CN109517781B - Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof - Google Patents
Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof Download PDFInfo
- Publication number
- CN109517781B CN109517781B CN201811402090.1A CN201811402090A CN109517781B CN 109517781 B CN109517781 B CN 109517781B CN 201811402090 A CN201811402090 A CN 201811402090A CN 109517781 B CN109517781 B CN 109517781B
- Authority
- CN
- China
- Prior art keywords
- gel
- superparamagnetic
- coating
- cell
- microspheres
- 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
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
- C12N5/0075—General culture methods using substrates using microcarriers
-
- 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
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
Abstract
The invention provides a cell-microsphere with an elutable superparamagnetic gel coating and a preparation method thereof. The invention also provides the application of the cell-microsphere in the co-culture and the later separation of the co-culture of the cells containing the cell gel microsphere for clinical implantation repair. The invention can solve the problems that the prior microcarrier can not realize the removal of magnetic materials after the co-culture is finished, can not separate different cells in a co-culture system, can not be used for clinical implantation repair, and the like.
Description
Technical Field
The invention belongs to the technical field of biomedical high polymer materials, and relates to a cell-microsphere with an elutable superparamagnetic gel coating, and a preparation method and application thereof.
Background
The existing magnetic microcarrier is obtained by processing the surface of a magnetic material, coating a macromolecular material, combining with biological macromolecular substances such as antigens, antibodies, nucleic acids and the like, wherein the magnetic material is positioned inside the microcarrier, most of the magnetic microcarrier is planted with cells in the later period, and the cells grow on the surface of the microcarrier. For example, antibody-coated magnetic beads are allowed to sufficiently react with cells to be separated, and the antibody magnetic beads are bonded to the corresponding cells in the form of cell-antibody-magnetic bead complexes, whereby the cells can be specifically separated by utilizing the difference between their movement in a magnetic field and other cells. However, the existing magnetic microcarrier still has certain limitations in the application aspect, on one hand, cells grow on the surface of the microcarrier and cannot meet the requirement of tissue engineering three-dimensional culture, and on the other hand, the magnetic material is positioned in the microcarrier and cannot be removed, so that the magnetic material cannot be used for clinical repair. Thus, the function of existing magnetic microcarriers is cell culture and sorting, and is generally used only as an in vitro study.
A large number of studies have shown that a three-dimensional culture environment is more conducive to cell growth to the relevant tissue. The co-culture technology is widely used in a plurality of tissue engineering, the co-culture technology co-cultures the target cells and the seed cells in the same environment, and the target cells can secrete a plurality of cytokines which are beneficial to the differentiation of the seed cells to promote the differentiation of the seed cells. The commonly used co-culture at present comprises 2D-2D cell co-culture, 2D-3D cell-scaffold co-culture and 3D-3D scaffold co-culture, and the separation of target cells and seed cells in the later period is realized by utilizing the initial spatial distribution, so that the space between different cells is far, and related factors secreted by the target cells cannot be quickly and effectively obtained by the seed cells.
CN102286422A discloses a composite microcapsule model for in vitro co-culture, which takes natural polysaccharide sodium alginate as a matrix material, and the polysaccharide sodium alginate is dropped into a multivalent cation solution to carry out gelation treatment twice to form a ball-in-ball type composite microcapsule model consisting of an inner layer and an outer layer of sodium alginate gel microspheres, wherein one layer of the gel microspheres can contain one cell, and the other layer can contain the other cell. Although the inner layer gel microspheres in the microcapsule model realize the growth of cells in a three-dimensional environment and can also solve the problem that the space between target cells and seed cells is far, the composite microcapsule model cannot realize the separation of different cells in the outer layer gel and the inner layer gel, and in addition, the inner layer gel and the outer layer gel are made of the same material, the cells in the inner layer gel and the outer layer gel can migrate between the two layers of gels, which further increases the separation difficulty of different cells after the co-culture is completed, so that the composite microcapsule model can only be used for in-vitro research and cannot be used for clinical repair.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a cell-microsphere with an elutable superparamagnetic gel coating, a preparation method and application thereof, so as to solve the problems that the conventional microcarrier cannot remove a magnetic material after co-culture is finished, and cannot be used for clinical implantation repair because different cells in a co-culture system are separated.
The cell-microsphere with the elutable superparamagnetic gel coating comprises a gel microsphere and a superparamagnetic gel coating for coating the gel microsphere, wherein the gel microsphere contains cells, the superparamagnetic gel coating contains superparamagnetic particles, the superparamagnetic gel coating can be eluted and removed, and the superparamagnetic gel coating and the gel microsphere cannot be simultaneously eluted and removed.
In the above technical scheme of the cell-microsphere with the elutable superparamagnetic gel coating, the gel material constituting the gel microsphere is a biocompatible temperature-sensitive material, photosensitive material or ion-sensitive material, the gel material constituting the superparamagnetic gel coating is a biocompatible temperature-sensitive material or ion-sensitive material, and the gel microsphere is different from the gel material constituting the superparamagnetic gel layer.
Preferably, the gel material forming the gel microsphere is a temperature-sensitive material or a photosensitive material, and the gel material forming the superparamagnetic gel coating is an ion-sensitive material, and the superparamagnetic gel coating is removed by dissolving in a solution, for example, the gel material forming the superparamagnetic gel coating can adopt calcium alginate gel, and the calcium alginate gel can be removed by dissolving in a citric acid solution; or the gel material forming the gel microsphere is a photosensitive material, a temperature-sensitive material or an ion-sensitive material, the gel material forming the superparamagnetic gel coating is a temperature-sensitive material which can be reversibly converted between a liquid state and a gel state along with the change of the environmental temperature, the superparamagnetic gel coating is removed by changing the environmental temperature, and when the gel materials forming the gel microsphere and the superparamagnetic gel coating are both temperature-sensitive materials, the selection principle of the temperature-sensitive material is to ensure that the gel microsphere and the gel material forming the superparamagnetic gel coating are not removed under the same temperature condition, for example, the gel material forming the superparamagnetic gel coating can adopt gelatin gel, and the gelatin gel can be liquefied and removed after being heated to about 30 ℃.
