CN111690590B - Three-dimensional gel with quantitatively-controllable local microstructure and preparation and application methods thereof - Google Patents
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Abstract
The invention discloses a preparation method of a three-dimensional gel with a quantitatively controllable local microstructure, which comprises the following steps of S1, dividing a gel trunk macromolecular solution into a first trunk macromolecular solution part and a second trunk macromolecular solution part; s2, mixing the first part of the main macromolecular solution and the second part of the main macromolecular solution with the adhesive peptide chains with different amounts of substances respectively, and incubating to graft the mixture to obtain a first product and a second product; s3, adding site blocking molecules with different material amounts into the first product and the second product respectively to obtain a third product and a fourth product which have different amounts of vacant crosslinking sites; s4, incubating the product III and the product IV at room temperature for 2-10 minutes, and mixing to obtain a mixture V; s5, adding the cell suspension into the mixture V, and uniformly stirring to obtain a precursor solution containing cells; s6, adding a cross-linking molecule solution to the inner bottom surface of the container, and then mixing the cell-containing precursor solution with the cross-linking molecule solution to obtain the three-dimensional gel with non-uniform cross-linking density and adhesive peptide chains.
Description
Technical Field
The invention relates to a three-dimensional gel with a local microstructure capable of being regulated and controlled quantitatively, and a preparation method and an application method thereof, and belongs to the technical field of three-dimensional gels.
Background
The in vitro three-dimensional culture or four-dimensional culture of cells is an important technical means in the field of tissue engineering and regenerative medicine, different biological materials and technical methods are used for constructing an environment close to in vivo growth and utilizing the inherent self-organizing property of cell populations to enable the cells to present a three-dimensional growth state, and the internal structure or physicochemical property of extracellular matrix materials is adjusted to guide the change of the adhesion, extension, proliferation and differentiation capacities and characteristics of the cells. The construction of the three-dimensional extracellular matrix with excellent properties and flexible regulation is one of the key points of 3D or 4D culture, while the currently adopted materials are various natural or synthetic hydrogels, but all the components in the common hydrogel are homogeneous, and the rigidity and the distribution of adhesion sites of the hydrogel are uniform. When the distribution of adhesion sites within the hydrogel is non-uniform, the amount of stress that can be experienced by each pseudopodal adhesion site of a cell varies, which affects the morphological characteristics of the cell in terms of adhesion site selection, as well as in terms of extension. On the other hand, when the local rigidity values in the hydrogel are different, the material properties of the whole gel can be changed, and if the non-uniform distribution state of the cross-linked molecules can be formed on a scale which is obviously smaller than that of the cells, the adhesion, extension, migration and even differentiation rules of the cells can be influenced.
Disclosure of Invention
The purpose is as follows: in order to overcome the defects in the prior art, the invention provides the three-dimensional gel with the local microstructure capable of being quantitatively regulated and controlled, and the preparation and application methods thereof, so that the internal structure of the hydrogel can be flexibly regulated and controlled, the distribution of cross-linking molecules and adhesion molecules of the hydrogel can be quantitatively regulated and controlled, and the way of controlling the growth and self-organization of cells in the three-dimensional gel is further promoted.
The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a preparation method of three-dimensional gel with a quantitatively regulated local microstructure comprises the following steps,
step S1, dividing a gel trunk macromolecular solution into two parts, namely a first part of the trunk macromolecular solution and a second part of the trunk macromolecular solution;
s2, mixing the first part of the main macromolecular solution and the second part of the main macromolecular solution with the adhesive peptide chains with different amounts of substances respectively, and incubating to graft the two parts to obtain a first product and a second product;
s3, adding site blocking molecules with different substance amounts into the product I and the product II respectively to obtain a product III and a product IV, wherein the product III and the product IV have different numbers of vacant crosslinking sites;
s4, respectively incubating the product III and the product IV at room temperature for 2-10 minutes, and then mixing together to obtain a mixture V;
s5, adding the cell suspension into the mixture V, and uniformly stirring to obtain a precursor solution containing cells;
and S6, adding a cross-linking molecule solution to the inner bottom surface of the container, and then mixing the precursor solution containing the cells with the cross-linking molecule solution to obtain the three-dimensional gel with non-uniform cross-linking density and non-uniform adhesive peptide chain.
