阅读说明：本技术 一种含有二硫键的双重响应功能分子、水凝胶微球的制备方法及应用 (Preparation method and application of disulfide bond-containing dual-response functional molecule and hydrogel microsphere ) 是由 包春燕 薛源 汪晨曦 周耀武 王学斌 杨会婷 项艳鑫 冯梦婷 朱麟勇 于 2020-06-18 设计创作，主要内容包括：本发明公开了一种含有二硫键的双重响应功能分子,结构通式如下所示：<Image he="129" wi="635" file="DDA0002545042120000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明还提供了一种由含有二硫键双重响应功能分子制备的双重响应水凝胶微球,直径可在50-500μm之间调控。本发明还提供了一种所述双重响应水凝胶微球作为细胞培养载体,能够用于粘附型细胞的黏附、扩增和非酶收获,实现在大规模培养细胞中的应用。(The invention discloses a double-response functional molecule containing a disulfide bond, which has the following structural general formula: the invention also provides a preparation method of the double-response functional molecule containing the disulfide bondThe diameter of the dual-response hydrogel microsphere can be regulated and controlled between 50 and 500 mu m. The invention also provides a dual-response hydrogel microsphere as a cell culture carrier, which can be used for adhesion, amplification and non-enzymatic harvesting of adhesion cells and realizes application in large-scale cell culture.)

1. A dual response functional molecule containing disulfide bonds, characterized by the following general structural formula:

wherein X, Y are each independently selected from one of the following structures: alkylene, -O (CH)₂)_m-、-(CH₂)_mO(CH₂)_n-、-(CH₂CH₂O)_m-、-(CH₂)_m(CH₂CH₂O)_n-、-(CH₂)_m(CH₂CH₂O)_nO-、-(CH₂)_m(OCH₂CH₂)_nO-、-COO(CH₂)_m-、-OCO(CH₂)_m-、-(CH₂)_mCOO(CH₂)_n-、-(CH₂)_mOCO(CH₂)_n-、-CO₃(CH₂)_m-、-(CH₂)_mCO₃(CH₂)_n-、-NHCO(CH₂)_m-、-CONH(CH₂)_m-、-(CH₂)_mNHCO(CH₂)_m-、-(CH₂)_mCONH(CH₂)_n-、-NHCONH(CH₂)_m-、-(CH₂)_mNHCONH(CH₂)_n-、-(CH₂)_mNHCO(CH₂)_nO-；

X, Y may be the same or different;

m is more than or equal to 0, n is more than or equal to 0; m and n are integers;

R₁one selected from the following structures: acrylate, methacrylate, thiol, amino, carboxyl, azido, styryl, propynyl, maleimido, succinimidyl, tetrazoyl, vinyl, carboxaldehyde, CH₂CHCONH-、CH₂CHCH₂CONH-, C4-C18 aryl, C4-C18 aryl containing at least one hydroxyl group, alkyl or nitro group, C1-C18 alkyl substituted or unsubstituted C2-C8 heterocyclic group and ether group containing C2-C8 heterocyclic group;

R₂one selected from the following structures:

R₃、R₄、R₅、R₆、R₇each independently selected from one of the following structures: hydrogen, halogen, hydroxyl, sulfydryl, amino, nitro, cyano, formaldehyde, ketone, ester, amide, phosphate, sulfonic acid, sulfonate, sulfone, sulfoxide, alkyl and alkoxy.

2. The disulfide bond-containing dual response functional molecule of claim 1, wherein R is represented by the general formula₁One selected from the following structures:

x is selected from the following structures:

y is selected from one of the following structures:

x, Y, n5, n6 and n7 are all integers of 1 to 8,

R₂is one of the following structures:

3. the disulfide bond-containing dual response functional molecule of claim 2, wherein said disulfide bond-containing dual response functional molecule is one of the following structures:

wherein n5, n6 and n7 are integers of 1 to 8.