As an alternative mode, the temperature-sensitive material can be a gel material formed by materials with the gel temperature lower than 40 ℃ such as collagen, gelatin, poly isopropyl acrylamide and derivatives thereof; the photosensitive material can be a gel material formed by the reaction of water-soluble photosensitive polysaccharide such as methacrylic anhydride or maleic anhydride modified hyaluronic acid derivative, chondroitin sulfate derivative, pullulan derivative and the like under the conditions of photoinitiator and proper light, wherein the photoinitiator can be 2-hydroxy-4' - (hydroxyethoxy) -2-methyl propiophenone (I2959), 1-hydroxycyclohexyl phenyl acetone (I184) or 2, 2-dimethoxy-phenyl acetophenone (I651); the ion-sensitive material can be gel material formed by cross-linking alginic acid derivative and calcium ions.
Further, the temperature-sensitive material capable of reversibly switching between a liquid state and a gel state with the change of the environmental temperature includes gelatin gel and poly (N-isopropylacrylamide) gel.
Furthermore, when the gel materials forming the gel microspheres and the superparamagnetic gel coating are temperature-sensitive materials, the gel materials forming the superparamagnetic gel coating are temperature-sensitive materials which can be mutually and reversibly converted between a liquid state and a gel state along with the change of the environmental temperature, the temperature-sensitive materials forming the gel microspheres can be temperature-sensitive materials which can be mutually and reversibly converted between the liquid state and the gel state along with the change of the environmental temperature, or the temperature-sensitive materials which cannot be converted into the liquid state along with the change of the environmental temperature after the gel state is formed, and the selection principle of the temperature-sensitive materials forming the gel microspheres and the superparamagnetic gel coating is to ensure that the gel materials forming the gel microspheres and the superparamagnetic gel coating are not removed under the same temperature condition. For example, according to the form of the gel microsphere-coating, the combination form of the temperature sensitive material can be a collagen microsphere-gelatin coating, a collagen microsphere-poly (N-isopropylacrylamide) coating.
In the above technical solution of cell-microsphere with an elutable superparamagnetic gel coating, the size of the superparamagnetic particles in the superparamagnetic gel coating is selected according to the pores of the gel microsphere and the gel network structure of the superparamagnetic gel coating, the size of the superparamagnetic particles in the superparamagnetic gel coating should be larger than the gel network size of the superparamagnetic gel coating, and meanwhile, the size of the superparamagnetic particles in the superparamagnetic gel coating is larger than the gel network size of the gel microsphere, so as to prevent the superparamagnetic particles from permeating into the gel microsphere and leaking into the culture medium for cell co-culture. The method is favorable for improving the stability of later separation, and simultaneously avoids the biological toxicity of the superparamagnetic particles to the gel microsphere coated cells. Preferably, in the superparamagnetic gel coating, the size of superparamagnetic particles is 1-50 μm, more preferably 5-40 μm, 10-30 μm or 15-25 μm, and the content of superparamagnetic particles in the superparamagnetic gel coating is 0.1-10 mg/mL.
In the technical scheme of the cell-microsphere with the elutable superparamagnetic gel coating, the thickness of the superparamagnetic gel coating is 10-150 μm.
In the technical scheme of the cell-microsphere with the elutable superparamagnetic gel coating, the cell-microsphere is spherical or approximately spherical, in order to facilitate nutrient enrichment of cells in the gel microsphere and diffusion and acquisition of relevant factors secreted by the cells, the size of the cell-microsphere is determined according to actual application requirements, and the size of the cell-microsphere is usually 30-3000 μm, preferably 30-1000 μm.
The invention also provides a preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, which comprises the following steps:
(1) adjusting the pH value of the gel material solution to 6.5-7.5, removing bubbles in the gel material solution, preparing the counted cells into cell suspension by using a culture medium or a buffer solution, and then adding the cell suspension into the gel material solution from which the bubbles are removed to obtain a cell-gel material mixed solution;
(2) dispersing the cell-gel material mixed solution into an oil phase liquid, performing emulsification and gelation treatment to obtain cell-microspheres, and regulating the surface environment of the cell-microspheres to the condition required by gelation of the coating material in the step (3); for example, the temperature or ion concentration of the cell-microsphere surface is regulated to reach the conditions required by the gelation of the coating material in the step (3);
(3) uniformly mixing the gel material and the superparamagnetic particles to form a coating material, uniformly coating the coating material on the surfaces of the cells and the microspheres, gelatinizing the coating material coated on the surfaces of the cells and the microspheres to convert the coating material into a gel coating, removing the redundant coating material through centrifugation or standing, and cleaning to obtain the cells and the microspheres with the elutable superparamagnetic gel coating.
In the technical scheme of the preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, the thickness of the elutable superparamagnetic gel coating covering the gel microsphere is determined according to the actual application requirement, and the times of coating the coating material and the gelation treatment operation in the step (3) are increased, so that the thickness of the superparamagnetic gel coating can be increased. Specifically, step (3) may be performed as follows:
uniformly mixing a gel material and superparamagnetic particles to form a coating material, uniformly coating the coating material on the surfaces of the cells and the microspheres, gelatinizing the coating material coated on the surfaces of the cells and the microspheres to convert the coating material into a gel coating, removing the redundant coating material through centrifugation or standing, and cleaning;
removing water on the surfaces of the microspheres obtained after cleaning in the step I, then uniformly coating a coating material on the surfaces of the microspheres, gelatinizing the coating material coated on the surfaces of the cells and the microspheres to convert the coating material into a gel coating, removing redundant coating material through centrifugation or standing, and cleaning;
thirdly, repeating the operation of the second step for a plurality of times, for example, 0 to 5 times according to the requirement of the thickness of the superparamagnetic gel coating in practical application to obtain the cell-microsphere with proper thickness and capable of eluting the superparamagnetic gel coating.
