CN116426003A - 3D hydrogel for cell expansion culture and preparation method thereof - Google Patents
3D hydrogel for cell expansion culture and preparation method thereof Download PDFInfo
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
- C12N5/0659—Satellite cells
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C—CHEMISTRY; METALLURGY
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a 3D hydrogel for cell expansion culture and a preparation method thereof. Filtering after GMA functional material and photo-curing agent to obtain sterile precursor solution; the sterile precursor solution is exposed to a 405nm light source for photocuring to obtain a cell-free or cell-containing 3D hydrogel. The 3D cell culture hydrogel system provided by the invention contains a large amount of hydrophilic groups, can keep higher water permeability and water retention capacity, can store a large amount of cell nutrient solution to provide nutrient substances for cells in real time, and has good air permeability, thus providing a good three-dimensional microenvironment for efficient cell expansion and having an important effect on cell expansion culture.
Description
Technical field:
the invention belongs to the technical field of bioengineering, and particularly relates to a 3D hydrogel for cell expansion culture and a preparation method thereof.
The background technology is as follows:
conventional two-dimensional (2D) cells are often tested on flat, rigid materials (e.g., polystyrene, glass). Although these materials have convenience and economy, such culture conditions may alter the metabolism and gene expression patterns of cells, thereby causing abnormal cell behavior, and three-dimensional (3D) cell culture enhances cell-to-cell and cell-to-environment communication and links by simulating in vivo microenvironment, and has similar growth morphology, function, gene expression, topology, etc. Due to the large difference in function exhibited by 2D and 3D cultured cells, cell culture techniques are gradually shifted from 2D culture to 3D culture.
The biomaterial scaffold is one of the important factors for 3D culture of cells, and the carrier scaffold should give the cells long-term three-dimensional support, so that the cells proliferate rapidly and develop into complete functional tissues. The ideal vector needs to have the following characteristics: nontoxic, good biocompatibility, proper mechanical property and degradability. Among them, a hydrogel polymer network with high water content has become one of the best candidates for 3D cell culture, they mimic the mechanical properties of extracellular matrix and can provide support for cell adhesion. The physical and biochemical properties of hydrogels depend to a large extent on their composition, crosslinking method and crosslinking density.
The cross-linking of hydrogels can be physical cross-linking, chemical cross-linking, and combinations thereof. While one representative crosslinking method among chemical crosslinking is photocrosslinking. Photocrosslinking is a rapid, gentle crosslinking method. Ultraviolet or visible light may be used to induce chemical reactions of the photoactive monomers in the presence of photoinitiators. Photo-crosslinked hydrogels have many advantages over other crosslinking methods: 1) Photocrosslinking can occur under mild conditions (e.g., room temperature, neutral pH, etc.); 2) The hydrogel with adjustable mechanical/physical and chemical properties can be prepared by adjusting the intensity of a light source and the dosage of a photoinitiator; 3) The method can be combined with different processing methods to manufacture the multifunctional hydrogel with complex shape and 3D structure.
At present, a preparation method of a cell-containing hydrogel with high biocompatibility and rapidness is highly needed, so that it is necessary to design a photo-curing hydrogel material with high biocompatibility to construct a novel 3D cell culture system suitable for technical development of regenerative medicine, biological medicine and the like.
The invention comprises the following steps:
the technical problem to be solved by the invention is that a preparation method of the cell-containing hydrogel which is rapid in preparation and high in biocompatibility is not available at present.
In order to solve the problems, the invention provides a preparation method of a photocuring material for 3D cell culture, by using the preparation method, the uniform batch production of the photocuring material for 3D cell culture can be realized, the gelling speed of the photocuring material is high, the cell activity can be maintained at a higher level, and the cell function is promoted; in addition, the invention also provides the 3D hydrogel for cell expansion culture prepared by the preparation method of the light-cured material, and the 3D hydrogel cell culture system has good biocompatibility and can be applied to 3D cell culture.
In order to achieve the above object, the present invention is realized by the following technical scheme, which is a preparation method of a 3D hydrogel for cell expansion culture, comprising the following steps:
(1) Dissolving a GMA (glycidyl methacrylate) functional material and a photocuring agent in pure water or ion buffer, if the functional material is difficult to dissolve, using oscillation or ultrasonic auxiliary dissolution in the heating process, and filtering the dissolved functional material by using a sterile filter of 0.22 mu m to obtain a sterile precursor solution;
(2) 3D hydrogels for cell expansion culture can be made in two ways: 1) Pre-preparing 3D hydrogel: exposing the sterile precursor solution to a 405nm light source for photocuring, and freeze-drying to obtain a 3D hydrogel material for 3D cell culture; 2) Cells were directly encapsulated in 3D hydrogel: the sterile precursor solution preheated at 37 ℃ is mixed with the cell suspension and then rapidly injected into a cell culture plate, and the cell culture plate is exposed to a 405nm light source for photocuring to obtain the 3D hydrogel containing cells. The mechanical strength of the hydrogel can be controlled by illumination time and strength. Wherein the ultraviolet irradiation can cause cell injury and skin canceration. Photopolymerization using 405nm was safer and more biocompatible.