4. The disulfide bond-containing dual response functional molecule of claim 3, wherein said disulfide bond-containing dual response functional molecule is one of the following structures:

5. a method for preparing hydrogel microspheres containing the dual response functional molecules of any one of claims 1 to 4, comprising the steps of:

dissolving 2-40 mg of dual response functional molecules into 0.1-1000 mL of deionized water, adding 20-400 mg of gelling components, and uniformly mixing to obtain 0.1-30% of hydrogel precursor solution containing 0.0001-0.1 mM of dual response functional molecules;

or dissolving 2-40 mg of water-soluble macromolecules marked with dual response functional molecules into 0.1-1000 mL of deionized water, and uniformly mixing to obtain 0.1-30% of hydrogel precursor solution containing 0.0001-0.1 mM of dual response functional molecules;

in nitrogen atmosphere, adding an initiator or an initiation aid, mixing uniformly in a vortex manner, dripping the mixture into paraffin oil containing span 80 for reaction, and filtering and dialyzing the polymerized microspheres to obtain the hydrogel microspheres containing the dual-response functional molecules.

6. The method for preparing hydrogel microspheres with dual response functional molecules as claimed in claim 5, wherein the gel-forming component is at least one of water-soluble ethylene, propylene monomers and polymerizable water-soluble macromolecules;

the water-soluble macromolecule marked with the dual-response functional molecule is a transparent acid water-soluble macromolecule marked with the dual-response functional molecule.

7. The method for preparing hydrogel microspheres with dual response functional molecules according to claim 5, wherein the initiator or the initiation aid is at least one of potassium persulfate, ammonium persulfate and tetramethylethylenediamine;

the volume ratio of the span 80 in the paraffin oil is 0.1-1%;

the particle size of the hydrogel microsphere containing the dual response functional molecules is controlled to be 50-500 mu m.

8. Use of hydrogel microspheres containing dual response functional molecules prepared by the method of claims 5 to 7 in large scale culture of adherent cells.

9. The use of hydrogel microspheres comprising dual response functional molecules according to claim 8 in the large scale culture of adherent cells, wherein said use comprises the steps of:

the surface of the hydrogel microsphere containing the dual-response functional molecules is covalently fixed with bioactive factors through illumination, cells are adhered to the surface of the hydrogel microsphere through the bioactive factors and realize large-scale amplification, and the cells are subjected to enzyme-free mild release from the surface of the hydrogel microsphere through the addition of a reducing agent, so that the large-scale cell culture and capture are realized.

10. The use of hydrogel microspheres comprising dual response functional molecules according to claim 9 in the large scale culture of adherent cells,

the surface of the hydrogel microsphere containing the dual-response functional molecules is covalently immobilized with bioactive factors through illumination: quantitatively illuminating the hydrogel microspheres containing the molecules with the dual-response function, soaking the illuminated hydrogel microspheres containing the molecules with the dual-response function in a solution in which bioactive factors are dissolved, taking out the material after soaking, and washing or replacing the material with a buffer solution to remove the bioactive molecules which are combined on the surface of the material in a non-covalent manner, thereby obtaining the hydrogel microspheres containing the molecules with the dual-response function and fixed with the bioactive factors;

the cells are adhered to the surfaces of the hydrogel microspheres through the bioactive factors and realize large-scale amplification, the hydrogel microspheres containing double-response functional molecules and fixing the bioactive factors are placed in a serum-free DMEM medium for balancing, the balanced hydrogel microspheres are added into a 24-anti-adhesion pore plate, and dispersed adhesive HUVEC cells are added to ensure that the number of the cells in the system is 2 × 10⁴Culturing in DMEM high-sugar medium, 10% fetal calf serum, 1% double antibody, 5% CO at 37 deg.C₂The method is characterized by comprising the following steps of (1) performing an aseptic culture environment, wherein cell experiments are performed under an aseptic condition;

the addition of the reducing agent enables cells to obtain enzyme-free mild release from the surfaces of hydrogel microspheres: CO-culturing reducing culture medium and hydrogel microsphere with cell adhered to surface and containing dual response functional molecules at 37 deg.C and 5% CO₂(ii) a The released cells are collected and cultured with fresh cell growth medium or frozen for subsequent use.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a disulfide bond-containing dual-response functional molecule, a dual-response hydrogel microsphere and application of the disulfide bond-containing dual-response functional molecule as a cell carrier in large-scale cell culture.