In the above technical solution of the method for preparing cell-microsphere with an elutable superparamagnetic gel coating, the gelation method specifically adopted in the emulsification and gelation process in step (2) is related to the gel material of the gel microsphere adopted in the device, and the gelation process means may be temperature control, ionic crosslinking, photoinitiated crosslinking, and other gelation methods, for example: the temperature-sensitive material needs to form gel in a specific temperature environment, for example, the temperature is increased from 35 ℃ to above the gel temperature and is kept for 1-45 minutes; the ion sensitive material needs to form gel in the presence of cross-linking ions, for example, a divalent cation solution, calcium chloride, zinc chloride, magnesium chloride and the like are added dropwise to form gel, and the photosensitive material needs to form gel in a photoinitiator and a specific illumination environment, for example, ultraviolet light with a specific wavelength is used for irradiating for 10-360 seconds.
In the above technical solution of the method for preparing cell-microsphere with an elutable superparamagnetic gel coating, the conditions for gelation of the coating material coated on the surface of the cell-microsphere in step (3), and the conditions for gelation of the coating material in step (2) for regulating the environment of the surface of the cell-microsphere to step (3) are related to the gel material of the gel coating specifically adopted, for example: the temperature-sensitive material needs to form gel in a specific temperature environment, for example, the temperature is increased from 35 ℃ to above the gel temperature and is kept for 1-45 minutes; the ion sensitive material needs to form gel in the presence of cross-linking ions, such as dripping divalent cation solution, calcium chloride, zinc chloride, magnesium chloride and the like to realize gelling.
In the technical scheme of the preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, the step (1) removes air bubbles in the gel material solution in a centrifugal or standing mode.
In the technical scheme of the preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, the operation of the step (2) is specifically as follows:
adding a surfactant with the volume percentage concentration of 0-10% (v/v) into an oil phase liquid, then adding a cell-gel material mixed solution under the action of mechanical stirring to uniformly disperse a gel material into the oil phase liquid to form water-in-oil microspheres, selecting corresponding gel conditions to realize the gelation of the microspheres while stirring the two-phase mixed solution, and separating the gel microspheres by adopting a common separation mode such as centrifugation or filtration.
Preferably, the oil phase liquid can be one or a mixture of mineral oil such as methyl silicone oil and paraffin oil, or vegetable oil such as olive oil, castor oil, corn oil and rapeseed oil, and the oil phase liquid with the viscosity of 20-200 mPas is preferably selected. The surfactant can be one of Tween-20, Tween-40, Tween-60, Tween-80, Span-20, Span-40, Span-60, Span-80 and Triton X-100.
Preferably, the ratio of the cell-gel material mixed solution (aqueous phase solution) to the oil phase liquid is 0.5-40%, preferably 1-5%, and the mixture is continuously stirred at a rotation speed of 100-1500 rpm for 1-30 min to disperse the aqueous phase into microspheres.
In the technical scheme of the preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, the step (2) further comprises a washing separation operation after the gelation treatment, and the oil phase liquid is fully removed through the washing separation step. The washing operation is specifically as follows: dispersing and washing the separated gel microspheres in a culture medium or a buffer solution containing 0-0.1% (v/v) Tween 80 for 2-4 times. The culture medium can be RPMI1640 culture medium, MEM culture medium or alpha MEM culture medium, and the buffer can be physiological saline, PBS buffer or HEPES buffer.
The invention also provides application of the cell-microsphere with the elutable superparamagnetic gel coating in cell co-culture and co-culture later-stage separation of the cell-containing gel microsphere for clinical implantation repair.
One possible application is as follows:
(1) preparing cell-microspheres loaded with target cells and provided with an elutable superparamagnetic gel coating according to the method of the invention;
(2) preparing a cell-microsphere loaded with seed cells and provided with an elutable gel coating by the method, wherein the gel coating of the microsphere does not contain superparamagnetic particles;
(3) co-culturing the microspheres prepared in the step (1) and the step (2);
(4) and after the co-culture is finished, a magnetic field is applied to separate the two microspheres, and the microspheres loaded with seed cells do not contain superparamagnetic particles and target cells, so that the gel coating can be removed and used for clinical implantation repair.
A second possible application is as follows:
(1) preparing cell-microspheres loaded with seed cells and provided with an elutable superparamagnetic gel coating according to the method of the invention;
(2) preparing a cell-microsphere with an elutable gel coating loaded with target cells according to the method of the present invention, wherein the gel coating of the microsphere does not contain superparamagnetic particles;
(3) co-culturing the microspheres prepared in the step (1) and the step (2);
(4) and after the co-culture is finished, a magnetic field is applied to separate the two microspheres, the superparamagnetic gel coating of the microspheres loaded with the seed cells can be eluted and removed, and the superparamagnetic gel coating can be used for clinical implantation repair after being removed.
A third possible application is as follows:
(1) preparing cell-microspheres loaded with first seed cells having an elutable superparamagnetic gel coating according to the method of the present invention;
(2) preparing a cell-microsphere with an elutable gel coat loaded with a second seed cell according to the method of the present invention, wherein the gel coat of the microsphere does not contain superparamagnetic particles;
(3) co-culturing the microspheres prepared in the step (1) and the step (2);
(4) after the co-culture is finished, applying a magnetic field to separate the two microspheres, wherein the microspheres loaded with the second seed cells do not contain superparamagnetic particles, and the gel coating is removed to be used for clinical implantation repair; the superparamagnetic gel coating of the microsphere loaded with the first seed cells can be eluted and removed, and the obtained gel microsphere loaded with the first seed cells can also be used for clinical implantation repair after the superparamagnetic gel coating is removed.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the cell-microsphere with the elutable superparamagnetic gel coating comprises a cell-containing gel microsphere and a superparamagnetic gel coating for coating the cell-containing gel microsphere, wherein the superparamagnetic gel coating can be eluted and removed, and the superparamagnetic gel coating and the gel microsphere cannot be simultaneously eluted and removed. On one hand, the cells loaded in the gel microspheres can provide a 3D culture environment for the cells, and the 3D culture environment is more beneficial to the growth of the cells to related tissues; on the other hand, the small size of the microspheres is beneficial to nutrient proliferation of cells, and the blending of the microspheres with micron-sized size is beneficial to the diffusion and acquisition of related factors secreted by the cells; meanwhile, the superparamagnetic gel coating has the characteristics of magnetism and being capable of being eluted and removed, the two microspheres wrapping different cells or micro-tissues are co-cultured, the two microspheres can be separated by utilizing the magnetism of the superparamagnetic gel coating after the in vitro co-culture is finished, and then the microspheres are eluted and removed by utilizing the characteristic that the superparamagnetic gel coating can be removed, so that the magnetic substance residue is avoided, in addition, the gel coating can prevent the cells from migrating to the outside of the microspheres during the co-culture, and the problem that the different cells cannot be separated due to the mutual migration between the two microspheres during the co-culture is avoided. The characteristics enable the cell-microsphere with the elutable superparamagnetic gel coating provided by the invention to be applied to cell co-culture and co-culture later-stage separation of cell-containing gel microspheres for clinical implantation repair, and can solve the problems that the removal of magnetic materials and the separation of different cells in a co-culture system cannot be realized after the co-culture of the existing microcarrier is finished, and the microcarrier cannot be used for clinical implantation repair.