Further, the specific operation process of step S1 is as follows:
s11, taking out the distilled water and the carbonate buffer solution which are frozen to be solid at the temperature of minus 80 ℃, and melting the distilled water and the carbonate buffer solution to be liquid at room temperature to obtain liquid distilled water and liquid carbonate buffer solution;
s12, taking out the maleimide-glucose polymer which is frozen at the temperature of minus 80 ℃ and is in a solid state, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain a maleimide-glucose polymer solution;
step S13, uniformly mixing the liquid distilled water and the liquid buffer solution obtained in the step S11 and the maleimide-glucose polymer solution obtained in the step S12 in a centrifugal tube to obtain a gel backbone macromolecule solution;
step S14, dividing the gel trunk macromolecular solution obtained in the step S13 into two parts, namely a first part of the trunk macromolecular solution with the volume of V1 and a second part of the trunk macromolecular solution with the volume of V2.
Further, the volume V1 of the first portion of the stem macromolecule solution is equal to the volume V2 of the second portion of the stem macromolecule solution.
Further, the specific operation process of step S2 is as follows:
the adhesion peptide chain is an RGD peptide chain containing sulfydryl;
s21, taking out the RGD peptide chain solid substance containing the sulfhydryl frozen at the temperature of-20 ℃ or-80 ℃, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain RGD peptide chain aqueous solution containing the sulfhydryl;
s22, adding a water solution containing sulfhydryl RGD peptide chain with the volume of V3 into the first part of the main macromolecular solution in the step S1, uniformly mixing by blowing, and standing at room temperature for 5-6 minutes to obtain a first product;
and S22, adding a V4-volume water solution containing a sulfhydryl RGD peptide chain into the second part of the main macromolecular solution in the step S1, uniformly mixing by blowing, and standing at room temperature for 5-6 minutes to obtain a second product.
Further, preparing a plurality of parts of water solutions containing the sulfhydryl RGD peptide chain with different concentrations according to the operation of the step S21, wherein the mass concentration of substances containing the sulfhydryl RGD peptide chain in the water solutions containing the sulfhydryl RGD peptide chain is less than 100 mu M.
Further, the step S3 includes a process of,
the site-blocking molecule is thioglycerol;
s31, taking out the thioglycerol which is frozen at the temperature of minus 80 ℃ and is in a solid state, adding distilled water to dissolve the thioglycerol, and oscillating the thioglycerol by a vortex oscillator during dissolution to obtain a thioglycerol molecular solution;
step S32, adding a thioglycerol molecule solution with the volume of V5 into the product I in the step S2, uniformly mixing by blowing, and standing at room temperature for T 0 Taking minutes to obtain a third product;
s33, adding a thioglycerol molecule solution with the volume of V6 into the product II in the step S2, uniformly mixing by blowing, and standing at room temperature for T 0 After a few minutes, the product four is obtained.
Wherein, T is more than or equal to 3 0 ≤5。
The three-dimensional gel with the local microstructure capable of being quantitatively regulated and controlled is prepared by the method.
The application method of the three-dimensional gel capable of being quantitatively regulated and controlled based on the local microstructure comprises the following processes,
covering the gel sample with a fresh culture medium, and then transferring the culture plate to an incubator; in the culture process, the culture medium is replaced every two days at the first two times, and then the culture medium is replaced every day;
the cultured three-dimensional gel is used for constructing a large-scale gel matrix gradient structure.
Further, the application process of the cultured three-dimensional gel in the construction of a large-scale gel matrix gradient structure is as follows:
the bottom layer gel structure is printed by three-dimensional gel 3D with low micro-heterogeneity of cross-linked molecules, and the micro-heterogeneity of the cross-linked molecules of the gel in the bottom layer upward gel layer structure is gradually improved, namely the rigidity of the gel material is gradually reduced from the bottom layer upward.