Background

With the continuous development of the biomedical fields such as cell biology, biochemical preparations, tissue engineering and the like, the demand for cell matrixes is more and more great, and the development of a large-scale cell culture technology is urgently needed. The traditional laboratory cell culture methods have various forms and can achieve better experimental culture purposes, but have many disadvantages when applied to industrial cell culture. For example, in the roller culture of cells, the cells can only adhere to one layer on the inner wall of the roller, and the area of the roller wall greatly limits the culture scale of the cells. Meanwhile, the separation method of the cells uses enzyme digestion or mechanical separation, and dryness loss of the passaged cells is easily caused. In particular, enzymatic digestion, which causes cell shedding by removing surface proteins and ions required for cell adhesion, is relatively damaging to cells (Nikul G.Patel., at el. (2012) Acta biomaterials 8(7): 2559-2567). Therefore, in order to improve the large-scale culture of cells, improvements in cell culture materials and separation methods are required.

In response to the disadvantages of two-dimensional cell culture, three-dimensional cell culture provides a larger culture space, which makes cells more like in vivoAs well as in three dimensions. Hydrogels are considered the most competitive cell scaffold material due to their high water content and microstructure similar to the extracellular matrix. But limited by oxygen diffusion (max 200 microns, De Vos p., et al (2012). adv. drug deliv. rev.67-68C: 15-34), the hydrogel interior is often unsuitable for large scale cell culture due to low porosity. Therefore, hydrogel microsphere surface cell culture technology is produced. The microsphere technology can simulate a 3D cell culture environment, has high specific surface area, provides adhesion sites for cells to realize large-scale cell culture, and enables the cell adhesion rate to be as high as 10⁸one/mL (Peng L., et al. (2011). Hepatology 54(3): 820-828). Temperature-sensitive poly-N-isopropylacrylamide (PNIPAAM) hydrogel microspheres are by far the most commonly used culture material for cell adhesion and isolation (a method for large-scale culture of stem cells, published as CN 104894063A; tamura a. et al biomaterials,2012,33,3803-. By utilizing the characteristic of temperature-sensitive reversible phase transition, cell adhesion can be realized by hydrophobic effect above the phase transition critical temperature, and then cell desorption can be realized by hydrophilic transition when the temperature is reduced to be below the phase transition critical temperature. Although the materials can realize cell adhesion and cell detachment only through temperature regulation without the action of enzyme, in order to meet the survival temperature of cells, the phase transition temperature of the materials needs to be strictly regulated. Therefore, a microgel material with stimulation response is urgently needed to realize large-scale cell culture so as to make up for the defects of the temperature-sensitive microgel technology.

Mild reduction and photostimulation response have been widely applied to the functionalization of biological materials. Therefore, the property of the surface of the hydrogel microsphere is regulated and controlled by utilizing the mild stimulation of light and reduction, so that the adhesion, growth and detachment of cells can be realized, and the possibility is provided for the large-scale culture of three-dimensional cells.

Disclosure of Invention

It is a first object of the present invention to provide a dual response functional molecule containing disulfide bonds and photoreactive groups.

The second purpose of the invention is to provide a preparation method of the hydrogel microsphere containing the molecule with the double response function, the prepared hydrogel microsphere with the double response function can regulate and control the adhesion and the separation of cells on the surface of the hydrogel microsphere by utilizing the double responsiveness of light and reduction, and the material can simulate the extracellular matrix to mediate the adhesion and the separation of the cells by regulating and controlling the adsorption and the dissociation of adhesion protein so as to solve the technical problem of cell damage caused by enzyme digestion in the existing cell separation process.