2. The gel microspheres of the cell-microspheres with the elutable superparamagnetic gel coating and the gel materials of the superparamagnetic gel coating are biocompatible environment-sensitive gel materials, have good biocompatibility and biodegradability, so that the cell-microspheres provided by the invention have the characteristics of safety, degradability, good biocompatibility and the like when being used for clinical implantation repair.
3. The invention also provides a preparation method of the cell-microsphere with the elutable superparamagnetic gel coating, which has the characteristics of simple operation, mild process conditions and low cost and is beneficial to realizing industrial production.
Drawings
Fig. 1 is an optical microscope photograph of cell-microspheres with an elutable superparamagnetic gel coating of collagen gel microspheres prepared in example 1 and microspheres prepared in example 3.
Fig. 2 is an optical microscope photograph of the fluorescent collagen gel microspheres prepared in example 2, and the fluorescent collagen gel microspheres with elutable superparamagnetic gel coating prepared in examples 5 and 6.
Fig. 3 is a bar graph of the gel coat thickness of fluorescent collagen gel microspheres with an elutable gel coat prepared in examples 5 and 6.
FIG. 4 is a scanning electron micrograph of the collagen gel microspheres prepared in example 1.
Fig. 5 is a scanning electron micrograph of the cell-microspheres with an elutable gel coating prepared in example 4.
FIG. 6 is a photograph of the purple-colored microspheres of example 7 in a suspended state under magnetic field conditions.
FIG. 7 is a photograph of the separation of microspheres before and after application of the magnetic field in example 7.
FIG. 8 is a photograph of example 8 before and after elution to remove the superparamagnetic gel coat.
FIG. 9 shows the results of the cell viability assay in example 9.
Fig. 10 is an optical micrograph of the bottom of a culture dish for culturing the mesenchymal stem cell-collagen gel microspheres and the mesenchymal stem cell-collagen gel microspheres with an elutable superparamagnetic gel coating in example 10.
FIG. 11 is a schematic diagram of co-cultivation in example 11.
Detailed Description
The cell-microsphere with an elutable superparamagnetic gel coating, and the preparation method and application thereof provided by the present invention are further described by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.
Example 1
In this example, the preparation of collagen gel microspheres loaded with bone marrow mesenchymal stem cells comprises the following steps:
(1) taking a collagen solution with the concentration of 15mg/mL, adjusting the pH value to 7.4 by using a 1mol/L sodium hydroxide solution in an ice water bath, and then diluting the collagen solution until the collagen concentration is 10 mg/mL; counting the prepared bone marrow mesenchymal stem cells in advance, preparing a cell suspension by using an alpha-MEM culture medium, adding the cell suspension into a collagen solution, and uniformly dispersing to obtain a cell-collagen mixed solution, wherein the final concentration of collagen in the cell-collagen mixed solution is 6.5mg/mL, and the cell density is 5 multiplied by 106one/mL.
(2) Taking 70mL of methyl silicone oil with the viscosity of 50 mPas, adding 0.05% (v/v) of Span-80 to form an oil phase, stirring with a magnetic stirrer at the rotating speed of 500rpm, dropwise adding a cell-collagen mixed solution (water phase) into the oil phase under the condition of ice-water bath, continuously stirring for 30 minutes, then heating a water phase-oil phase mixed system to 37 ℃, continuously stirring for 20 minutes, centrifuging the obtained mixed solution after stirring is finished, dispersing a bottom layer precipitate into an alpha-MEM culture medium, cleaning, and washing with the alpha-MEM culture medium for 3 times to obtain the mesenchymal dry cell-collagen gel microspheres.
Example 2
In this example, the preparation of the collagen gel microspheres loaded with fluorescent particles comprises the following steps:
(1) taking a collagen solution with the concentration of 15mg/mL, adjusting the pH value to 7.4 by using a 1mol/L sodium hydroxide solution in an ice water bath, and then diluting the collagen solution until the collagen concentration is 6 mg/mL; uniformly dispersing red fluorescent particles and green fluorescent particles (v/v:1/50) into a collagen solution to obtain a fluorescent particle-collagen mixed solution.
(2) Taking 60mL of methyl silicone oil with the viscosity of 50 mPas, stirring the mixture by a magnetic stirrer at the rotating speed of 550rpm, dropwise adding a fluorescent particle-collagen mixed solution (water phase) into the oil phase under the condition of ice-water bath, continuously stirring the mixture for 30 minutes, then heating a water phase-oil phase mixed system to 37 ℃, continuously stirring the mixture for 20 minutes, centrifuging the obtained mixed solution after stirring is finished, dispersing a bottom layer precipitate into a PBS buffer solution for cleaning, and washing the bottom layer precipitate for 3 times by the PBS buffer solution to obtain the fluorescent collagen gel microspheres.
Example 3
In this example, cell-microspheres with an elutable superparamagnetic gel coating were prepared as follows:
(1) the mesenchymal stem cell-collagen gel microspheres prepared in example 1 are taken and placed in a centrifuge tube, washed for 3 times by using a sodium chloride solution, then placed in a sodium chloride solution containing calcium chloride for soaking for 3 minutes, and the liquid is sucked dry for standby.
(2) Diluting 6% sodium alginate solution by one time with solution containing 4mg/mL superparamagnetic particles with the diameter of 1 μm, and mixing uniformly to obtain a coating material for later use.