Further, the application process of the cultured three-dimensional gel in the construction of a large-scale gel matrix gradient structure is as follows:
the bottom layer gel structure is printed by three-dimensional gel 3D with low micro-heterogeneity of cross-linked molecules, the second layer gel structure above the bottom layer is spliced by a plurality of gel blocks, and the gel blocks are formed by 3D printing of gel materials with different micro-heterogeneity of cross-linked molecules.
Has the beneficial effects that: the preparation method of the three-dimensional gel with the local microstructure capable of being quantitatively regulated and controlled is simple and easy to operate, and by quantitatively mixing products with different component contents according to design values, clustering aggregation of adhesion molecules and crosslinking molecules can be simultaneously realized on a nanoscale, so that a microscopic non-uniform state of a gel material is realized, and a quantitative forming process of complex non-uniform gel is avoided; the microscopically inhomogeneous state refers to the inhomogeneous distribution of a certain component or several component molecules within the scale of several nanometers to several hundred nanometers. The micro-heterogeneity degree of each component in the three-dimensional gel prepared by the invention can be independently and quantitatively controlled, and the three-dimensional gel has positive effects on quantitatively researching the cell response characteristics of specific components under different micro-heterogeneity degrees, regulating and controlling the interaction between cells and materials, promoting the cell extension and migration and the like.
Drawings
FIG. 1 is a schematic diagram of a three-dimensional gel preparation process of the present invention;
FIG. 2 is a schematic diagram of a two-layer gel structure in example 1 of the application of the three-dimensional gel of the present invention;
FIG. 3 is a schematic diagram of a two-layer gel structure in three-dimensional gel application example 2 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
The method comprises the steps of constructing the gel with the structural components aggregated in local clusters so that the concentration of a certain component in the gel has a micro gradient change characteristic in microscopic local parts, specifically, respectively grafting high-density small molecules and low-density small molecules onto polymer molecular chains, and then mixing the high-density small molecules and the low-density small molecules, so that the microscopic local non-uniform distribution of the small molecules dispersed in the gel is obtained. The gradient distribution of the density of the small molecules at microscopic positions can change the spatial distribution of the interaction stress between the cells and the gel material, and the concentration distribution of the cytokines in the space is also changed.
The three-dimensional gel with a quantifiable local microstructure and the preparation and application methods thereof are specifically described by combining with the graph shown in FIG. 1.
A preparation method of three-dimensional gel with a quantitatively regulated local microstructure comprises the following steps:
s1, taking out the solid distilled water and the carbonate buffer solution which are frozen at the temperature of minus 80 ℃, and melting the solid distilled water and the carbonate buffer solution into liquid at room temperature to obtain liquid distilled water and liquid carbonate buffer solution; and (3) taking the maleimide-glucose polymer which is frozen to be in a solid state at the temperature of-80 ℃, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain a maleimide-glucose polymer solution.
Uniformly mixing the liquid distilled water and the liquid buffer solution with the maleimide-glucose polymer solution in a centrifugal tube to obtain a gel trunk macromolecular solution 01; dividing the gel main macromolecule solution 01 into a main macromolecule solution first part 021 with the volume of V1 and a main macromolecule solution second part 022 with the volume of V2; v1= V2.
S2, taking out the RGD peptide chain solid substance containing the sulfhydryl group frozen at the temperature of-20 ℃ or-80 ℃, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain RGD peptide chain aqueous solution containing the sulfhydryl group; the quantity concentration of the substance containing the sulfhydryl RGD peptide chain is less than 100 mu M, and a plurality of portions of sulfhydryl RGD peptide chain-containing water solutions with different concentrations can be prepared according to requirements.