The third purpose of the invention is to provide the application of the hydrogel microspheres containing the dual-response functional molecules in large-scale culture of adherent cells.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a double-response functional molecule containing disulfide bonds, which has the following structural general formula:

wherein X, Y are each independently selected from one of the following structures: alkylene, -O (CH)₂)_m-、-(CH₂)_mO(CH₂)_n-、-(CH₂CH₂O)_m-、-(CH₂)_m(CH₂CH₂O)_n-、-(CH₂)_m(CH₂CH₂O)_nO-、-(CH₂)_m(OCH₂CH₂)_nO-、-COO(CH₂)_m-、-OCO(CH₂)_m-、-(CH₂)_mCOO(CH₂)_n-、-(CH₂)_mOCO(CH₂)_n-、-CO₃(CH₂)_m-、-(CH₂)_mCO₃(CH₂)_n-、-NHCO(CH₂)_m-、-CONH(CH₂)_m-、-(CH₂)_mNHCO(CH₂)_m-、-(CH₂)_mCONH(CH₂)_n-、-NHCONH(CH₂)_m-、-(CH₂)_mNHCONH(CH₂)_n-、-(CH₂)_mNHCO(CH₂)_nO-；

X, Y may be the same or different;

m is more than or equal to 0, n is more than or equal to 0; m and n are integers;

R₁is a functional group which can chemically react with the hydrogel microsphere component and is selected from one of the following structures: acrylate, methacrylate, thiol, amino, carboxyl, azido, styryl, propynyl, maleimido, succinimidyl, tetrazoyl, vinyl, carboxaldehyde, CH₂CHCONH-、CH₂CHCH₂CONH-, C4-C18 aryl, C4-C18 aryl containing at least one hydroxyl group, alkyl or nitro group, C1-C18 alkyl substituted or unsubstituted C2-C8 heterocyclic group and ether group containing C2-C8 heterocyclic group;

R₂is a photoreactive group that can be covalently bonded to a biologically active factor and is selected from one of the following structures:

R₃、R₄、R₅、R₆、R₇each independently selected from one of the following structures: hydrogen, halogen, hydroxyl, sulfhydryl, amino, nitro, cyano, formaldehyde, keto (such as-COCH)₃) Ester group (e.g.: -COOCH₃) Amide groups (e.g.: -CONHCH₃) Phosphoric acid groups, phosphoric ester groups (such as: -PO₄(CH₃)₂) Sulfonic acid group (-SO)₃H) Sulfonate groups (e.g.: -SO₂OCH₃) Sulfone group (e.g.: -SO₂CH₃) Sulfoxide groups (e.g.: -SOCH₃) Alkyl, alkoxy.

In the definitions of the formulae given above, the terms used are generally defined as follows:

the term alkylene is a radical having from 1 to 20 carbon atomsSaturated or unsaturated aliphatic branched or branched alkylene radicals of radicals, e.g. CH₂-、-CH₂CH₂-and the like.

The term C4-C18 aryl refers to mono-, di-, or tricyclic hydrocarbon compounds in which at least one ring is aromatic and each ring contains up to 7 carbon atoms, e.g., phenyl, and the like.

The term C4-C18 aryl having at least one hydroxyl, alkyl or nitro group means an aryl group in which at least one hydrogen atom of the aryl group is replaced by a hydroxyl, alkyl or nitro group, e.g.

The term C1-C18 alkyl substituted C2-C18 heterocyclyl; such as 2-methyloxirane, 2-methylfuryl, and the like.

The term unsubstituted C2-C18 heterocyclyl is intended to include ethylene oxide, furyl, and the like.

The term ether containing a C2-C8 heterocyclic group such as

The term halogen means F, Cl, Br, I.

The term alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon group containing 1 to 18 carbon atoms, for example: methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and the like.

The term alkoxy refers to a group having an oxygen atom attached to the terminal of an alkyl group containing 1 to 18 carbon atoms, for example: methoxy, ethoxy, n-propoxy, isopropoxy, and the like.

Preferably, in the general formula of the disulfide bond-containing dual-response functional molecule, R is₁One selected from the following structures:

x is selected from the following structures:

y is selected from one of the following structures:

x, Y, n5, n6 and n7 are all integers of 1-8, n5 is preferably 2, n6 is preferably 2, and n7 is preferably 1 or 2;

R₂is one of the following structures:

more preferably, the disulfide bond containing dual response functional molecule is one of the following structures:

wherein n5, n6 and n7 are integers of 1 to 8.

Most preferably, the disulfide bond containing dual response functional molecule is one of the following structures:

the second aspect of the invention provides a preparation method of hydrogel microspheres containing the dual-response functional molecules, which comprises the following steps:

dissolving 2-40 mg of dual response functional molecules into 0.1-1000 mL of deionized water, adding 20-400 mg of gelling components, and uniformly mixing to obtain 0.1-30% of hydrogel precursor solution containing 0.0001-0.1 mM of dual response functional molecules;

or dissolving 2-40 mg of water-soluble macromolecules marked with dual response functional molecules into 0.1-1000 mL of deionized water, and uniformly mixing to obtain 0.1-30% of hydrogel precursor solution containing 0.0001-0.1 mM of dual response functional molecules;

in nitrogen atmosphere, adding an initiator or an initiation aid, mixing uniformly in a vortex manner, dripping the mixture into paraffin oil containing span 80 for reaction, and filtering and dialyzing the polymerized microspheres to obtain the hydrogel microspheres containing the dual-response functional molecules.

The gel-forming component is at least one of water-soluble ethylene, propylene monomers and polymerizable water-soluble macromolecules; preferably at least one or a mixture of polyethylene glycol methacrylate (PEGMA), hydroxyethyl methacrylate (HEMA) and polyethylene glycol diacrylate (PEGDA). The polymerizable water-soluble macromolecule is selected from vinyl, acrylate functionalized hyaluronic acid, polyethylene glycol, chitosan, agarose, cellulose, glucan, alginic acid and the like, wherein the labeling rate of the vinyl and the acrylate is 5-90%.

The water-soluble macromolecule marked with the dual-response functional molecule is transparent acid water-soluble macromolecule (HANB) marked with the dual-response functional molecule.

The preparation method of the transparent acid water-soluble macromolecule (HANB) marked with the dual response functional molecule comprises the following steps: mixing double-response functional molecules containing disulfide bonds, acrylate functionalized hyaluronic acid (HAMA), N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a molar ratio of 1:1 (1-5), dissolving the mixture in anhydrous dichloromethane at 0 ℃, recovering to room temperature for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain the hyaluronic acid water-soluble macromolecules (HANB) marked with the double-response functional molecules.

The preparation method of the acrylate functionalized hyaluronic acid (HAMA) comprises the following steps:

dissolving a hyaluronic acid compound HA and a methacrylic anhydride compound a21 in a molar ratio of 1:1 in deionized water, dropwise adding a sodium hydroxide solution to keep the pH value of a hyaluronic acid system at 7-9, reacting at 30-50 ℃ for 1-12 h, dialyzing in deionized water, and freeze-drying to obtain the acrylate functionalized hyaluronic acid (HAMA).

The initiator or the initiation aid is at least one of potassium persulfate, ammonium persulfate and tetramethylethylenediamine, and preferably a mixture of ammonium persulfate and tetramethylethylenediamine with a molar ratio of 1: 1.

The volume ratio of the span 80 in the paraffin oil is 0.1-1%.

The volume ratio of the hydrogel precursor solution to the paraffin oil containing span 80 is 0.01-0.4.

The rotating speed of the reaction dropped into the span 80-containing paraffin oil is 100-1000rpm, and the time is 30-120 min.

The hydrogel microsphere containing the dual response functional molecules has the advantages of particle size control of 50-500 mu m, uniform particle size distribution, good monodispersity (the dispersity index PDI is less than 0.1), excellent mechanical property and long-term storage in a refrigerator of-20 ℃.

The third aspect of the invention provides an application of the hydrogel microsphere containing the dual response functional molecule in large-scale culture of adhesion type cells.