(3) And (2) dropwise adding the coating material prepared in the step (2) onto the mesenchymal stem cell-collagen gel microspheres processed in the step (1), uniformly mixing, performing crosslinking reaction between the coating material dropwise added to the surfaces of the gel microspheres and calcium ions on the surfaces of the gel microspheres to perform gelation to form a gel coating, removing redundant coating material on the surfaces of the microspheres by centrifugation, and then placing the gel coating in an alpha-MEM culture medium for cleaning for 3 times to remove redundant alginate and superparamagnetic particles to obtain the cell-microspheres with the elutable superparamagnetic gel coating.
Example 4
In this example, cell-microspheres with an elutable superparamagnetic gel coating were prepared as follows:
(1) the mesenchymal stem cell-collagen gel microspheres prepared in example 1 are taken and placed in a centrifuge tube, washed for 3 times by using a sodium chloride solution, then placed in a sodium chloride solution containing calcium chloride for soaking for 3 minutes, and the liquid is sucked dry for standby.
(2) Diluting 6% sodium alginate solution by one time with solution containing 4mg/mL superparamagnetic particles with the diameter of 1 μm, and mixing uniformly to obtain coating material for use.
(3) And (2) dropwise adding the coating material prepared in the step (2) onto the mesenchymal stem cell-collagen gel microspheres processed in the step (1), uniformly mixing, performing crosslinking reaction between the coating material dropwise added to the surfaces of the gel microspheres and calcium ions on the surfaces of the gel microspheres to perform gelation to form a gel coating, removing redundant coating material on the surfaces of the microspheres by centrifugation, and then placing the gel coating in an alpha-MEM culture medium for cleaning for 3 times to remove redundant alginate and superparamagnetic particles to obtain the cell-microspheres with the elutable superparamagnetic gel coating.
(4) And (4) taking the cell-microsphere with the elutable superparamagnetic gel coating obtained in the step (3), and repeating the operations in the steps (1) and (3) for 1 time to obtain the cell-microsphere coated with two layers of elutable superparamagnetic gel coatings.
Example 5
In this example, the fluorescent collagen gel microspheres with an elutable gel coating were prepared by the following steps:
(1) the fluorescent collagen gel microspheres prepared in example 2 were placed in a centrifuge tube, washed 3 times with sodium chloride solution, then placed in sodium chloride solution containing calcium chloride for 3 minutes, and the liquid was blotted dry for use.
(2) Diluting 6% sodium alginate solution by one time with deionized water, and mixing uniformly to obtain the coating material for later use.
(3) And (3) dropwise adding the coating material prepared in the step (2) onto the fluorescent collagen gel microspheres processed in the step (1), uniformly mixing, performing crosslinking reaction between the coating material dropwise added to the surfaces of the gel microspheres and calcium ions on the surfaces of the gel microspheres to gelatinize to form a gel coating, removing redundant coating material on the surfaces of the microspheres through centrifugation, and then putting the microspheres into PBS buffer solution to clean for 3 times to remove redundant alginate, thus obtaining the fluorescent collagen gel microspheres with the washable gel coating.
Example 6
In this example, the fluorescent collagen gel microspheres with an elutable gel coating were prepared by the following steps:
(1) the fluorescent collagen gel microspheres prepared in example 2 were placed in a centrifuge tube, washed 3 times with sodium chloride solution, then placed in sodium chloride solution containing calcium chloride for 3 minutes, and the liquid was blotted dry for use.
(2) Diluting 6% sodium alginate solution by one time with deionized water, and mixing uniformly to obtain the coating material for later use.
(3) And (3) dropwise adding the coating material prepared in the step (2) onto the fluorescent collagen gel microspheres processed in the step (1), uniformly mixing, performing crosslinking reaction between the coating material dropwise added to the surfaces of the gel microspheres and calcium ions on the surfaces of the gel microspheres to gelatinize to form a gel coating, removing redundant coating material on the surfaces of the microspheres through centrifugation, and then putting the microspheres into PBS buffer solution to clean for 3 times to remove redundant alginate, thus obtaining the fluorescent collagen gel microspheres with the washable gel coating.
(4) And (4) taking the cell-microsphere with the elutable superparamagnetic gel coating obtained in the step (3), and repeating the operations in the steps (1) and (3) for 5 times to obtain the fluorescent collagen gel microsphere coated with six layers of elutable superparamagnetic gel coatings.
The optical microscope photographs of the collagen gel microspheres prepared in example 1 and the cell-microspheres with an elutable superparamagnetic gel coating prepared in example 3 are sequentially shown in fig. 1 (a) and (B). The optical micrographs of the fluorescent collagen gel microspheres prepared in example 2 and the fluorescent collagen gel microspheres with the elutable gel coat prepared in examples 5 and 6 are shown in fig. 2 (a), (B) and (C) in sequence, wherein the dotted lines in the two graphs (B) and (C) indicate the outer boundary of the collagen gel microspheres and the outer boundary of the gel coat. FIG. 3 is a bar graph of the thickness of the gel coating of the fluorescent collagen gel microspheres with elutable gel coatings prepared in examples 5 and 6, and it can be seen from FIG. 3 that the thickness of the gel coating can be adjusted and controlled within a range of 10-150 nm according to different coating times.
The scanning electron micrograph of the collagen gel microspheres prepared in example 1 is shown in fig. 4, and the images (a), (B), and (C) of fig. 4 are scanning electron micrographs at different magnifications. The scanning electron micrographs of the cell-microspheres with elutable superparamagnetic gel coating prepared in example 4 are shown in fig. 5, and the images (a), (B), (C) of fig. 5 are scanning electron micrographs at different magnifications. As can be seen from FIG. 5, the cell-microsphere with the elutable superparamagnetic gel coating prepared in example 4 is approximately spherical and has a size of about 700-800 μm.
Example 7
In this example, cell-microspheres with an elutable superparamagnetic gel coating were separated from cell-microspheres without an elutable superparamagnetic gel coating according to the following procedure.
(1) The cell-microspheres prepared in example 1 and the cell-microspheres with an elutable superparamagnetic gel coating prepared in example 4 were stained with TB. The cell-microspheres with elutable superparamagnetic gel coating prepared in example 4 are labeled purple. Since the mesenchymal stem cell-collagen gel microspheres prepared in example 1 did not contain the sodium alginate gel coating (polysaccharide component), they could not be stained purple by TB.
(2) Adding the two microspheres into a centrifugal tube, adding water, shaking the centrifugal tube until the two microspheres are in a suspended state, vertically placing the centrifugal tube, applying a magnetic field on the wall of the centrifugal tube by using a magnet, and finding out that the purple microspheres can be in a suspended state under the condition of the magnetic field through experiments, wherein the suspended state is shown in fig. 6.
(3) Adding the two microspheres into a centrifugal tube, adding water, shaking the centrifugal tube until the two microspheres are in a suspension state, transversely placing the centrifugal tube, applying a magnetic field on the wall of the centrifugal tube by using a magnet, and finding out that the purple microspheres can be sucked on the wall of the centrifugal tube by an experiment, and moving the microspheres which are not sucked into another centrifugal tube by using a dropper. Photographs before and after the magnetic field application are shown in both (a) and (B) of fig. 7.
(4) After transferring to a new centrifuge tube, applying a magnetic field to the wall of the new centrifuge tube by using a magnet, and experimentally, finding that the purple microspheres are attracted to the wall of the centrifuge tube, and transferring the uninspired microspheres to another centrifuge tube by using a dropper.
(5) And (5) repeating the operation of the step (4) for a plurality of times to complete the separation of the two microspheres.
Example 8
In this example, the coating on the surface of the cell-microsphere with the elutable superparamagnetic gel coating, prepared in example 4, was eluted.
(1) The cell-microspheres with the elutable superparamagnetic gel coating prepared in example 4 were put into a centrifuge tube, added with an aqueous solution of sodium citrate with a concentration of 55mmol/L, soaked for 5 minutes, filtered, blotted to remove the aqueous solution of sodium citrate on the surface of the microspheres, and then washed in α -MEM medium for 3 times.
(2) And (3) taking the microspheres processed in the step (1), and repeating the operation of the step (1) for 2 times.
(3) And (3) dyeing the microspheres treated in the step (2) by using TB, and finding that no purple heterodyeing occurs, which indicates that the superparamagnetic gel coating on the surfaces of the microspheres is completely removed by elution.
The photographs before and after the elution removal of the superparamagnetic gel coating on the cell-microsphere surface with the elutable superparamagnetic gel coating are respectively shown in (A) and (B) of FIG. 8, wherein the purple differential staining in (A) is that the superparamagnetic gel coating is stained by TB, and the purple differential staining is not seen in (B), which indicates that the superparamagnetic gel coating is completely eluted and removed.
Example 9
In this example, it is examined whether the addition of the superparamagnetic gel coating on the surface of the mesenchymal stem cell-collagen gel microsphere prepared in example 1 may affect the activity of the mesenchymal stem cells in the collagen gel microsphere.
The microspheres prepared in example 1 and example 3 were taken and subjected to cell viability assay. And (3) washing the microspheres for 3 times by using PBS, adding the microspheres into FDA-PI staining solution prepared in advance, washing the microspheres for 3 times by using PBS after staining for 5 minutes, and observing and taking pictures by using a laser confocal microscope. As shown in fig. 9, it can be seen from fig. 9 that the addition of the superparamagnetic gel coating on the surface of the cell-gel microspheres did not affect the activity of the cells in the collagen gel microspheres.
Example 10
In this example, it is examined whether the addition of a superparamagnetic gel coating on the surface of the mesenchymal stem cell-collagen gel microsphere prepared in example 1 helps to prevent the migration of mesenchymal stem cells in the collagen gel microsphere out of the microsphere.
After three days of culturing the microspheres prepared in example 1 and example 3 in a culture dish, the bottom surface of the culture dish was observed under an optical microscope to see whether cells were adhered. Because the culture dish has good cell adhesion capability, if cells in the microspheres migrate out, adherent cells can be observed on the bottom surface of the culture dish. The results are shown in fig. 10, wherein A, B of fig. 10 are optical micrographs of the bottom of a culture dish for culturing the mesenchymal stem cell-collagen gel microspheres prepared in example 1, B is a partially enlarged view of a drawing, C, D of fig. 10 is an optical micrograph of the bottom of a culture dish for culturing the mesenchymal stem cell-collagen gel microspheres with an elutable superparamagnetic gel coating prepared in example 3, and D is a partially enlarged view of C. As can be seen from fig. 10, the addition of a superparamagnetic gel coating on the surface of the cell-gel microspheres helps to prevent the cells in the gel microspheres from migrating out of the microspheres.
Example 11
In this example, the application of the cell-microsphere with an elutable superparamagnetic gel coating in the co-culture and post-separation of cells containing the cell gel microsphere for clinical implantation repair is provided, and the schematic diagram is shown in fig. 11.
(1) Preparing collagen gel microspheres loaded with seed cells (seed cell-collagen gel microspheres) according to the method of example 1; preparing collagen gel microspheres loaded with seed cells (target cells-collagen gel microspheres) by the method of example 1;
(2) taking the seed cell-collagen gel microspheres, and preparing the seed cell-microspheres with the elutable superparamagnetic gel coating by referring to the method of example 4;
(3) the target cell-collagen gel microspheres were prepared with the elution gel coating according to the method of example 4. Note that superparamagnetic particles are not added during preparation, and the gel coating of the prepared microsphere does not contain superparamagnetic particles.
(4) And (3) blending the seed cell-microspheres with the elutable superparamagnetic gel coating prepared in the step (2) and the target cell-microspheres with the elutable gel coating prepared in the step (3), performing co-culture, and adjusting the distance between the microspheres by adjusting the concentration of the two microspheres according to actual needs.
(5) And (3) after the co-culture is finished, applying a magnetic field to separate the two microspheres, eluting and removing the superparamagnetic gel coating on the surfaces of the seed cells and the microspheres by referring to the method of the embodiment 8 after the separation, and removing the superparamagnetic gel coating to be used for clinical implantation repair.