Further, the GMA functionalized material is a GMA functionalized protein and/or a GMA functionalized polysaccharide.
Further, the protein is one or more than one mixture of gelatin, gelatin derivative, collagen, silk fibroin, sericin, glycoprotein, laminin and matrigel; the polysaccharide is one or more of alginate, hyaluronic acid, chondroitin sulfate, chitosan, agar, and proteoglycan.
Further, the preparation method of the GMA functionalized protein comprises the following steps: dissolving soluble protein in pure water or ion buffer solution to prepare protein solution with certain concentration, regulating pH to 9.0 with sodium bicarbonate, adding 8mLGMA reagent after the protein solution is fully dissolved and transparent, and stirring at 45deg.C for 5 hr under conditions that can allow protein and GMA reagent to react fully; the reaction of the GMA and the protein requires proper temperature and reaction time, the GMA can effectively react with the protein or the polysaccharide between 20 ℃ and 60 ℃, and the efficient reaction of the GMA and the protein or the polysaccharide can be realized at 45 ℃ for 5 hours, so that the vinyl modification of the protein and the polysaccharide is realized. And (3) dialyzing the GMA functionalized protein solution in distilled water for 3 days after the reaction is finished, changing water for 3 times a day, removing unreacted GMA, and freeze-drying after the dialysis is finished to obtain the GMA functionalized protein material.
Further, the preparation method of the GMA functionalized polysaccharide material comprises the following steps: dissolving soluble polysaccharide in pure water or ion buffer solution to prepare polysaccharide solution with certain concentration; adding tetraethylammonium bromide (TEAB) of the same mass as the polysaccharide after the polysaccharide solution is fully dissolved, wherein the TEAB is used as a phase transfer catalyst, and adding N, N-Dimethylformamide (DMF) after the polysaccharide solution is fully dissolved, wherein DMF is added for completely dissolving GMA in the reaction mixture without generating polysaccharide precipitation; the GMA is insoluble in water, and the DMF is added to promote the mixing and removal of the GMA and the polysaccharide in an aqueous solution system, so that the reaction efficiency is improved. The addition amount of DMF is one third of the solvent; adding GMA (1:14w/w) into the polysaccharide solution, stirring at 25 ℃ for 6 days, dialyzing the GMA functionalized polysaccharide solution in distilled water for 3 days after the reaction is finished, changing water for 3 times a day, removing unreacted GMA, and freeze-drying after the dialysis is finished to obtain the GMA functionalized polysaccharide material.
Further, the molecular weight of the dialysis bag was 3500Da.
Further, the photo-curing agent is one of phenyl-2, 4,6-trimethylbenzoyl lithium phosphite (lithium phenyl (2, 4, 6-trimethylphenyl) phosphate, LAP), irgacure2959, eosin y, riboflavin, fluorescein, camphorquinone.
The 3D hydrogel for cell expansion culture prepared by the method. The light-cured material can be glued in 10-15 seconds, the glue-forming speed is high, the cell activity can be maintained at a higher level, and the cell function is promoted.
An application of the 3D hydrogel in 3D cell culture, comprising the following steps:
(1) Cell-free 3D hydrogel: placing the 3D hydrogel in a cell culture plate, injecting the cell suspension into the 3D hydrogel from the top, and then supplementing a culture medium for long-term 3D cell culture;
(2) Cell-containing 3D hydrogels: the medium was directly supplemented for long-term 3D cell culture.
The invention has the beneficial effects that:
(1) The photocuring material used for preparing the hydrogel can be produced in batch uniformly, the gelling speed of the photocuring material is high, the cell activity can be maintained at a higher level, and the cell function is promoted.
(2) The 3D cell culture hydrogel system contains a large amount of hydrophilic groups, so that higher water permeability and water retention can be maintained, and the protein/polysaccharide used in the 3D cell culture hydrogel system is a high hydrophilic substance which can be dissolved in an aqueous solution; the hydrophobic GMA used is of a smaller molecular weight than the protein and does not affect the hydrophilicity of the final sample; the formed hydrogel has very high hydrophilicity; in addition, the low-concentration hydrogel forms a macroporous network in which cells grow, so that the exchange of nutrient substances and wastes required by the cells can be realized, and better permeability is shown. The hydrogel system can store a large amount of cell nutrient solution to provide nutrient substances for cells in real time, and good air permeability provides good three-dimensional microenvironment for efficient cell expansion and has important effect on cell expansion culture.
Drawings
Fig. 1: gelatin-based photo-setting lyophilized material.
Fig. 2: gelatin-based photocurable hydrogels.
Fig. 3: gelatin-based photocurable hydrogels were compared to Matrigel-cultured garrupa muscle stem cells.
The specific embodiment is as follows:
for the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
a method for preparing a 3D hydrogel for cell expansion culture, comprising the steps of:
(1) Adding 10g of gelatin into 100mL of pure water, heating, stirring and dissolving, adjusting pH to 9.0 by using sodium bicarbonate, adding 8mLGMA reagent after the gelatin solution is clear and transparent, stirring at 45 ℃ for 5 hours, dialyzing the GMA functionalized gelatin (GMA gel) solution in distilled water for 3 days (dialysis bag molecular weight 3500 Da) after the reaction is finished, changing water for 3 times a day, removing unreacted GMA, and freeze-drying after the dialysis is finished to obtain GMA gel.
