[go: up one dir, main page]

CN116162290B - Porous microcarrier, preparation method and application thereof - Google Patents

Porous microcarrier, preparation method and application thereof Download PDF

Info

Publication number
CN116162290B
CN116162290B CN202310221076.6A CN202310221076A CN116162290B CN 116162290 B CN116162290 B CN 116162290B CN 202310221076 A CN202310221076 A CN 202310221076A CN 116162290 B CN116162290 B CN 116162290B
Authority
CN
China
Prior art keywords
porous microcarrier
hyperbranched
reaction
acid
porous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202310221076.6A
Other languages
Chinese (zh)
Other versions
CN116162290A (en
Inventor
周光前
李智立
王凯旋
周德重
乔昕明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Abbe Cell Technology Co ltd
Shenzhen Danlun Gene Technology Co ltd
Original Assignee
Shenzhen Abbe Cell Technology Co ltd
Shenzhen Danlun Gene Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Abbe Cell Technology Co ltd, Shenzhen Danlun Gene Technology Co ltd filed Critical Shenzhen Abbe Cell Technology Co ltd
Priority to CN202310221076.6A priority Critical patent/CN116162290B/en
Publication of CN116162290A publication Critical patent/CN116162290A/en
Application granted granted Critical
Publication of CN116162290B publication Critical patent/CN116162290B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • 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
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/0061—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof characterized by the use of several polymeric components
    • 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
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • 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
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • 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
    • C08J2435/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical, and containing at least one other carboxyl radical in the molecule, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Derivatives of such polymers
    • 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
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2533/80—Hyaluronan
    • 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
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Inorganic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Rheumatology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Polymers & Plastics (AREA)
  • Microbiology (AREA)
  • Materials Engineering (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention discloses a porous microcarrier, a preparation method and application thereof, wherein the porous microcarrier comprises a hyperbranched polymer and a natural biological material which are mutually crosslinked; the hyperbranched polymer comprises hyperbranched polyacrylate or hyperbranched polyacrylamide; natural biological materials include: at least one of gelatin, gelatin derivatives, hyaluronic acid, collagen, glycoprotein, laminin, fibronectin, alginate, and alginate derivatives; the preparation method of the porous microcarrier comprises the steps of carrying out cross-linking reaction on hyperbranched polymer and natural biological material, dividing the hyperbranched polymer into micron-sized spherical particles through a microfluidic technology, and then carrying out treatment through a pore-forming technology to obtain the porous microcarrier; the porous microcarrier has good biocompatibility and mechanical property and wide application.