Example 12
In this example, chondrocyte-hyaluronic acid gel microspheres with an elutable superparamagnetic gel coating were prepared as follows:
(1) taking a proper amount of methacrylic acid hyaluronic acid, and adding the methacrylic acid hyaluronic acid into a PBS (phosphate buffer solution) containing a photoinitiator I2959 prepared in advance; counting cartilage cells prepared in advance, preparing cell suspension by using an alpha-MEM culture medium, adding the cell suspension into a hyaluronic acid solution, and uniformly dispersing to obtain a cell-hyaluronic acid mixed solution, wherein the final concentration of methacrylated hyaluronic acid in the cell-hyaluronic acid mixed solution is 10mg/mL, and the cell density is 5 multiplied by 106one/mL.
(2) Taking 70mL of methyl silicone oil with the viscosity of 50 mPas, adding 0.01% (v/v) of Span-80 to form an oil phase, stirring at the rotating speed of 500rpm by using a magnetic stirrer, dropwise adding a cell-hyaluronic acid mixed solution (water phase) into the oil phase under the condition of ice-water bath, continuously stirring for 30 minutes, inserting an ultraviolet light emission probe into the mixed solution, irradiating for 1 minute to complete the gelation treatment of the microspheres, centrifuging the obtained mixed solution, dispersing a bottom layer precipitate into an alpha-MEM culture medium, cleaning, and washing for 3 times by using the alpha-MEM culture medium to obtain the chondrocyte-hyaluronic acid gel microspheres.
(3) And (3) putting the chondrocyte cell-hyaluronic acid gel microspheres prepared in the step (2) into a centrifuge tube, washing the chondrocyte cell-hyaluronic acid gel microspheres for 3 times by using PBS (phosphate buffer solution), then putting the cleaned chondrocyte cell-hyaluronic acid gel microspheres into the PBS buffer solution at 4 ℃ for soaking for 5 minutes, sucking the liquid dry, and keeping the liquid in an ice bath at 4 ℃.
(4) Diluting 8% gelatin water solution at 30 deg.C with 4mg/mL solution containing 2 μm superparamagnetic particles, mixing well to obtain coating material, and keeping at 30 deg.C for use.
(5) And (3) dropwise adding the coating material prepared in the step (4) onto the chondrocyte-hyaluronic acid gel microspheres processed in the step (3), uniformly mixing, wherein the coating material dropwise added to the surfaces of the gel microspheres is converted into a gel state when contacting the gel microspheres at 4 ℃ to form a gelatin gel coating, removing redundant coating material on the surfaces of the microspheres through centrifugation, and then placing the microspheres in a culture medium for washing for 3 times to remove redundant gelatin and superparamagnetic particles, so that the cell-hyaluronic acid gel microspheres with the elutable superparamagnetic gel coating are obtained.
(6) And (4) taking the cell-hyaluronic acid gel microspheres with the elutable superparamagnetic gel coating obtained in the step (5), and repeating the operations in the steps (3) and (5) for 3 times to obtain the cell-hyaluronic acid gel microspheres coated with four layers of elutable superparamagnetic gel coatings.
The cell-hyaluronic acid gel microspheres with the elutable superparamagnetic gel coating prepared in the example were used for co-culture, the co-culture temperature was controlled at 20 ℃, after the co-culture was completed, the microspheres were separated and washed in 35 ℃ PBS buffer to remove the superparamagnetic gel coating.
The gelatin gel of the superparamagnetic gel coating in this embodiment may also be replaced with poly (N-isopropylacrylamide) gel, and after the superparamagnetic gel coating is formed in an appropriate manner, the poly (N-isopropylacrylamide) gel coating may also be removed in a manner of changing the ambient temperature.
Claims (6)
1. The cell-microsphere with the elutable superparamagnetic gel coating is characterized by comprising a gel microsphere and a superparamagnetic gel coating for coating the gel microsphere, wherein the gel microsphere contains cells, the superparamagnetic gel coating contains superparamagnetic particles, the superparamagnetic gel coating can be eluted and removed, and the superparamagnetic gel coating and the gel microsphere cannot be simultaneously eluted and removed;
the gel microsphere is different from the gel material forming the superparamagnetic gel coating; the gel material forming the gel microsphere is a temperature-sensitive material or a photosensitive material, and the gel material forming the superparamagnetic gel coating is an ion-sensitive material, and the superparamagnetic gel coating is removed by dissolving in a solution; or the gel material forming the gel microsphere is a photosensitive material, a temperature-sensitive material or an ion-sensitive material, the gel material forming the superparamagnetic gel coating is a temperature-sensitive material which can be reversibly converted between a liquid state and a gel state along with the change of the environmental temperature, the superparamagnetic gel coating is removed by changing the environmental temperature, and when the gel materials forming the gel microsphere and the superparamagnetic gel coating are both temperature-sensitive materials, the selection principle of the temperature-sensitive materials is to ensure that the gel material forming the gel microsphere and the gel material forming the superparamagnetic gel coating are not removed under the same temperature condition;
the size of the superparamagnetic particles in the superparamagnetic gel coating is larger than the size of a gel network of the superparamagnetic gel coating, and the size of the superparamagnetic particles in the superparamagnetic gel coating is larger than the size of the gel network of the gel microspheres, so that the superparamagnetic particles are prevented from permeating into the gel microspheres and leaking into a culture medium for cell co-culture; the size of the superparamagnetic particles is 1-50 mu m; the thickness of the superparamagnetic gel coating is 10-150 mu m.
2. The cell-microsphere with an elutable superparamagnetic gel coat according to claim 1, wherein said temperature sensitive material capable of reversibly switching between a liquid state and a gel state with a change in environmental temperature comprises gelatin gel and poly (N-isopropylacrylamide) gel.
3. The cell-microsphere with the elutable superparamagnetic gel coat according to claim 1, wherein the content of superparamagnetic particles in the superparamagnetic gel coat is 0.1-10 mg/mL.
4. Cell-microsphere with elutable superparamagnetic gel coating according to any of claims 1 to 3, wherein said cell-microsphere is spherical or nearly spherical and has a size of 30 to 3000 μm.