The application comprises the following steps:

the surface of the hydrogel microsphere containing the dual-response functional molecules is covalently fixed with bioactive factors through illumination, cells are adhered to the surface of the hydrogel microsphere through the bioactive factors and realize large-scale amplification, and the cells are subjected to enzyme-free mild release from the surface of the hydrogel microsphere through the addition of a reducing agent, so that the large-scale cell culture and capture are realized.

The bioactive factor can be cell adhesion protein (such as collagen, fibronectin, laminin, vitronectin, gelatin, etc.), polysaccharide (such as chitosan, etc.), polypeptide (such as arginine-glycine-aspartic acid RGD adhesion peptide, etc.), polylysine, antibody, enzyme, cell growth factor, etc. with good biocompatibility. Preferably, the bioactive factor is adhesion protein and peptide capable of performing affinity action with cell surface integrin, such as collagen, gelatin, RGD short peptide, etc.

The reducing agent can be a reducing molecule with good biocompatibility, such as reducing Glutathione (GSH), tris (2-carbonylethyl) phosphate (TCEP), dithiothreitol, cysteine and the like, preferably, the reducing agent is reducing glutathione or tris (2-carbonylethyl) phosphate with high efficiency and high biocompatibility, and particularly, in use, a reducing agent solution is neutralized by using a sodium hydroxide aqueous solution.

The surface of the hydrogel microsphere containing the dual-response functional molecules is obtained by covalently bonding the light-stimulated laser affinity group and the incubated bioactive factor through the illumination covalent fixation of the bioactive factor, and the optimal implementation mode can be realized by adopting the following method:

quantitatively illuminating the hydrogel microspheres containing the molecules with the dual-response function, soaking the illuminated hydrogel microspheres containing the molecules with the dual-response function in a solution in which bioactive factors are dissolved, taking out the material after soaking, and washing or replacing the material with a buffer solution to remove the bioactive molecules which are combined on the surface of the material in a non-covalent manner, thereby obtaining the hydrogel microspheres containing the molecules with the dual-response function and fixed with the bioactive factors.

The buffer solution can be DPBS solution, PBS solution, Tris-buffer solution, CBS solution or deionized water, and the DPBS solution is preferred.

The wavelength of the light source is determined according to the absorption of the selected photoaffinity group, and can be 250-500nm, preferably 300-400nm, and the light intensity is preferably 5-50mW/cm²More preferably 10mW/cm²The irradiation time is 1 to 15 minutes, preferably 2 minutes.

The concentration of the solution dissolved with the bioactive factors is 0.005-0.5mg/mL, and the soaking time is 0.5-30h, preferably 3 h.

The cells are adhered to the surfaces of the hydrogel microspheres through the bioactive factors and realize large-scale amplification, the hydrogel microspheres containing double-response functional molecules and fixing the bioactive factors are placed in a serum-free DMEM medium for balancing, the balanced hydrogel microspheres are added into a 24-anti-adhesion pore plate, and dispersed adhesive HUVEC cells are added to ensure that the number of the cells in the system is 2 × 10⁴Culturing in DMEM high-sugar medium, 10% fetal calf serum, 1% double antibody, 5% CO at 37 deg.C₂The culture environment is sterile, and the cell experiments are carried out under the sterile condition.

In the process of obtaining enzyme-free mild release of cells from the surfaces of hydrogel microspheres by adding the reducing agent, disulfide bonds on the hydrogel microspheres containing the molecules with dual response functions are broken by adding the reducing agent, and then the bioactive factors covalently fixed on the surfaces of the hydrogel microspheres containing the molecules with dual response functions are separated from the surfaces, so that the cells adhered to the surfaces of the hydrogel microspheres containing the molecules with dual response functions are released, and the preferable realization mode of releasing the cells on the surfaces of the hydrogel microspheres containing the molecules with dual response functions stimulated by the reducing agent can be realized by adopting the following method:

CO-culturing reducing culture medium and hydrogel microsphere with cell adhered to surface and containing dual response functional molecules at 37 deg.C and 5% CO₂(ii) a The released cells are collected and cultured with fresh cell growth medium or frozen for subsequent use.