Uniformly mixing the first part 021 of the main macromolecular solution with a sulfhydryl RGD peptide chain-containing aqueous solution with the volume of V3, standing at room temperature for 5-6 minutes, and incubating to graft the mixture to obtain a product I031; and uniformly mixing the second part 022 of the main macromolecular solution and the hydrosulfuryl RGD peptide chain-containing aqueous solution with the volume of V4 in another tube, standing for 5-6 minutes at room temperature, and incubating to graft the mixture to obtain a product of two 032, wherein V3 is more than V4. Product one 031 is the subsequent low crosslinking moiety (i.e., L moiety, which may be 0 in volume) and product two 032 is the subsequent high crosslinking moiety (i.e., H moiety).
And S3, taking out the thioglycerol which is frozen at the temperature of-80 ℃ and is in a solid state, adding distilled water to dissolve the thioglycerol, and oscillating the thioglycerol by a vortex oscillator during dissolving to obtain a thioglycerol molecular solution.
Adding a thioglycerol molecular solution with the volume of V5 into the product I031, uniformly mixing by blowing, and standing at room temperature for 4.5 minutes to obtain a product III 05; and adding a thioglycerol molecular solution with the volume of V6 into the second 032 product, uniformly mixing by blowing, and standing at room temperature for 4.5 minutes to obtain a fourth 06 product, wherein V5 is more than V6. Because thiol-containing thioglycerol can occupy the cross-linking sites of maleimide-dextran, different amounts of thioglycerol are added to leave different amounts of control cross-linking sites, i.e., more thioglycerol is added to product one 031, so that the rate of maleimide-dextran cross-linking is relatively reduced, and product three 05 with fewer vacant cross-linking sites is obtained; and adding less thioglycerol into the second product 032 to relatively improve the crosslinking rate of the maleimide-glucan and obtain a fourth product 06 with more vacant crosslinking sites.
S4, product three 05 and product four 06 were each incubated at room temperature for 5-6 minutes and then mixed together to give mixture five 07.
And S5, adding the cell suspension into the mixture five 07, and uniformly stirring to obtain a precursor solution containing the cells 082.
S6, adding a polyethylene glycol-oligopeptide conjugate solution to the inner bottom surface of a hole of a 96-hole plate, and then mixing a precursor solution containing cells 082 and cross-linking molecules 081 in the 96-hole plate to obtain the three-dimensional gel 09 with non-uniform cross-linking density and non-uniform adhesion peptide chain. As shown in fig. 1, the region with a lower cross-linked molecule density is 0811, and the region with a higher cross-linked molecule density is 0812; the region with lower density of the adhesion peptide chain is 0201, and the region with higher density of the adhesion peptide chain is 0202.
The application process of the three-dimensional gel prepared by the method is as follows:
covering the hydrogel sample with 120-150 mu L of fresh culture medium, and then moving the culture plate to an incubator; the volume of the hydrogel sample was maintained at 30 μ L. In the culture process, the culture medium is replaced every two days at the first two times, and then the culture medium is replaced every day; the cultured three-dimensional gel is used for constructing a large-scale gel matrix gradient structure.
Application example 1
As shown in fig. 2, a two-layer gel structure was constructed by 3D printing. The microscopic unevenness of the cross-linked molecules in the bottom layer gel 11 is low, that is, the difference between the densities of the cross-linked molecules in the microscopic regions 0811 and 0812 of the material is small, so that the overall stronger rigidity is achieved, and the stable support of the base is realized. The degree of microscopic heterogeneity of the cross-linked molecules in the layer above the bottom layer, i.e. the second layer gel 12, is increased, i.e. the difference between the cross-linked molecule densities in the microscopic regions 0811 and 0822 of the gel molecules in the second layer is increased, and the overall stiffness of the second layer is reduced compared to the bottom layer gel. Therefore, a more diversified gradient structure with larger scale is constructed, and the rigidity and the stability of the whole structure can be kept larger.