Description

Porous microcarrier, preparation method and application thereof
Technical Field
The invention relates to a porous microcarrier, a preparation method and application thereof, belonging to the technical field of biomedical materials.
Background
Stem cells are a type of pluripotent cells with proliferation and differentiation potential and self-renewing replication capacity, and have great application prospects in providing new cell therapies for a plurality of diseases which cannot be treated at present, modeling human diseases or drug discovery, such as blood system diseases, nervous system diseases, cardiovascular diseases, diabetes, bone joints and the like. In recent years, the clinical application of cell therapy is increasingly wide, and statistical research shows that tens of millions to billions of stem cells are needed for each patient per kilogram of body weight, and methods based on cell therapy and tissue regeneration for these diseases require in vitro expansion and induced differentiation of stem cells to meet clinical demands. Culturing cells in conventional monolayers for conventional two-dimensional (2D) monolayer culture may result in loss of specific morphology and phenotype during passage, and may alter secretion of cell-specific extracellular matrix (ECM), etc. The development and function of cells depend on intermolecular interactions in the microenvironment of the cells, but traditional monolayer culture is not a natural state of cell growth, cells may have differences from the natural state in terms of gene expression, signal transduction and morphology, and in terms of spatial structure, extracellular matrix, cytokine addition and the like, three-dimensional (3D) hematopoietic microenvironment in vivo cannot be simulated, thus limiting the clinical application of stem cell therapy. Over the past few decades, various methods for stem cell expansion and differentiation have been developed to meet the rapidly growing clinical demands, such as biological scaffolds, microcarriers, microcapsules, cellulose membranes, and a variety of three-dimensional technologies. One of the most widely accepted techniques at present is the use of Microcarriers (MCs) for the mass expansion of stem cells in bioreactor systems, which have high magnification potential, reduced space, biological process steps, pollution risk and overall process costs, and also allow for the intuitive and conditional controllability of cell culture, direct observation of the morphological structure of the cells, detection of whether they function normally, convenient investigation of physiological and pathological conditions of the human body, and prevention or treatment of diseases, providing a safer and more effective material basis for the research of regenerative medicine and tissue engineering and the well-established conditions according to the current good manufacturing practice (cGMP), worthy of further investigation.
Disclosure of Invention
In order to overcome the defects of the prior art, the first aim of the invention is to provide a porous microcarrier which is a hydrogel system, has good biocompatibility and mechanical properties and has wide application.
The second aim of the invention is to provide a preparation method of the porous microcarrier, which is simple and efficient, easy to control and good in stability.
A third object of the present invention is to provide the use of a porous microcarrier as described above.
The first object of the invention can be achieved by adopting the following technical scheme: a porous microcarrier comprising a hyperbranched polymer and a natural biological material that are cross-linked to each other; the hyperbranched polymer comprises hyperbranched polyacrylate or hyperbranched polyacrylamide; natural biological materials include: at least one of gelatin, gelatin derivatives, hyaluronic acid, collagen, glycoprotein, laminin, fibronectin, alginate and alginate derivatives.
Further, the mass ratio of the hyperbranched polymer to the natural biological material is (0.2-5): 1.
Further, the hyperbranched polymer is prepared by the following method:
Dissolving a polymer monomer and a chain transfer agent in a solvent, and deoxidizing to obtain a mixed solution; under the condition of introducing argon, adding an initiator into the mixed solution to obtain a reaction mixed system; the reaction mixed system generates reversible addition fragmentation chain transfer free radical polymer reaction under the anaerobic condition, gel permeation chromatography is used for monitoring the molecular weight of the polymer, when the molecular weight of the reaction reaches a set value of 10000-100000g/mol, the reaction is stopped, and then purification treatment is carried out to obtain hyperbranched polymer;
