CN114796099A - Cell-loaded zwitterionic microgel and preparation method and application thereof - Google Patents
Cell-loaded zwitterionic microgel and preparation method and application thereof Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
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- 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
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
The invention provides a cell-loaded zwitterionic microgel and a preparation method and application thereof. The preparation method provided by the invention is simple, can prepare microgel with uniform size, can keep high survival rate of cells coated in the microgel, and can keep the dryness of stem cells when the stem cells are coated in the microgel.
Description
Technical Field
The invention relates to the technical field of preparation of cell-loaded microgel, in particular to cell-loaded zwitterionic microgel and a preparation method and application thereof.
Background
The microgel is hydrogel with the size of micron grade, has certain mechanical property and a three-dimensional network structure with high water content, can diffuse and exchange nutrient substances, gas and metabolites with the surrounding environment, and is beneficial to the survival of cells loaded in the microgel. Meanwhile, due to the size effect, the microgel can easily pass through the needle head of the syringe and has injectability. In addition, the microgel has larger specific surface area and can improve the interaction of cells and matrixes. The zwitterionic material carries a pair of positive and negative charges on the same molecule and maintains an overall electrically neutral charge. The zwitterionic material has excellent antifouling properties. Since they have opposite charges that can electrostatically induce a surrounding high water layer, thereby creating a high energy hydration barrier to overcome non-specific protein adsorption. These properties have led to the study of zwitterionic-based materials in various fields, particularly in stem cell culture. Hydrogels formed from pure zwitterionic polycarboxybetaines are reported to inhibit foreign body reactions and resist collagen encapsulation when implanted in mice, as well as to protect proteins from immunogenic reactions in the blood. Furthermore, stem cells are encapsulated in zwitterionic polycarboxybetaine gels, and the cells retain their therapeutic pluripotency and avoid non-specific differentiation.
Disclosure of Invention
The invention overcomes the defects in the prior art, and provides the zwitter-ion microgel loaded with cells, a preparation method and an application thereof.
The purpose of the invention is realized by the following technical scheme.
The cell-loaded zwitterionic microgel and a preparation method thereof are carried out according to the following steps:
and 3, pouring the reacted solution into a dialysis bag, dialyzing, and freeze-drying the dialyzed solution to obtain the zwitterionic polymer macromonomer (PCB-OAA), and dissolving the zwitterionic polymer macromonomer (PCB-OAA) and stem cells into PBS solution to form PBS solution of the zwitterionic polymer macromonomer (PCB-OAA), wherein the mass percent of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 1-20%, and the mass percent of the cells in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 0-1 × 10 9 The mass percentage of the cells is not equal to zero, and the stem cells are embryonic stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retina stem cells or pancreas stem cells;
step 4, respectively filling the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) prepared in the step 3 and the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) into different injectors, and respectively introducing the two solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases, wherein the mass percent of the dimercapto polyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) is 0.5-10%;
step 5, adding span 80 into the mineral oil, and introducing the mineral oil mixed with span 80 into a micro-fluidic chip after uniform mixing to serve as a continuous phase, wherein the mass percentage of span 80 in the mineral oil mixed with span 80 is 0.5% -5%;
step 6, mixing PBS solution of zwitterionic polymer macromonomer (PCB-OAA) and PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) in a microchannel of the microfluidic chip, then forming monodisperse liquid drops under the shearing action of mineral oil of a continuous phase, namely the span 80, and flowing out of the microchannel along with the continuous phase from a delivery pipe, wherein the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.1-20 muL/min, the flow rate of the PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) is 0.1-20 muL/min, and the flow rate of the mineral oil mixed with span 80 is 0.1-50 muL/min;
and 7, reacting the zwitterionic polymer macromonomer (PCB-OAA) and dimercapto polyethylene glycol (HS-PEG-SH) in the liquid drops to obtain the cell-loaded zwitterionic microgel (PCB-PEG).
In the step 1, the azo initiator adopts azodicyano valeric acid, 3- [ [2- (methacryloyloxy) ethyl ] dimethyl ammonium ] propionate and azo free radical initiator in the ratio of 175 to 1, the polymerization temperature is 70 ℃, and the polymerization time is 24 h.
In the step 2, sodium bicarbonate is used as the base, acrylic anhydride is used as the double-bond-containing acid monomer, and the reaction time is 24 hours.
In step 3, the mass percentage of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 2% -10%, and the mass percentage of the cells in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 1X 10 5 -1×10 8 /ml。
In step 4, the mass percent of the dimercaptopolyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercaptopolyethylene glycol (HS-PEG-SH) is 1% -5%, preferably 2% -4%.
In step 5, the weight percentage of span 80 in the mineral oil mixed with span 80 is 1-3%.
In step 6, the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.5-5 μ L/min, the flow rate of the PBS solution of dimercaptopolyethylene glycol (HS-PEG-SH) is 0.5-5 μ L/min, and the flow rate of the mineral oil mixed with span 80 is 0.3-10 μ L/min.
The invention has the beneficial effects that: the preparation method is simple, the size of the prepared microgel is uniform, and the size of the microgel is adjustable; the cells loaded in the microgel can keep high survival rate, and can have higher survival rate after long-term culture, in addition, because the super-hydrophilicity of zwitterions and polyethylene glycol can resist the adsorption of nonspecific protein, the stem cells are coated in the microgel, and the stem cells coated in the microgel can keep the dryness and retain the characteristic of multi-differentiation.
Drawings
FIG. 1 is a schematic diagram of the preparation of zwitterionic polymer macromonomers (PCB-OAA) prepared according to the present invention;
FIG. 2 is a photograph of zwitterionic microgel (PCB-PEG) prepared in accordance with the present invention;
FIG. 3 is a photograph of the cell-loaded zwitterionic microgel (PCB-PEG) prepared in accordance with the present invention;
FIG. 4 is a photograph showing the detection of cell activity of the cell-loaded zwitterionic microgel (PCB-PEG) prepared in accordance with the present invention;
FIG. 5 is the oil red O staining pattern and the PCR characterization result of the related genes of the stem cells in the cell-loaded zwitterionic microgel (PCB-PEG) prepared by the invention after being cultured in the adipogenic differentiation medium;
FIG. 6 is a PCR characterization result of alizarin red staining pattern and related genes of stem cells in the zwitterionic microgel (PCB-PEG) loaded with cells prepared by the invention after culturing in osteogenic differentiation induction medium;
FIG. 7 shows the PCR characterization of the relevant genes of stem cells in the cell-loaded zwitterionic microgel (PCB-PEG) prepared in the present invention after culturing in the myoblast differentiation-inducing medium;
FIG. 8 is a photograph of zwitterionic microgel (PCB-PEG) of various sizes prepared in accordance with the present invention;
FIG. 9 is a photograph of cell-loaded zwitterionic microgels of different cell loadings prepared in accordance with the present invention.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Example 1
and 2, after the reaction is finished, cooling the reaction solution to room temperature, adding 8.4g of sodium bicarbonate to neutralize acid in the solution, and carrying out ice-water bath on the solution for 30 min. Then dropwise adding 10ml of acrylic anhydride, reacting at room temperature for 24 hours to finally obtain a zwitterionic polymer macromonomer solution;
and 4, dissolving the prepared zwitter-ion macromonomer PCB-OAA in the PBS solution by using a balance to prepare a solution with a certain mass fraction. The mass fraction is 4.5%;
step 5, weighing a proper amount of sulfhydryl-substituted polyethylene glycol by using balance to prepare PBS solution with the concentration of 2.5%;
step 6, respectively loading the PCB-OAA solution and the HS-PEG-SH solution into an injector, and then introducing the solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases;
and 8, mixing the PCB-OAA solution and the HS-PEG-SH solution in the microchannel at the speed of 0.7 mu L/min, setting the speed of the continuous phase to be 1.5 mu L/min, forming monodisperse liquid drops under the shearing action of the continuous phase, and flowing out of the microchannel along with the continuous phase from the delivery pipe. The PCB-OAA and HS-PEG-SH react in the droplets to form microgels, as shown in figure 2;
step 9, a large amount of cells are mixed in advance in the PCB-OAA solution to obtain the cell-loaded microgel, as shown in FIG. 3. Taking mesenchymal stem cells as an example, obtaining the PCB-PEG microgel loaded with the mesenchymal stem cells, wherein the PCB-PEG microgel can keep high survival rate and dryness;
step 10, culturing the PCB-PEG microgel loaded with the mesenchymal stem cells in a complete culture medium, respectively carrying out cell survival test after culturing for 1,7 and 14 days, and determining by using a live and dead cell staining kit. As shown in fig. 4, the results show that the mesenchymal stem cells in the microgel survived well and survived well within 14 days of continuous culture;
step 11, culturing the PCB-PEG microgel loaded with the mesenchymal stem cells in a complete culture medium, adding the adipogenic differentiation medium into the complete culture medium for differentiation culture after culturing for 1 day, and taking the mesenchymal stem cells on the tissue culture plate as a control group. Differentiation results were determined by oil red O staining analysis and RT-PCR analysis 21 days after differentiation. As shown in fig. 5a, it was found that the mesenchymal stem cells loaded in the microgel substantially maintained their undifferentiated state, while the mesenchymal stem cells on the tissue culture plate underwent differentiation in the adipogenic direction as shown in fig. 5 b; the PCR result also shows that the RNA related to adipocyte differentiation is inhibited to be expressed, and the corresponding RNA is expressed in a higher manner.
And step 12, culturing the PCB-PEG microgel loaded with the mesenchymal stem cells in a complete culture medium, adding an osteogenic differentiation culture medium into the complete culture medium for differentiation culture after culturing for 1 day, and taking the mesenchymal stem cells on the tissue culture plate as a control group. Differentiation results were determined by alizarin red staining analysis and RT-PCR analysis 21 days after differentiation. As shown in fig. 6a, it was found that the mesenchymal stem cells loaded in the microgel substantially maintained their undifferentiated state, while the mesenchymal stem cells on the tissue culture plate underwent differentiation into an osteogenic direction as shown in fig. 6 b; the PCR result also shows that the osteoblast differentiation related RNA is inhibited from being expressed, and the corresponding xerosis RNA is highly expressed.
Step 13, culturing the PCB-PEG microgel loaded with the mesenchymal stem cells in a complete culture medium, adding a myogenic differentiation culture medium containing 10 mu M of 5-azacytidine into the complete culture medium after culturing for 1 day, and performing differentiation culture by taking the mesenchymal stem cells on a tissue culture plate as a control group. Differentiation results were determined by PCR analysis 21 days after differentiation. As shown in fig. 7, it was found that the mesenchymal stem cells loaded in the microgel substantially maintained their undifferentiated state, while the mesenchymal stem cells on the tissue culture plate underwent differentiation in the myogenic direction.
Example 2
and 2, after the reaction is finished, cooling the reaction solution to room temperature, adding 8.4g of sodium bicarbonate to neutralize acid in the solution, and carrying out ice-water bath on the solution for 30 min. Then dropwise adding 10ml of acrylic anhydride, reacting at room temperature for 24 hours to finally obtain a zwitterionic polymer macromonomer solution;
and 4, dissolving the prepared zwitter-ion macromonomer PCB-OAA in the PBS solution by using a balance to prepare a solution with a certain mass fraction. The mass fraction is 4.5%;
step 5, weighing a proper amount of sulfhydryl-substituted polyethylene glycol by using balance to prepare PBS solution with the concentration of 2.5%;
step 6, respectively loading the PCB-OAA solution and the HS-PEG-SH solution into an injector, and then introducing the solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases;
and 8, mixing the PCB-OAA solution and the HS-PEG-SH solution in the microchannel at the speed of 1 mu L/min, setting the speed of the continuous phase to be 3 mu L/min, forming monodisperse liquid drops under the shearing action of the continuous phase, and flowing out of the microchannel along with the continuous phase from the delivery pipe. The PCB-OAA and HS-PEG-SH reacted in the droplets to form a microgel, as shown in fig. 8.
Example 3
and 2, after the reaction is finished, cooling the reaction solution to room temperature, adding 8.4g of sodium bicarbonate to neutralize acid in the solution, and carrying out ice-water bath on the solution for 30 min. Then dropwise adding 10ml of acrylic anhydride, reacting at room temperature for 24 hours to finally obtain a zwitterionic polymer macromonomer solution;
and 4, dissolving the prepared zwitter-ion macromonomer PCB-OAA in the PBS solution by using a balance to prepare a solution with a certain mass fraction. The mass fraction is 4.5%;
step 5, weighing a proper amount of sulfhydryl-substituted polyethylene glycol by using balance to prepare PBS solution with the concentration of 2.5%;
step 6, respectively loading the PCB-OAA solution and the HS-PEG-SH solution into an injector, and then introducing the solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases;
step 8, a small amount of cells is mixed in advance in the PCB-OAA solution to obtain the cell-loaded microgel, as shown in FIG. 9. Taking mesenchymal stem cells as an example, the PCB-PEG microgel loaded with the mesenchymal stem cells is obtained, and the PCB-PEG microgel can keep high survival rate and dryness.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (10)
1. The cell-loaded zwitterionic microgel is characterized in that: the method comprises the following steps:
step 1, dissolving a monomer 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate in water, uniformly mixing, adding an azo free radical initiator, and heating to initiate polymerization reaction in a nitrogen protection atmosphere to obtain a zwitterionic polymer reaction solution, wherein the azo initiator adopts azobisisobutyramidine hydrochloride, azobisisopropylimidazoline hydrochloride or azobiscyanovaleric acid, and the dosage proportion range of the 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate to the azo free radical initiator is (50-400): 1, the polymerization temperature is 50-90 ℃, and the polymerization time is 10-36 h;
step 2, after the temperature of the zwitterionic polymer reaction solution prepared in the step 1 is reduced to room temperature of 20-25 ℃, adding an alkali aqueous solution into the reaction solution to neutralize the reaction solution until the pH value is 8-10, placing the mixed solution into an ice-water bath, dropwise adding an acid monomer containing double bonds into the mixed solution, and reacting to obtain a zwitterionic polymer macromonomer solution, wherein the alkali adopts sodium hydroxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate, the acid monomer containing the double bonds adopts acrylic acid, methacrylic acid, acrylic anhydride or methacrylic anhydride, the adding amount of the acid monomer is 5-20ml, and the reaction time is 10-36 h;
step 3, pouring the reacted solution into a dialysis bag, dialyzing, and freeze-drying the dialyzed solution to obtain the zwitterionPolymer macromonomer (PCB-OAA), and dissolving the zwitterionic polymer macromonomer (PCB-OAA) and the stem cells in PBS solution to form PBS solution of the zwitterionic polymer macromonomer (PCB-OAA), wherein the mass percent of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 1% -20%, and the mass percent of the cells in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 0-1 × 10 9 The mass percentage of the cells is not equal to zero, and the stem cells are embryonic stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retina stem cells or pancreas stem cells;
step 4, respectively filling the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) prepared in the step 3 and the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) into different injectors, and respectively introducing the two solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases, wherein the mass percent of the dimercapto polyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) is 0.5-10%;
step 5, adding span 80 into the mineral oil, and introducing the mineral oil mixed with span 80 into a micro-fluidic chip after uniform mixing to serve as a continuous phase, wherein the mass percentage of span 80 in the mineral oil mixed with span 80 is 0.5% -5%;
step 6, mixing PBS solution of zwitterionic polymer macromonomer (PCB-OAA) and PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) in a microchannel of the microfluidic chip, then forming monodisperse liquid drops under the shearing action of mineral oil of a continuous phase, namely the span 80, and flowing out of the microchannel along with the continuous phase from a delivery pipe, wherein the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.1-20 muL/min, the flow rate of the PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) is 0.1-20 muL/min, and the flow rate of the mineral oil mixed with span 80 is 0.1-50 muL/min;
and 7, reacting the zwitterionic polymer macromonomer (PCB-OAA) and dimercapto polyethylene glycol (HS-PEG-SH) in the liquid drops to obtain the cell-loaded zwitterionic microgel (PCB-PEG).
2. The cell-loaded zwitterionic microgel of claim 1, wherein: in the step 1, the azo initiator adopts azodicyano valeric acid, 3- [ [2- (methacryloyloxy) ethyl ] dimethyl ammonium ] propionate and azo free radical initiator with the dosage ratio of 175:1, the polymerization temperature of 70 ℃ and the polymerization time of 24 hours; in the step 2, sodium bicarbonate is used as the base, acrylic anhydride is used as the double-bond-containing acid monomer, and the reaction time is 24 hours.
3. The cell-loaded zwitterionic microgel of claim 1, wherein: in step 3, the mass percent of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 2% -10%; the mass percentage of cells in PBS solution of zwitterionic Polymer macromonomer (PCB-OAA) was 1X 10 5 -1×10 8 Per ml; in step 4, the mass percent of the dimercaptopolyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercaptopolyethylene glycol (HS-PEG-SH) is 1% -5%, preferably 2% -4%.
4. The cell-loaded zwitterionic microgel of claim 1, wherein: in the step 5, the weight percentage of the span 80 in the mineral oil mixed with the span 80 is 1-3 percent; in step 6, the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.5-5 μ L/min, the flow rate of the PBS solution of dimercaptopolyethylene glycol (HS-PEG-SH) is 0.5-5 μ L/min, and the flow rate of the mineral oil mixed with span 80 is 0.3-10 μ L/min.
5. The method for preparing a cell-loaded zwitterionic microgel of any one of claims 1 to 4, wherein: the method comprises the following steps:
step 1, dissolving a monomer 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate in water, uniformly mixing, adding an azo free radical initiator, and heating to initiate polymerization reaction in a nitrogen protection atmosphere to obtain a zwitterionic polymer reaction solution, wherein the azo initiator adopts azobisisobutyramidine hydrochloride, azobisisopropylimidazoline hydrochloride or azobiscyanovaleric acid, and the dosage proportion range of the 3- [ [2- (methacryloyloxy) ethyl ] dimethylammonium ] propionate to the azo free radical initiator is (50-400): 1, the polymerization reaction temperature is 50-90 ℃, and the polymerization reaction time is 10-36 h;
step 2, after the temperature of the zwitterionic polymer reaction solution prepared in the step 1 is reduced to room temperature of 20-25 ℃, adding an alkali aqueous solution into the reaction solution to neutralize the reaction solution until the pH value is 8-10, placing the mixed solution into an ice-water bath, dropwise adding an acid monomer containing double bonds into the mixed solution, and reacting to obtain a zwitterionic polymer macromonomer solution, wherein the alkali adopts sodium hydroxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate, the acid monomer containing the double bonds adopts acrylic acid, methacrylic acid, acrylic anhydride or methacrylic anhydride, the adding amount of the acid monomer is 5-20ml, and the reaction time is 10-36 h;
and 3, pouring the reacted solution into a dialysis bag, dialyzing, and freeze-drying the dialyzed solution to obtain the zwitterionic polymer macromonomer (PCB-OAA), and dissolving the zwitterionic polymer macromonomer (PCB-OAA) and stem cells into PBS solution to form PBS solution of the zwitterionic polymer macromonomer (PCB-OAA), wherein the mass percent of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 1-20%, and the mass percent of the cells in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 0-1 × 10 9 The mass percentage of the cells is not equal to zero, and the stem cells are embryonic stem cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retina stem cells or pancreas stem cells;
step 4, respectively filling the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) prepared in the step 3 and the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) into different injectors, and respectively introducing the two solutions into the microfluidic chip through a laboratory micro-injection pump to serve as discontinuous phases, wherein the mass percent of the dimercapto polyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercapto polyethylene glycol (HS-PEG-SH) is 0.5-10%;
step 5, adding span 80 into the mineral oil, and introducing the mineral oil mixed with span 80 into a micro-fluidic chip after uniform mixing to serve as a continuous phase, wherein the mass percentage of span 80 in the mineral oil mixed with span 80 is 0.5% -5%;
step 6, mixing PBS solution of zwitterionic polymer macromonomer (PCB-OAA) and PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) in a microchannel of the microfluidic chip, then forming monodisperse liquid drops under the shearing action of mineral oil of a continuous phase, namely the span 80, and flowing out of the microchannel along with the continuous phase from a delivery pipe, wherein the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.1-20 muL/min, the flow rate of the PBS solution of dimercapto polyethylene glycol (HS-PEG-SH) is 0.1-20 muL/min, and the flow rate of the mineral oil mixed with span 80 is 0.1-50 muL/min;
and 7, reacting the zwitterionic polymer macromonomer (PCB-OAA) and dimercapto polyethylene glycol (HS-PEG-SH) in the liquid drops to obtain the cell-loaded zwitterionic microgel (PCB-PEG).
6. The method of preparing a cell-loaded zwitterionic microgel of claim 5, wherein: in the step 1, the azo initiator adopts azodicyano valeric acid, 3- [ [2- (methacryloyloxy) ethyl ] dimethyl ammonium ] propionate and azo free radical initiator with the dosage ratio of 175:1, the polymerization temperature of 70 ℃ and the polymerization time of 24 hours; in the step 2, sodium bicarbonate is used as the base, acrylic anhydride is used as the double-bond-containing acid monomer, and the reaction time is 24 hours.
7. The cell-loaded zwitterionic microgel of claim 5The preparation method of the glue is characterized by comprising the following steps: in step 3, the mass percentage of the zwitterionic polymer macromonomer (PCB-OAA) in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 2% -10%, and the mass percentage of the cells in the PBS solution of the zwitterionic polymer macromonomer (PCB-OAA) is 1X 10 5 -1×10 8 Per ml; in step 4, the mass percent of the dimercaptopolyethylene glycol (HS-PEG-SH) in the PBS solution of the dimercaptopolyethylene glycol (HS-PEG-SH) is 1% -5%, preferably 2% -4%.
8. The method of preparing a cell-loaded zwitterionic microgel of claim 5, wherein: in step 5, the weight percentage of span 80 in the mineral oil mixed with span 80 is 1-3%.
9. The method of preparing a cell-loaded zwitterionic microgel of claim 5, wherein: in step 6, the flow rate of the PBS solution of zwitterionic polymer macromonomer (PCB-OAA) is 0.5-5 μ L/min, the flow rate of the PBS solution of dimercaptopolyethylene glycol (HS-PEG-SH) is 0.5-5 μ L/min, and the flow rate of the mineral oil mixed with span 80 is 0.3-10 μ L/min.
10. Use of the cell-loaded zwitterionic microgel of any one of claims 1 to 4 in stem cell culture, filler materials and tissue engineering.
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