Application example 2
As shown in fig. 3, a two-layer gel structure was constructed by 3D printing. The microscopic nonuniformity of the cross-linked molecules in the bottom layer gel 11 is low, that is, the difference between the cross-linked molecule densities of the material microscopic regions 0811 and 0812 is small, so that the overall stronger rigidity is favorably achieved, and the stable support of the base is realized. The second layer of gel above the bottom layer of gel 11 constitutes a macrostructure gradient in the horizontal direction, and the second layer of gel is composed of 3D-printed gel blocks 121, 122, 123, 124, and the three-dimensional gel materials adopted by the gel blocks 121, 122, 123, 124 have different microscopic non-uniformity of cross-linked molecules and microscopic non-uniformity of adhered peptide chains, and as shown in fig. 3, the microscopic non-uniformity of cross-linked molecules and the microscopic non-uniformity of adhered peptide chains can show a gradually increasing or decreasing trend in the order from left to right. Thus, a gel matrix gradient structure with a larger scale and complex internal structure characteristics is constructed, so that the mechanical signal stimulation of diversification and gradient of internal cells is realized.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention, and such modifications and adaptations are intended to be within the scope of the invention.
Claims (9)
1. A preparation method of three-dimensional gel with a local microstructure capable of being regulated and controlled quantitatively is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
step S1, uniformly mixing liquid distilled water and liquid buffer solution with a maleimide-glucose polymer solution to obtain a gel backbone macromolecule solution (01), dividing the gel backbone macromolecule solution (01) into two parts, namely a backbone macromolecule solution first part (021) with the volume of V1 and a backbone macromolecule solution second part (022) with the volume of V2, wherein V1= V2;
s2, preparing a sulfhydryl-containing RGD peptide chain with the substance quantity concentration of less than 100 mu M, uniformly mixing a first part (021) of a main macromolecular solution with a sulfhydryl-containing RGD peptide chain aqueous solution with the volume of V3, standing at room temperature for 5-6 minutes, incubating to graft to obtain a first product (031), uniformly mixing a second part (022) of the main macromolecular solution with a sulfhydryl-containing RGD peptide chain aqueous solution with the volume of V4 in another tube, standing at room temperature for 5-6 minutes, incubating to graft to obtain a second product (032), wherein V3 is greater than V4;
s3, adding a thioglycerol molecule solution with the volume of V5 into the product I (031), adding a thioglycerol molecule solution with the volume of V6 into the product II (032), uniformly mixing, standing at room temperature for 3-5 minutes to respectively obtain a product III (05) and a product IV (06), wherein V5 is larger than V6, and the product III (05) and the product IV (06) have different numbers of vacant crosslinking sites;
step S4, respectively incubating the product III (05) and the product IV (06) for 2-10 minutes at room temperature, and then mixing together again to obtain a mixture V (07);
s5, adding the cell suspension into the mixture V (07), and uniformly stirring to obtain a precursor solution containing cells (082);
s6, adding a cross-linking molecule solution (081) to the inner bottom surface of the container, and then mixing a precursor solution containing cells with the cross-linking molecule solution (081) to obtain the three-dimensional gel (09) with non-uniform cross-linking density and non-uniform adhesion peptide chain.
2. The method for preparing the three-dimensional gel with the quantitative regulation and control local microstructure according to claim 1, wherein the method comprises the following steps: the specific operation process of the step S1 is as follows:
s11, taking out the solid distilled water and the carbonate buffer solution which are frozen at the temperature of minus 80 ℃, and melting the solid distilled water and the carbonate buffer solution into liquid at room temperature to obtain liquid distilled water and liquid carbonate buffer solution;
s12, taking out the maleimide-glucose polymer which is frozen at the temperature of minus 80 ℃ and is in a solid state, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain a maleimide-glucose polymer solution;
step S13, uniformly mixing the liquid distilled water and the liquid buffer solution obtained in the step S11 and the maleimide-glucose polymer solution obtained in the step S12 in a centrifugal tube to obtain a gel backbone macromolecule solution (01);
step S14, dividing the gel backbone macromolecule solution (01) obtained in the step S13 into two parts, namely a backbone macromolecule solution first part (021) with the volume of V1 and a backbone macromolecule solution second part (022) with the volume of V2.