(2) Adding 1g of GMAGel and 5mg of LAP into 10mL of pure water, dissolving with the assistance of ultrasonic waves, filtering the sterile precursor solution with a sterile filter of 0.22 μm after dissolving, and suspending 50 mu L of the precursor solution and equal volume of the muscle stem cells of the Epinephelus fuscoguttatusLiquid (1X 10) 5 After mixing per mL), the mixture was rapidly poured into 96-well plates, gelled by irradiation with a 405nm light source for 15s, 1mL of medium was supplemented to each well, and 3D cell culture was performed in an incubator. As shown in FIG. 3, the muscle stem cells of the Epinephelus fuscoguttatus can be rapidly propagated on the 3D hydrogel, the density of the living cells is greatly increased in the culture process from 1 day to 3 days along with the time, and the living cells are stained by using calcein on the third day, so that the 3D hydrogel is covered with more cells. The gel is shown to be effective in promoting cell proliferation.
Example 2:
a method for preparing a 3D hydrogel for cell expansion culture, comprising the steps of:
(1) 2g of Hyaluronic Acid (HA) was added to 100mL of pure water, after being sufficiently dissolved, 2g of TEAB was added and stirred uniformly, 30mL of LDMF was added, 28mL of LGMA was added, stirring was performed at room temperature for 6 days, after the reaction was completed, dialysis was performed in distilled water for 3 days, water was changed 3 times a day, and freeze-drying was performed after the dialysis was completed to obtain GMA functionalized hyaluronic acid (GMAHA).
(2) Adding 2g of GMAHA and 20mg of camphorquinone into 10mL of pure water, heating, shaking for dissolution, filtering with a 0.22 μm sterile filter, irradiating with 468nm light source for 10min to obtain GMAHA hydrogel, lyophilizing under sterile condition, placing lyophilized sample in 6-well plate, adding 1mL of grass carp muscle stem cell suspension (1×10) 5 And 3/mL) is injected from the top of the freeze-dried hydrogel material, placed in an incubator for incubation for 3 hours, and 2mL of culture medium is supplemented after the cells are fully attached, and placed in the incubator for long-term 3D cell culture.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (8)
1. A method for preparing a 3D hydrogel for cell expansion culture, comprising the steps of:
(1) Filtering after GMA functional material and photo-curing agent to obtain sterile precursor solution;
(2) Exposing the sterile precursor solution to a 405nm light source for photocuring, and freeze-drying to obtain a cell-free 3D hydrogel for 3D cell culture;
or directly packaging the cells in the 3D hydrogel, mixing a sterile precursor solution preheated at 37 ℃ with the cell suspension, quickly injecting the mixture into a cell culture plate, and exposing the mixture to a 405nm light source for photo-curing to obtain the 3D hydrogel containing the cells.
2. The method of manufacturing according to claim 1, wherein: the GMA functional material is GMA functional protein and/or GMA functional polysaccharide.
3. The method of manufacturing as claimed in claim 2, wherein: the protein is one or more than one of gelatin, gelatin derivative, collagen, silk fibroin, sericin, glycoprotein, laminin and matrigel; the polysaccharide is one or more of alginate, hyaluronic acid, chondroitin sulfate, chitosan, agar, and proteoglycan.
4. A process according to claim 2 or 3, characterized in that the GMA-functionalized protein is prepared as follows: dissolving protein, regulating pH to 9.0, adding GMA reagent, stirring at 45deg.C for 5 hr, dialyzing the GMA functionalized protein solution in distilled water, and lyophilizing to obtain GMA functionalized protein.
5. A process according to claim 2 or 3, characterized in that the GMA-functionalized polysaccharide material is prepared by: adding tetraethylammonium bromide with the same mass as the polysaccharide after the polysaccharide is fully dissolved, and adding N, N-dimethylformamide after the polysaccharide is fully dissolved, wherein the adding amount of DMF is one third of that of the solvent; adding GMA into the solution, stirring at 25 ℃ for 6 days, after the reaction, putting the GMA functionalized polysaccharide solution into distilled water, and freeze-drying after the dialysis is finished to obtain the GMA functionalized polysaccharide material.
6. The method of manufacturing according to claim 1, wherein: the photocuring agent is one of phenyl-2, 4,6-trimethyl benzoyl lithium phosphite, irgacure2959, eosin y, riboflavin, fluorescein and camphorquinone.
7. A 3D hydrogel for cell expansion culture prepared by the method of any one of claims 1-6.
8. Use of the 3D hydrogel of claim 7 in 3D cell culture, comprising the steps of:
(1) Cell-free 3D hydrogel: placing the 3D hydrogel in a cell culture plate, injecting the cell suspension into the 3D hydrogel from the top, and then supplementing a culture medium for long-term 3D cell culture;
(2) Cell-containing 3D hydrogels: the medium was directly supplemented for long-term 3D cell culture.
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