the polymer monomers include acrylate monomers and acrylamide monomers;
The acrylate monomer comprises: at least one of polyethylene glycol diacrylate, diethylene glycol dimethacrylate, tetraethylene glycol diacrylate, butylene glycol diacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, methoxypolyethylene glycol acrylate, hydroxypropyl methacrylate, trimethylolpropane triacrylate, glycidyl methacrylate, methyl acrylate, isocyanoethyl methacrylate, 2- (dimethylamino) ethyl acrylate, 2-methyl-2- (2-methoxyethoxy) ethyl acrylate, hydroxyethyl acrylate, disulfide acrylate, and 2-1, 10-decanediyl 2-acrylate; the acrylamide monomer comprises: at least one of acrylamide, N-dimethyl bisacrylamide, and N, N' -bis (propionyl) cystamine.
Further, the reaction molar ratio of the vinyl monomer, the chain transfer agent and the initiator is (1-100): 1:0.5.
Further, the chain transfer agent is at least one of 4-cyano-4- [ (dodecylthiocarbonyl) thio ] pentanoic acid, 4-cyanopentanoic acid dithiobenzoate (CPADB), 2- (dodecyltrithiocarbonate group) -2-methylpropanoic acid (DCMA), 2-cyano-2-propyldodecyltrithiocarbonate, 2-cyano-2-propylbenzodithio, 2- (dodecylthiocarbonylthiocarbonylthio) propanoic acid, 4- ((((2-carboxyethyl) thio) thiocarbonyl) thio) -4-cyanopentanoic acid, 4-cyano-4- [ (dodecylthiocarbamic) thio) sulfonamide ] pentanol, cyanomethyldodecyl trithiocarbonate.
Further, the initiator is Azobisisobutyronitrile (AIBN), 4' -azobis (4-cyanovaleric acid) (ACVA) or benzoyl peroxide.
Further, the temperature of the reversible addition fragmentation chain transfer radical polymer reaction is 50-100 ℃.
The second object of the invention can be achieved by adopting the following technical scheme: the preparation method of the porous microcarrier comprises the steps of carrying out cross-linking reaction on hyperbranched polymer and natural biological material, dividing the hyperbranched polymer into micron-sized spherical particles through a microfluidic technology, and carrying out treatment through a pore-forming technology to obtain the porous microcarrier.
The third object of the invention can be achieved by adopting the following technical scheme: the use of a porous microcarrier as described above for the cultivation of stem cells or for the preparation of a medicament.
Compared with the prior art, the invention has the beneficial effects that:
1. the porous microcarrier is a hydrogel system, and has good biocompatibility and mechanical property;
2. the preparation method of the porous microcarrier is simple and efficient, is easy to control and has good stability;
3. the porous microcarrier has wide application, and can be used for stem cell culture, medicine preparation, protein purification, delivery and the like.
Drawings
FIG. 1 is a schematic diagram of the synthesis of hyperbranched polyacrylamide of example 1;
FIG. 2 is a gel permeation chromatograph of hyperbranched polyacrylamide;
FIGS. 3-4 are optical microscope pictures of microporous supports;
FIGS. 5-6 are electron microscope pictures of microporous supports;
FIGS. 7-8 are fluorescence micrographs of iMSCs;
FIGS. 9-10 are activity diagrams of iMSCs.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description below:
A porous microcarrier comprising a hyperbranched polymer and a natural biological material that are cross-linked to each other, the hyperbranched polymer comprising hyperbranched polyacrylate or hyperbranched polyacrylamide; the mass ratio of the hyperbranched polymer to the natural biological material is (0.2-5): 1; natural biological materials include: at least one of gelatin, gelatin derivatives, hyaluronic acid, collagen, glycoprotein, laminin, fibronectin, alginate and alginate derivatives.
Wherein the hyperbranched polymer is prepared by the following method:
Dissolving a polymer monomer and a chain transfer agent in a solvent, and deoxidizing to obtain a mixed solution; under the condition of introducing argon, adding an initiator into the mixed solution to obtain a reaction mixed system; the reaction mixed system generates reversible addition fragmentation chain transfer free radical polymer reaction under the conditions of no oxygen and 50-100 ℃, no metal and ligand compound exist in the reaction process, good biocompatibility is realized, good controllable polymerization can be realized at the temperature, and the required polymer structure is easy to obtain; monitoring the molecular weight of the polymer by using gel permeation chromatography, stopping the reaction when the molecular weight of the reaction reaches a set value of 10000-100000g/mol, wherein the molecular weight of the polymer is easy to control in the range; then, diethyl ether and normal hexane are used for 3 times of precipitation and purification treatment to obtain hyperbranched polymer; the molar ratio of the polymer monomer, chain transfer agent and initiator is (1-100): 1:0.5, in which case hyperbranched polymers having different branch lengths and degrees of branching can be readily designed.