The reducing culture medium is obtained by adding a sterile reducing agent into a serum-free culture medium and adjusting the pH to be neutral; the concentration of the reducing agent is determined according to the added reducing agent, and is preferably 10-60 mg/mL; the incubation time after addition of the reducing agent is determined according to the reducing agent added, and is preferably 120 minutes.

The collection method of the released cells can collect the released cells by centrifugation or a cell filter, and in a preferred embodiment, the collection method of the cells is centrifugation.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the invention takes disulfide bond as a fixed structure of a dual response functional molecule, and introduces a light affinity functional group at one end of the molecule, so that the molecule has response effects on light stimulation and reduction stimulation simultaneously, and introduces a group which can generate chemical reaction with a hydrogel microsphere component at the other end of the molecule, so that the dual response molecule can be modified on the hydrogel forming component or can be copolymerized with the forming component to form the hydrogel microsphere.

The method for culturing the cells on a large scale by using the dual-response hydrogel microspheres can realize the large-scale in-vitro proliferation of the cells and can collect and apply the proliferated cells. The cells collected in large scale by the method of the present invention have high activity and proliferation properties and can be applied to the repair of damaged tissues and organs. The cells collected in large scale by the invention have complete shapes, and the cells with special functions can be cultured in large scale to prepare functional proteins and the like.

The double-response hydrogel microspheres prepared by the invention have good biocompatibility; the cell adhesion mechanism in the extracellular matrix is simulated, the adhesion and the amplification of the cells are realized by connecting the bioactive molecules through light stimulation bonds, the release of the cells is realized by dissociating the bioactive molecules through mild reduction reaction, the whole cell adhesion and release scheme is mild and effective, the cells are harvested without enzyme digestion, and the damage of the proteins on the surfaces of the cells is avoided.

The dual-response hydrogel microspheres prepared by the invention realize three-dimensional cell culture, increase the cell growth space and improve the cell culture efficiency, and the optimal result is that the cell attachment rate is 2 × 10 after 5 days of culture⁴The volume/mL is increased to 3.6-6.8 × 10⁷At one/mL, the amplification was 3400-fold at 1800-. Compared with the traditional temperature-sensitive microsphere culture material, all the operating conditions can be carried out at the cell culture temperature without temperature regulation.

The dual-response hydrogel microsphere prepared by the invention can control cell adhesion and cell detachment, and is prepared by introducing dual-response functional molecules into the hydrogel microsphere. The adsorption of microsphere surface adhesion protein is realized by utilizing a first heavy response light coupling reaction, so that the adhesion and proliferation of cells are mediated; then, the dissociation of the adhesion protein is realized through the second heavy disulfide bond reduction response, so that the cell detachment is mediated; finally, the cell three-dimensional space is cultured on a large scale by simply regulating and controlling the adhesion, proliferation and separation of cells on the surface of the microsphere with large specific surface area. The raw materials adopted by the invention have good biocompatibility, the adhesion and the falling of cells are realized through the adsorption and the dissociation of protein, the pancreatin is avoided, the cell damage is reduced, and the invention has great application value in the fields of large cell culture and tissue repair.

Drawings

FIG. 1 is a schematic representation of hydrogel microspheres containing ANB-1 dual-responsive functional molecules prepared in example 7.

FIG. 2 is a photograph showing hydrogel microspheres having Rho-Gel proteins immobilized on the surfaces thereof in example 9.

FIG. 3 is a photograph showing the hydrogel microspheres obtained after 5 days of cell proliferation culture in example 9, with the fluorescent field on the left and the bright field on the right.

FIG. 4 is a photograph showing the surface of hydrogel microspheres in example 9 after cells are released by GSH reduction.

FIG. 5 is a schematic diagram showing the results of the test of the cell proliferation activity of CCK-8 on the surface of hydrogel microspheres in example 9.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The synthesis of the dual-response functional molecule and the preparation of the hydrogel microsphere are as follows: