 1 hour to obtain the composite hydrogel microspheres.
(6) Dividing the hydrogel microsphere sample in the step (5) into 6 groups (G1-G6) at random, liquefying the G1 groups with sodium citrate solution for 10 minutes, removing the sodium citrate solution, and washing with PBS for 3 times to obtain hydrogel microspheres only with matrigel; the G2 group sample is the single ion crosslinking type composite hydrogel microsphere for standby; the G3 group sample reacts with single-arm-polyethylene glycol of maleimide group; the G4 group sample reacts with maleimide group modified 2-arm-polyethylene glycol; reacting the G5 group sample with maleimide group modified 4-arm-polyethylene glycol; group G6 samples were reacted with maleimide group modified 8-arm-polyethylene glycol. The G3-G6 group forms a covalent bond through Diels-Alder (DA) click chemistry reaction to prepare the DA click chemistry crosslinked ion-covalent double-crosslinked composite hydrogel sphere.
(7) Detecting matrix rigidity of the 6 groups of hydrogel balls prepared in the step (6) by using a mechanical tester, and taking an average value of 3 parallel samples in each group, wherein the matrix rigidity is G1-102Pa respectively; g2-2530Pa; g3-95kPa; g4-260kPa; g5-380kPa; g6-520kPa. The composite hydrogel microspheres with different hardness are obtained through the regulation and control of the concentration, the type and the like of the cross-linking agent.
Example 2
Preparing a hydrogel organoid sphere containing primary breast cancer cells, comprising the steps of:
(1) And (3) reacting sodium alginate molecules with glycidyl methacrylate, and grafting an acrylic ester group onto the hydroxyl of the sodium alginate to obtain the sodium alginate containing the acrylic ester group, wherein the substitution degree of the sodium alginate is 50% by nuclear magnetic mass spectrometry.
(2) And (3) preparing the modified sodium alginate prepared in the step (1) into a solution with the final concentration of 20mg/mL by using a calcium EDTA solution with the concentration of 0.1 mol/L.
(3) Uniformly mixing the sodium alginate solution prepared in the step (2) with a commercial matrigel solution according to a volume ratio of 1:2 at the temperature of 4 ℃, and uniformly mixing the mixture with primary separated breast cancer cells, wherein the cell content is 10 6/mL.
(4) And (3) taking the mixed solution prepared in the step (3) as a disperse phase, taking liquid paraffin as a mobile phase, preparing water-in-oil liquid drops according to a pipeline microfluidic device of the attached figure 1, then introducing liquid paraffin containing glacial acetic acid (the volume ratio of the glacial acetic acid to the liquid paraffin is 1:100) into a subsequent pipeline as the mobile phase, and preparing and collecting the alginate-matrigel composite hydrogel microsphere with the particle size of 300 microns.
(5) And (3) placing the microspheres prepared in the step (4) in a 37 ℃ incubator for 1 hour to obtain the composite hydrogel microspheres.
(6) The hydrogel microsphere in the step (5) is reacted with polyethylene glycol PEG (PEG-SH) containing sulfhydryl four arms, and PBS is washed for 3 times, so as to obtain alginate-PEG-matrigel composite hydrogel spheres carrying primary breast cancer cells (figure 3A).
(7) After the hydrogel pellets prepared in the step (6) were cultured in an incubator for 14 days, it was seen that the cells proliferated significantly within the composite hydrogel pellets, and the activity remained good (fig. 3B).
Example 3
Preparing a hydrogel organoid pellet containing stem cell-derived hepatocytes, comprising the steps of:
(1) The azido group is grafted to the hydroxyl of sodium alginate glycogen to obtain Alg (OH) -g-BAT material, the reaction process is shown in figure 4A, the infrared spectrum shows that a new grafting group is obtained (see figure 4B), and the grafting rate is 60% by nuclear magnetic mass spectrometry.
(2) And (3) mixing the modified sodium alginate prepared in the step (1) with unmodified sodium alginate according to the following formula 1:5, mixing in proportion, and preparing the sodium alginate mixed solution with the final concentration of 30mg/mL by using the calcium EDTA solution with the concentration of 0.1 mol/L.
(3) Uniformly mixing the sodium alginate mixed solution prepared in the step (2) with a commercial matrigel solution at the temperature of 4 ℃ according to the volume ratio of 1:2, and uniformly mixing the mixture with hiHep cells, wherein the cell content is 10 6/mL.
(4) And (3) taking the mixed solution prepared in the step (3) as a disperse phase, taking liquid paraffin as a mobile phase, preparing water-in-oil liquid drops according to a pipeline microfluidic device of the attached figure 1, then introducing liquid paraffin containing glacial acetic acid (the volume ratio of the glacial acetic acid to the liquid paraffin is 1:100) into a subsequent pipeline as the mobile phase, and preparing and collecting the alginate-matrigel composite hydrogel microsphere with the particle size of 400 microns.
(5) And (3) placing the microspheres prepared in the step (4) in a 37 ℃ incubator for 1 hour to obtain the composite hydrogel microspheres.
(6) And (3) reacting the hydrogel microsphere in the step (5) with 5-norbornene-2-alkynyl modified 2-arm-polyethylene glycol, liquefying and reacting for 10 minutes by using sodium citrate solution, and washing for 3 times by using PBS to obtain the cell-carrying crosslinked alginate-PEG-matrigel composite hydrogel sphere.