5. A process for the preparation of cell-microspheres with an elutable superparamagnetic gel coating according to any of claims 1 to 4, characterized in that it comprises the following steps:
(1) adjusting the pH value of the gel material solution to 6.5-7.5, removing bubbles in the gel material solution, preparing the counted cells into cell suspension by using a culture medium or a buffer solution, and then adding the cell suspension into the gel material solution from which the bubbles are removed to obtain a cell-gel material mixed solution;
(2) dispersing the cell-gel material mixed solution into an oil phase liquid, performing emulsification and gelation treatment to obtain cell-microspheres, and regulating the surface environment of the cell-microspheres to the condition required by gelation of the coating material in the step (3);
(3) uniformly mixing the gel material and the superparamagnetic particles to form a coating material, uniformly coating the coating material on the surfaces of the cells and the microspheres, gelatinizing the coating material coated on the surfaces of the cells and the microspheres to convert the coating material into a gel coating, removing the redundant coating material through centrifugation or standing, and cleaning to obtain the cells and the microspheres with the elutable superparamagnetic gel coating.
6. Use of the cell-microspheres with an elutable superparamagnetic gel coat according to any of claims 1 to 4 in cell co-culture and post-co-culture separation of cell-containing gel microspheres for clinical implant repair.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811402090.1A CN109517781B (en) | 2018-11-23 | 2018-11-23 | Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811402090.1A CN109517781B (en) | 2018-11-23 | 2018-11-23 | Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109517781A CN109517781A (en) | 2019-03-26 |
| CN109517781B true CN109517781B (en) | 2022-07-08 |
Family
ID=65777719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811402090.1A Active CN109517781B (en) | 2018-11-23 | 2018-11-23 | Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517781B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114621638B (en) * | 2020-12-10 | 2022-11-01 | 深圳先进技术研究院 | Hybrid surface coating and preparation method and application thereof |
| CN113304700B (en) * | 2021-06-11 | 2023-06-02 | 上海科技大学 | Method for preparing magnetic polymer microsphere and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102861541A (en) * | 2012-09-25 | 2013-01-09 | 陕西师范大学 | Preparation method of surface-modified fluorescent magnetic polymer composite microspheres |
| WO2013112224A3 (en) * | 2011-11-09 | 2013-10-31 | The Regents Of The University Of California | Superparamagnetic colloids with enhanced charge stability for high quality magnetically tunable photonic structures |
| CN104436306A (en) * | 2014-11-11 | 2015-03-25 | 四川大学 | Cell-gel material composite microsphere as well as preparation method and application thereof |
| EP2865394A4 (en) * | 2012-06-21 | 2016-01-27 | Consejo Superior Investigacion | Compounds having magnetic functionality, implants or gels derived from same, and use of both in order to determine the enzyme activity of an enzyme |
-
2018
- 2018-11-23 CN CN201811402090.1A patent/CN109517781B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013112224A3 (en) * | 2011-11-09 | 2013-10-31 | The Regents Of The University Of California | Superparamagnetic colloids with enhanced charge stability for high quality magnetically tunable photonic structures |
| EP2865394A4 (en) * | 2012-06-21 | 2016-01-27 | Consejo Superior Investigacion | Compounds having magnetic functionality, implants or gels derived from same, and use of both in order to determine the enzyme activity of an enzyme |
| CN102861541A (en) * | 2012-09-25 | 2013-01-09 | 陕西师范大学 | Preparation method of surface-modified fluorescent magnetic polymer composite microspheres |
| CN104436306A (en) * | 2014-11-11 | 2015-03-25 | 四川大学 | Cell-gel material composite microsphere as well as preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109517781A (en) | 2019-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yin et al. | Agarose particle-templated porous bacterial cellulose and its application in cartilage growth in vitro | |
| Wenz et al. | Bone matrix production in hydroxyapatite-modified hydrogels suitable for bone bioprinting | |
| Huang et al. | Porous chitosan microspheres as microcarriers for 3D cell culture | |
| Gong et al. | Microcavitary hydrogel-mediating phase transfer cell culture for cartilage tissue engineering | |
| Skardal et al. | The generation of 3-D tissue models based on hyaluronan hydrogel-coated microcarriers within a rotating wall vessel bioreactor | |
| US8815276B2 (en) | Three-dimensional nanostructured hybrid scaffold and manufacture thereof | |
| Huri et al. | Scaffold pore size modulates in vitro osteogenesis of human adipose-derived stem/stromal cells | |
| CN100464790C (en) | Micro rack for bone tissue engineering and its prepn process and application | |
| Heo et al. | Aspiration-assisted bioprinting of co-cultured osteogenic spheroids for bone tissue engineering | |
| CN108888803A (en) | A kind of biological support and preparation method thereof, purposes and aquogel system | |
| EP1624054A1 (en) | Cell culture method and cultured tissue | |
| Green | Tissue bionics: examples in biomimetic tissue engineering | |
| CN104436306B (en) | A kind of Cellular gels Material cladding microballoon and its preparation method and application | |
| AU2018250832B2 (en) | Hydrogel for cell culture and biomedical applications | |
| Lima et al. | Novel methodology based on biomimetic superhydrophobic substrates to immobilize cells and proteins in hydrogel spheres for applications in bone regeneration | |
| Yao et al. | Injectable cell/hydrogel microspheres induce the formation of fat lobule-like microtissues and vascularized adipose tissue regeneration | |
| CN109517781B (en) | Cell-microsphere with elutable superparamagnetic gel coating and preparation method and application thereof | |
| Sobreiro-Almeida et al. | Decellularized kidney extracellular matrix bioinks recapitulate renal 3D microenvironment in vitro | |
| US9334473B2 (en) | Three dimensional cell culture compositions and methods of use | |
| WO2005014774A1 (en) | Carrier for culturing animal cell, and method for culturing or transplanting animal cell using said carrier for culture | |
| JP2019506888A (en) | Interphase movement of scaffolding with cargo | |
| Yamamoto et al. | Generation of stable co-cultures of vascular cells in a honeycomb alginate scaffold | |
| Liu et al. | Egg white improves the biological properties of an alginate-methylcellulose bioink for 3D bioprinting of volumetric bone constructs | |
| Veiga et al. | Silk‐based microcarriers: current developments and future perspectives | |
| Wang et al. | Cell migration of preosteoblast cells on a clinical gelatin sponge for 3D bone tissue engineering |
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 |