The polymer monomers include acrylate monomers and acrylamide monomers;
The acrylate monomer comprises: at least one of polyethylene glycol diacrylate, diethylene glycol dimethacrylate, tetraethylene glycol diacrylate, butylene glycol diacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, methoxypolyethylene glycol acrylate, hydroxypropyl methacrylate, trimethylolpropane triacrylate, glycidyl methacrylate, methyl acrylate, isocyanoethyl methacrylate, 2- (dimethylamino) ethyl acrylate, 2-methyl-2- (2-methoxyethoxy) ethyl acrylate, hydroxyethyl acrylate, disulfide acrylate, and 2-1, 10-decanediyl 2-acrylate; the acrylamide monomer comprises: at least one of acrylamide, N-dimethyl bisacrylamide, and N, N' -bis (propionyl) cystamine.
The chain transfer agent is at least one of 4-cyano-4- [ (dodecylthiocarbonyl) thio ] pentanoic acid, 4-cyanopentanoic acid dithiobenzoate (CPADB), 2- (dodecyltrithiocarbonate) -2-methylpropanoic acid (DCMA), 2-cyano-2-propyldodecyltrithiocarbonate, 2-cyano-2-propylbenzodithio, 2- (dodecylthiocarbonylthiocarbonylthio) propanoic acid, 4- ((((2-carboxyethyl) thio) thiocarbonyl) thio) -4-cyanopentanoic acid, 4-cyano-4- [ (dodecylthiocarbamic) thio) sulfonamide ] pentanol, cyanomethyldodecyl trithiocarbonate;
the initiator is Azodiisobutyronitrile (AIBN), 4' -azobis (4-cyanovaleric acid) (ACVA) or benzoyl peroxide;
The solvent is 2-butanone.
The hyperbranched polyethylene glycol obtained by the method contains a large number of unreacted double bonds, can be modified and modified by a plurality of methods such as Michael addition, click chemistry and the like, and can further improve the function of the polymer, so that the adjustable porous microcarrier can be prepared, and the application field of the porous microcarrier is expanded.
Preparing a porous microcarrier by using the obtained hyperbranched polymer, wherein the preparation method comprises the following steps:
The hyperbranched polymer and the natural biological material are subjected to crosslinking reaction, the hyperbranched polymer and the natural biological material are divided into micron-sized spherical particles through a microfluidic technology, and then the micron-sized spherical particles are treated through a pore-forming technology, so that the porous microcarrier is obtained.
The microfluidic technology is to use a microfluidic device, wherein the microfluidic device is a microchip with dispersed flow and continuous flow, and a micron-sized carrier is designed by adjusting the speed ratio of two-phase flow; the dispersion flow is aqueous phase solution containing hyperbranched polyethylene glycol and natural biological material, and the continuous flow is organic phase solution containing organic solvent and surfactant.
The porous microcarrier can be applied to stem cell culture, including but not limited to cell growth, proliferation, encapsulation, etc., can also be applied to drug preparation, and can also be applied to regenerative medicine research, including but not limited to cell proliferation, cell encapsulation, etc. The porous microcarrier has good biocompatibility and mechanical property, and solves the problems that the existing microcarrier on the market has complex process, single function and particles exist after degradation so as to influence the cell quality. Polystyrene-based microcarriers used in the prior art have the advantages of strong mechanical properties, easy sterilization and approved for the production of clinically desirable cells, but they are not degradable, require filtration and other manipulations, and can affect yield and viability during purification; degradable microcarriers have good biocompatibility, but cannot be modified for microcarrier surface properties including elastic modulus, surface cell adhesion ligands, etc.
Example 1:
Preparing hyperbranched polyacrylamide:
4.5mmol of N, N-dimethyl bisacrylamide (DMAA, the molecular weight of which is 154 g/mol) and 0.5mmol of chain transfer agent 2- (dodecyl trithiocarbonate group) -2-methylpropanoic acid are added into a three-mouth bottle filled with 50mL of 2-butanone, the monomers are fully dissolved through magnetic stirring, and a mixed solution is obtained after deoxidizing for 30min by using a nitrogen bubbling method; under the condition of introducing argon, 0.25mmol of initiator 4,4' -azobis (4-cyano valeric acid) (ACVA) is added into the mixed solution to obtain a reaction mixed system; continuously introducing nitrogen for 5min, allowing the reaction mixed system to react for 10h under the conditions of no oxygen and 55 ℃ by reversible addition fragmentation chain transfer free radical polymer, monitoring the molecular weight of the polymer by using gel permeation chromatography, and stopping the reaction when the molecular weight of the reaction reaches a set value of 10000-100000 g/mol; then, diethyl ether is used for 3 times of precipitation and purification treatment, so that hyperbranched polyacrylamide with the molecular weight of 50000g/mol and p=200 and m=150 is obtained, and a synthetic schematic diagram of the hyperbranched polyacrylamide is shown in fig. 1.
Example 2:
The preparation method of the porous microcarrier comprises the following steps:
Respectively dissolving 20mg of hyperbranched polyacrylamide and 50mg of hyaluronic acid obtained in the example 1 in 1mL of Phosphate Buffer Solution (PBS) to prepare a disperse phase (aqueous phase), injecting different phases (the disperse phase and the continuous phase) into a microfluidic chip of a microfluidic device at a controlled flow rate by using a syringe pump, forming W/O emulsion in the microchip at a chemical crosslinking reaction temperature of 25-37 ℃ for 2-5min, regulating the flow rate ratio of the disperse phase to the continuous phase, forming uniform liquid drops by emulsifying the continuous phase, collecting and solidifying the liquid drops for at least 24h to enable the liquid drops to be fully crosslinked to form micron-sized spherical particles; and then the porous microcarrier is obtained by processing through a pore-forming process solvent evaporation method.
Example 3:
The preparation method of the porous microcarrier comprises the following steps:
Respectively dissolving 10mg of hyperbranched polyacrylamide and 20mg of hyaluronic acid obtained in the example 1 in 1mL of Phosphate Buffer Solution (PBS) to prepare a disperse phase (aqueous phase), injecting different phases (the disperse phase and the continuous phase) into a microfluidic chip of a microfluidic device at a controlled flow rate by using a syringe pump, forming W/O emulsion in the microchip at a chemical crosslinking reaction temperature of 25-37 ℃ for 5-10min, regulating the flow rate ratio of the disperse phase to the continuous phase, forming uniform liquid drops by emulsifying the continuous phase, collecting and solidifying the liquid drops for at least more than 24h to enable the liquid drops to be fully crosslinked to form micron-sized spherical particles; and then the porous microcarrier is obtained by processing through a pore-forming process solvent evaporation method.
From examples 1-3, it is known that hyperbranched polyacrylamides have good controllability and adjustability, and that by varying the ratio of N, N-dimethyl bisacrylamide, chain transfer agent and initiator, the molecular weight and branching degree of the hyperbranched polyacrylamides obtained are different, so that hyperbranched polyacrylamides of different compositions and branching degrees are obtained.
FIG. 2 is a gel permeation chromatogram of hyperbranched polyacrylamide of example 1.
The porous microcarrier has adjustable chemical composition and mechanical property, in particular to a porous microcarrier with different structures and properties, which is obtained by changing the cross-linking proportion of hyperbranched polyacrylamide and hyaluronic acid by changing the molecular weight and branching degree of hyperbranched polyacrylamide. FIGS. 3-4 are optical microscope pictures of microporous supports of examples 2-3, respectively, forming uniformly sized micron-sized spherical particles, having a size of 100-300 μm; FIGS. 5-6 are electron microscope pictures of microporous carriers of examples 2-3, respectively, and the microporous carriers formed have larger pore diameters and good connectivity, and the pore diameters are different from 3-8 μm.
The porous microcarriers were placed in a humidified incubator at 37 ℃ and 5% v/v CO 2 for at least 1h incubation in culture medium, after which they were cultured and expanded in admixture with induced mesenchymal stem cells iMSCs. FIGS. 7-8 are fluorescence micrographs of culture iMSCs using the porous microcarriers of examples 2-3, respectively, showing good cell adhesion and biocompatibility. FIGS. 9-10 are graphs showing that iMSCs shows higher survival and amplification performance by the porous microcarrier cultures iMSCs of examples 2-3.
Various other corresponding changes and modifications will occur to those skilled in the art from the foregoing description and the accompanying drawings, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Claims (8)