(7) After the hydrogel spheres prepared in the step (6) are cultured in an incubator for 14 days, live-dead staining is performed to observe the cell activity in the composite hydrogel microspheres, and the result is shown in fig. 5, so that the cells can be obviously proliferated in the composite hydrogel microspheres, and the activity is kept good.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (6)
1. The hydrogel organoid ball with adjustable hardness and containing matrigel is characterized in that the organoid ball mainly comprises matrigel containing cells and natural high polymer materials, and is an ion-covalent double-crosslinked hydrogel ball; the organoid sphere realizes covalent cross-linking through click chemistry reaction; the natural polymer material comprises alginate and alginate covalently modified by one group in a click chemistry reaction group pair; covalently-modified alginate of one group in the click chemistry reaction group pair is covalently crosslinked with polyethylene glycol molecules covalently modified by the other group in the click chemistry reaction group pair through click chemistry reaction; the hardness of the organoid ball is 100Pa-800kPa;
The preparation method of the hydrogel organoid ball with the matrix gel and adjustable hardness comprises the following steps:
(1) Covalently modifying one group of the click chemistry reaction group pair on the carboxyl group of the alginate molecule;
(2) Mixing the modified alginate obtained in the step (1) with unmodified alginate according to the mass ratio of 1:0-1:20, and preparing an alginate mixed solution with the final concentration of 3-100mg/mL by using a poorly soluble salt solution of an ionic cross-linking agent of the alginate;
(3) Preparing matrigel and collagen solution, wherein the concentration of matrigel protein in the solution is 10-20mg/mL, and the concentration of collagen protein is 0-20mg/mL;
(4) Mixing the solution prepared in the step (3) and the mixed solution of the alginate prepared in the step (2) into a uniform solution in a low-temperature environment, wherein the content of the alginate in the solution is 3-50mg/mL, the concentration of the insoluble salt in the solution is 2-100mmol/L, the content of the matrigel is 5-20mg/mL, and the content of the collagen is 0-10mg/mL;
(5) Mixing the mixed solution prepared in the step (4) with cells, wherein the cell density in the mixed solution is 1X 10 3-5×106/mL; through a microfluidic pipeline, firstly forming uniform and monodisperse water-in-oil droplets in a low-temperature environment, then introducing an oil-soluble acid solution into the pipeline to trigger release of insoluble salt ions in a water phase, forming ionic crosslinking hydrogel spheres by the droplets, and collecting the hydrogel spheres through water-oil layering;
(6) Incubating the hydrogel ball prepared in the step (5) for 10-120 minutes at the temperature of 30-38 ℃ to form a composite hydrogel ball;
(7) The other group in the click chemistry reaction group is covalently modified with polyethylene glycol to form 2-arm, 4-arm and 8-arm polyethylene glycol respectively;
(8) Mixing the hydrogel spheres collected in the step (5) or (6) with the modified polyethylene glycol solution prepared in the step (7) to perform covalent crosslinking reaction to form ion-covalent double-crosslinked hydrogel spheres;
(9) Placing the ion-covalent double-crosslinked hydrogel spheres prepared in the step (8) into a bioreactor for culture to form organoid spheres;
The ionic crosslinking agent in the step (2) is a divalent cation or a trivalent cation, wherein the divalent cation comprises Ca 2+、Cu2+、Fe2+、Sr2+、Zn2+ and Ba 2+, and the trivalent cation comprises Fe 3+、Ga3+;
The insoluble salt in the step (2) is insoluble calcium salt, and the insoluble calcium salt is one or more of EDTA calcium, calcium carbonate, calcium citrate, calcium oxalate, calcium tartrate and calcium phosphate.
2. The matrigel-containing, hardness-adjustable hydrogel organoid sphere of claim 1, wherein the cell load in the organoid sphere is 10-10 7 cells/organoid sphere; the particle size of the organoid sphere is 30-800 mu m.
3. The matrigel-containing hydrogel organoball of adjustable hardness according to claim 1, wherein the organoball comprises head and neck organoball, thyroid organoball, breast organoball, pancreas organoball, liver organoball, thymus organoball, lung organoball, kidney organoball, stomach organoball, intestinal organoball, brain organoball and tumor organoball of each of the above organs.
4. The matrix gel-containing, hardness-adjustable hydrogel organoball of claim 1, wherein the alginate in step (1) comprises sodium alginate, potassium alginate; the click chemistry reaction group pair comprises any one pair of azide-alkyne groups, thiol-olefin groups, mercapto-acrylate groups and conjugated diene-yoke-philic diene groups; the thiol-olefin is preferably thiol-maleimide and the conjugated dienophile-yoke-philic dienome is preferably furyl-maleimide.
5. The matrigel-containing, hardness-adjustable hydrogel organoball of claim 1, wherein the cells in step (5) comprise primary cells, immortalized cell lines, and stem cell-derived cells; the oil-soluble acid comprises glacial acetic acid; the microfluidic pipeline comprises a T-shaped, Y-shaped, cross-shaped, back-shaped and arrow-shaped microfluidic pipeline capable of realizing water-in-oil liquid drops.
6. Use of a matrigel-containing, hardness-adjustable hydrogel organoid sphere according to any of claims 1-5 in high throughput drug screening.
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