3. The method for preparing the three-dimensional gel with the quantitative regulation and control of the local microstructure according to claim 1, wherein the method comprises the following steps: the specific operation process of the step S2 is as follows:
s21, taking out the RGD peptide chain solid substance containing the sulfhydryl group frozen at the temperature of-20 ℃ or-80 ℃, adding distilled water for dissolving, and oscillating through a vortex oscillator during dissolving to obtain RGD peptide chain aqueous solution containing the sulfhydryl group;
s22, adding a sulfhydryl-containing RGD peptide chain aqueous solution with the volume of V3 into the first part (021) of the main macromolecular solution in the step S1, blowing, uniformly mixing, and standing at room temperature for 5-6 minutes to obtain a first product (031);
and S22, adding a sulfhydryl-containing RGD peptide chain aqueous solution with the volume of V4 into the second part (022) of the trunk macromolecular solution in the step S1, blowing, beating and uniformly mixing, and standing at room temperature for 5-6 minutes to obtain a second product (032).
4. The method for preparing the three-dimensional gel with the quantitative regulation and control local microstructure according to claim 3, wherein the method comprises the following steps: preparing a plurality of water solutions containing sulfhydryl RGD peptide chains with different concentrations according to the operation of the step S21.
5. The method for preparing the three-dimensional gel with the quantitative regulation and control local microstructure according to claim 1, wherein the method comprises the following steps: said step S3 comprises the following procedure,
s31, taking out the thioglycerol which is frozen at the temperature of minus 80 ℃ and is in a solid state, adding distilled water to dissolve the thioglycerol, and oscillating the thioglycerol by a vortex oscillator during dissolving to obtain a thioglycerol molecular solution;
step S32, adding a thioglycerol molecular solution with the volume of V5 into the product I (031) in the step S2, uniformly blowing, standing at room temperature and keeping the mixture T at room temperature 0 After minutes, the product, three (05), is obtained;
step S33, adding a thioglycerol molecular solution with the volume of V6 into the product II (032) obtained in the step S2, blowing, beating and uniformly mixing, and standing at room temperature for T 0 Min, product four (06);
wherein T is more than or equal to 3 0 ≤5。
6. A three-dimensional gel with a quantitatively controllable local microstructure, which is characterized in that: prepared by the process of any one of claims 1 to 5.
7. The method for applying the three-dimensional gel with the quantitative regulation and control of the local microstructure according to claim 6, wherein the method comprises the following steps: comprises the following steps of,
covering the gel sample with a fresh culture medium, and then transferring the culture plate to an incubator; in the culture process, the culture medium is replaced every two days for the first two times, and then the culture medium is replaced every day;
the cultured three-dimensional gel is used for constructing a large-scale gel matrix gradient structure.
8. The method for applying the three-dimensional gel with the quantitative regulation and control of the local microstructure according to claim 7, wherein the method comprises the following steps: the application process of the cultured three-dimensional gel in the construction of a large-scale gel matrix gradient structure is as follows:
the bottom layer gel structure is printed by three-dimensional gel 3D with low micro-heterogeneity of cross-linked molecules, and the micro-heterogeneity of the cross-linked molecules of the gel in the bottom layer upward gel layer structure is gradually improved, namely the rigidity of the gel material is gradually reduced from the bottom layer upward.
9. The method for applying the three-dimensional gel with the quantitative regulation and control local microstructure according to claim 7, wherein the method comprises the following steps: the application process of the cultured three-dimensional gel in the construction of a large-scale gel matrix gradient structure is as follows:
the bottom layer gel structure is printed by three-dimensional gel 3D with low microscopic non-uniformity of cross-linked molecules, the second layer gel structure above the bottom layer is spliced by a plurality of gel blocks, and the gel blocks are formed by 3D printing of gel materials with different microscopic non-uniformity of cross-linked molecules.
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