1. The preparation method of the porous microcarrier is characterized in that hyperbranched polyacrylamide and hyaluronic acid are subjected to a crosslinking reaction, the hyperbranched polyacrylamide and hyaluronic acid are divided into micron-sized spherical particles through a microfluidic technology, and then the micron-sized spherical particles are treated through a pore-forming technology to obtain the porous microcarrier;
The hyperbranched polyacrylamide is prepared by the following steps:
Dissolving an acrylamide monomer and a chain transfer agent in a solvent, and deoxidizing to obtain a mixed solution; under the condition of introducing argon, adding an initiator into the mixed solution to obtain a reaction mixed system; the reaction mixed system generates reversible addition fragmentation chain transfer free radical polymer reaction under the anaerobic condition, gel permeation chromatography is used for monitoring the molecular weight of the polymer, when the reaction molecular weight reaches a set value of 10000-100000g/mol, the reaction is stopped, and then purification treatment is carried out to obtain hyperbranched polyacrylamide;
the acrylamide monomer comprises: at least one of acrylamide, N-dimethyl bisacrylamide, and N, N' -bis (propionyl) cystamine;
the mass ratio of the hyperbranched polyacrylamide to the hyaluronic acid is (0.2-5) 1.
2. The method of preparing a porous microcarrier according to claim 1, wherein the reaction molar ratio of acrylamide monomer, chain transfer agent and initiator is (1-100): 1:0.5.
3. The method of preparing a porous microcarrier of claim 1, wherein the chain transfer agent is at least one of 4-cyano-4- [ (dodecylthiocarbonyl) thio ] pentanoic acid, 4-cyanopentanoic acid dithiobenzoate, 2- (dodecyltrithiocarbonate group) -2-methylpropanoic acid, 2-cyano-2-propyldodecyltrithiocarbonate, 2-cyano-2-propylbenzodithio, 2- (dodecylthiocarbonylthiochio) propanoic acid, 4- ((((2-carboxyethyl) thio) thiocarbonyl) thio) -4-cyanopentanoic acid, 4-cyano-4- [ (dodecylthiochiocarbonyl) sulfonamide ] pentanoic acid, and cyanomethyldodecyltrithiocarbonate.
4. The method of preparing a porous microcarrier of claim 1, wherein the initiator is azobisisobutyronitrile, 4' -azobis (4-cyanovaleric acid), or benzoyl peroxide.
5. The method of preparing a porous microcarrier of claim 1, wherein the reversible addition fragmentation chain transfer free radical polymer reaction temperature is 50-100 ℃.
6. A porous microcarrier, characterized in that it is obtained by the preparation method according to claim 1.
7. The use of a porous microcarrier according to claim 6, wherein the porous microcarrier is used in stem cell culture.
8. Use of a porous microcarrier according to claim 6, wherein the porous microcarrier is used in the preparation of a medicament.
CN202310221076.6A 2023-03-09 2023-03-09 Porous microcarrier, preparation method and application thereof Active CN116162290B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310221076.6A CN116162290B (en) 2023-03-09 2023-03-09 Porous microcarrier, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310221076.6A CN116162290B (en) 2023-03-09 2023-03-09 Porous microcarrier, preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN116162290A CN116162290A (en) 2023-05-26
CN116162290B true CN116162290B (en) 2024-05-28

Family

ID=86420033

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310221076.6A Active CN116162290B (en) 2023-03-09 2023-03-09 Porous microcarrier, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN116162290B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102229690A (en) * 2011-05-17 2011-11-02 浙江大学 Method for preparing starch grafted hyperbranched amide polymer
CN102492092A (en) * 2011-12-21 2012-06-13 浙江大学 Method for preparing hyperbranched ionic amide type polymer by inverse emulsion polymerization
CN104128136A (en) * 2013-05-03 2014-11-05 中国科学院化学研究所 Micro-nano gel particle, controllable preparation method and application thereof
CN109666109A (en) * 2017-10-16 2019-04-23 天津大学 Polyethylene glycol-glycidyl methacrylate dissaving polymer and preparation method thereof of epoxy group modification
CN109666107A (en) * 2017-10-16 2019-04-23 天津大学 Polyethylene glycol -3- methacryl dopamine the dissaving polymer and preparation method thereof of catechol modification
CN110156925A (en) * 2019-05-13 2019-08-23 浙江大学 A kind of preparation method of water-dispersed polyacrylamide polymer
CN112480417A (en) * 2019-09-11 2021-03-12 天津大学 Natural polymer-based injectable hydrogel and preparation method thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102229690A (en) * 2011-05-17 2011-11-02 浙江大学 Method for preparing starch grafted hyperbranched amide polymer
CN102492092A (en) * 2011-12-21 2012-06-13 浙江大学 Method for preparing hyperbranched ionic amide type polymer by inverse emulsion polymerization
CN104128136A (en) * 2013-05-03 2014-11-05 中国科学院化学研究所 Micro-nano gel particle, controllable preparation method and application thereof
CN109666109A (en) * 2017-10-16 2019-04-23 天津大学 Polyethylene glycol-glycidyl methacrylate dissaving polymer and preparation method thereof of epoxy group modification
CN109666107A (en) * 2017-10-16 2019-04-23 天津大学 Polyethylene glycol -3- methacryl dopamine the dissaving polymer and preparation method thereof of catechol modification
CN110156925A (en) * 2019-05-13 2019-08-23 浙江大学 A kind of preparation method of water-dispersed polyacrylamide polymer
CN112480417A (en) * 2019-09-11 2021-03-12 天津大学 Natural polymer-based injectable hydrogel and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A rapid crosslinking injectable hydrogel for stem cell delivery, from multifunctional hyperbranched polymers via RAFT homopolymerization of PEGDA;A rapid crosslinking injectable hydrogel for stem cell delivery, from multifunctional hyperbranched polymers via RAFT homopolymerization of PEGDA;Polymer Chemistry;20150630;6182-6192 *
Andre R. Studart.Hierarchical Porous Materials Made by Drying Complex Suspensions.2011,955-964. *
Fabrication of Microbeads with a Controllable Hollow Interior and Porous Wall Using a Capillary Fluidic Device;Sung-Wook Choi;Advanced Functional Mterials;20091231;2943-2949 *
Thermoresponsive hyperbranched copolymer with multi acrylatefunctionality for in situ cross-linkable hyaluronic acid composite semi-IPN hydrogel;Yixiao Dong;J Mater Sci;20111206;25–35 *

Also Published As

Publication number Publication date
CN116162290A (en) 2023-05-26

Similar Documents

Publication Publication Date Title
Gan et al. Thermogelable PNIPAM microgel dispersion as 3D cell scaffold: effect of syneresis
US6897064B2 (en) Cell or tissue-culturing carrier, and culturing method
EP2612902B1 (en) Temperature-responsive substrate for cell culture and method for producing same
JP7617604B2 (en) Microgel particles and related compositions, systems, and methods for use in 3D printing and 3D cell culture media
CN103409361A (en) Thermosensitive microcarrier as well as preparation technology and application method thereof
CN113577381A (en) Injectable cartilage constructed based on microgel scaffold material and application thereof
JP6375358B2 (en) Cell culture composition and cell culture vessel
TWI673103B (en) Injectable self-assembling microbead-gel, use thereof, and method for preparing injectable self-assembling microbead-gel
Wissemann et al. Pure gelatin microcarriers: synthesis and use in cell attachment and growth of fibroblast and endothelial cells
CN102161728A (en) Semi-interpenetrating-network nano composite hydrogel with biocompatibility and temperature sensitivity, and preparation method and application thereof
WO2014143871A2 (en) Thermoresponsive polymer applications for adherent cell culture and recovery
CN119350663B (en) Composite hydrogel microsphere with excellent cell adhesion performance, preparation method and application thereof
CN117467157A (en) Porous hydrogel microcarrier for 3D cell culture and preparation method and application thereof
CN116162290B (en) Porous microcarrier, preparation method and application thereof
US5173421A (en) Cell culture carriers
JP2013059312A (en) Bead for culturing temperature responsive cell, and method of manufacturing the same
CN115804758A (en) Method for preparing porous stem cell microcarrier, porous stem cell microcarrier prepared by the method and application thereof
JPH0833486A (en) Cell culture carrier and cell culture method
CN113201525B (en) Stem cell microsphere group, stem cell in-vitro amplification method and application
CN115369072A (en) Hydrogel fiber for realizing formation and passage of multicellular spheroids and preparation method thereof
Zhou et al. Microspheres for cell culture
Yang et al. In vitro study of the interactions of galactosylated thermo-responsive hydrogels with cells
Cer et al. Polyethylene glycol‐based cationically charged hydrogel beads as a new microcarrier for cell culture
US20040248290A1 (en) Culture systems for the sterile continuous cultivation of cells
JPWO2004078961A1 (en) Floating carrier and floating / collecting method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant