A porous scaffold with a cell adhesion layer, its preparation method and application

Abstract

This invention provides a porous scaffold with a cell adhesion layer, its preparation method, and its application. The preparation method involves designing reactive groups in the hydrogel precursor and cell adhesion layer material to successfully modify the cell adhesion layer onto the surface of the porous hydrogel (including the inner walls of the pores), forming a porous hydrogel scaffold with cell adhesion properties. This porous scaffold with a cell adhesion layer combines cell proliferation and spheroid culture recovery functions, showing broad prospects in cell culture and tissue engineering. When applied to cell culture, it enables cells to adhere to the inner walls of the pores of the porous scaffold during cell seeding. After the cells proliferate to a predetermined number, the cell adhesion layer is lysed, allowing the cells to form three-dimensional cell microspheres within the pores of the porous scaffold. Following the formation of the cell microspheres, the scaffold matrix is ​​lysed, thereby achieving the collection of the cell microspheres.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a porous scaffold with a cell adhesion layer, its preparation method, and its application. Background Technology

[0002] Multicellular spheroids are one of the most common 3D cell culture systems. Spheroids can be generated from a single cell type or combined through co-culture of different cell types. Cellular spheroids possess a cellular microenvironment more closely resembling that in vivo and exhibit a more vigorous state of cell secretion, and have been applied in various research fields such as tumor models, drug screening, angiogenesis, organoids, tissue regeneration, and 3D printing.

[0003] Traditional cell spheroid culture involves first obtaining a large number of cells, then seeding them onto low-adhesion surfaces, hydrogels, scaffolds, or other carriers, where the cells aggregate to form spheroids. This process requires prior 2D cell expansion, is cumbersome, and hydrogels and scaffolds cannot effectively recover the cell spheroids, limiting subsequent research applications.

[0004] Patent application CN202110982360.6 discloses a method for preparing a biomaterial scaffold containing mesenchymal stem cell spheres. To create space within the scaffold for culturing cell spheres, a water-in-water emulsion is prepared by exploiting the incompatibility between a dextran solution and a specific polymer material. After the hydrogel solidifies, its interior is rich in pores. While this invention creates a macroporous structure within the hydrogel scaffold for cell culture, subsequent cell recovery is not effective, and the poor connectivity between the pores hinders material transfer, making long-term culture unsuitable. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a porous scaffold with a cell adhesion layer and its preparation method. This method utilizes the controllable degradation of a specific component hydrogel to achieve the dissociation and outflow of the pore-forming agent and the controllable disintegration of the adhesion layer material and the matrix material during subsequent use. When this porous scaffold is applied to the recovery of cell spheroids from culture media, the cell adhesion layer on the surface of the porous scaffold promotes cell adhesion and proliferation after cell inoculation, thus playing a role in cell amplification. Subsequently, lysis is used to remove the adhesion layer, and the non-adhesive nature of the porous scaffold body inhibits cell adhesion and promotes cell aggregation to form spheroids. After the cells form spheroids, the porous scaffold matrix material is lysed using a scaffold lysis buffer, thereby achieving the recovery of the cell spheroids. The porous scaffold prepared by the method of this invention combines the functions of cell amplification, cell spheroid culture, and cell microsphere recovery, simplifying the cell microsphere culture process.

[0006] A method for preparing a porous scaffold with a cell adhesion layer includes the following steps:

[0007] (1) Pour an aqueous solution of hydrogel precursor, crosslinking agent and condensing agent into a mold containing a pore-forming agent until the pore-forming agent is covered, and then solidify and demold to obtain hydrogel;

[0008] (2) The obtained hydrogel was immersed in the pore-forming agent lysis solution to decompose the pore-forming agent, and then rinsed with water to obtain a porous hydrogel.

[0009] (3) The porous hydrogel is immersed in a cell adhesion layer modification solution for surface modification, washed with water, and freeze-dried to obtain the porous scaffold with a cell adhesion layer.

[0010] In step (1) above, the pore-forming agent is first densely packed into the mold, and then an aqueous solution of hydrogel precursor, crosslinking agent and condensing agent is poured into the mold to ensure that the pores in the obtained porous hydrogel are interconnected.

[0011] Preferably, the hydrogel precursor is a polymer containing carboxyl groups and double bond functional groups. More preferably, the polymer can be propylene-modified alginate, propylene-modified hyaluronic acid, etc. Even more preferably, acryloyl-modified hyaluronic acid (HAAC) is preferred.

[0012] Preferably, the crosslinking agent is cystamine dihydrochloride.

[0013] Preferably, the molar ratio of carboxyl groups to crosslinking agents in the hydrogel precursor is (1-10):1. More preferably, it is (1-4):1. Even more preferably, it is 2:1.

[0014] Preferably, the condensing agent is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS).

[0015] Preferably, the molar ratio of the crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(1-4):(1-4). More preferably, it is 1:2:2.

[0016] Preferably, the pore-forming agent is cross-linked dextran microspheres;

[0017] Accordingly, the pore-forming agent lysis solution is a dextranase solution.

[0018] As a further preferred embodiment, the dextranase solution contains 1-10% dextranase by mass, and the solvent can be water or PBS buffer. Even more preferably, the dextranase solution contains 5% dextranase by mass.

[0019] As a further preferred embodiment, the diameter of the cross-linked dextran microspheres is 50–500 μm. Even more preferably, it is 200–400 μm.

[0020] Preferably, the cell adhesion layer modification solution is a dextran (SH-Dex-RGD) solution modified with thiol and RGD peptide.

[0021] The carboxyl groups in the hydrogel precursor are used for cross-linking to form a gel; the double bonds therein are used to undergo a Macaddition reaction with the thiol groups in the cell adhesion layer, thereby modifying the cell adhesion layer with porous hydrogel; and the RGD peptides in the cell adhesion layer are used to provide adhesion for cell proliferation, enabling cells to adhere to the inner wall of the porous scaffold pores for proliferation.

[0022] As a further preferred embodiment, the mass fraction of thiol- and RGD peptide-modified dextran in the cell adhesion layer modification solution is 2-20%. More preferably, it is 2-10%. As an even more preferred embodiment, the mass fraction of thiol- and RGD peptide-modified dextran in the cell adhesion layer modification solution is 5%.

[0023] As a further preferred option, the thiol- and RGD peptide-modified dextran is obtained by sequentially coupling carboxylated dextran with cysteine ​​and RGD peptide.

[0024] Specifically, the preparation process of thiol- and RGD peptide-modified dextran is as follows:

[0025] Carboxymethyl dextran was dissolved in water, and cysteamine, EDC·HCl and NHS were added in sequence until fully dissolved. The mixture was stirred at room temperature. After the reaction was completed, the mixture was dialyzed with deionized water. After the dialysis reaction was completed, the thiolated dextran was obtained by freeze drying.

[0026] The above-mentioned thiolized dextran was dissolved in deionized water, and RGD peptide, EDC·HCl and NHS were added in sequence until fully dissolved. The reaction was stirred at room temperature. After the reaction was completed, the reaction was dialyzed with deionized water. After the dialyzed reaction was completed, the thiol- and RGD peptide-modified dextran was obtained by freeze drying.

[0027] Preferably, in step (3), when performing surface modification, the pH of the adhesion layer modification solution is 7-8 and the soaking time is greater than 2 hours.

[0028] A porous scaffold with a cell adhesion layer is prepared by any one of the methods described above. The porous scaffold with a cell adhesion layer prepared by the above methods has a regular porous structure and controllable disintegration characteristics. Both the outer surface and the inner surface of the pores of the porous scaffold are modified with cell adhesion peptides (RGD peptides in the cell adhesion layer). These cell adhesion peptides can be mediated to dissociate from the scaffold body using a reagent (adhesion layer lysis buffer). Before the cell adhesion peptides dissociate, inoculated cells can adhere and proliferate on the inner wall of the pores of the porous scaffold. After the adhesion peptides dissociate, the cells detach from the inner wall of the pores of the porous scaffold and aggregate inside the pores to form multicellular spheroids. The porous scaffold is cross-linked by disulfide bonds. Once the cell spheroids have formed inside, the scaffold can be disintegrated using a lysis reagent to harvest the cell spheroids.

[0029] A method for culturing and recovering cell spheroids includes the following steps:

[0030] a. Place the porous scaffold in the cell culture plate, add cell suspension and culture medium, and culture.

[0031] b. After cell proliferation, add adhesion layer lysis buffer to the well plate to decompose the adhesion layer and cause the cells to detach from the porous scaffold and aggregate in the pores to form multicellular spheres;

[0032] c. After the multicellular spheroids are formed, scaffold lysis buffer is added to the well plate. After the porous scaffold is lysed, the cell spheroids can be collected.

[0033] The porous scaffold is the aforementioned porous scaffold with a cell adhesion layer.

[0034] In step c above, the scaffold lysis buffer can be added at any time after the cell spheroids have formed.

[0035] Preferably, the adhesion layer lysis buffer is a dextranase solution; the adhesion layer lysis buffer corresponds to the cell adhesion layer described above. More preferably, the dextranase solution contains 1-10% dextranase by mass, and the solvent is PBS buffer. Even more preferably, it is 5%.

[0036] Preferably, the scaffold lysis buffer is a PBS solution of dithiothreitol (DTT), tri(2-chloroethyl) phosphate (TECP), or glutathione (GSH). More preferably, the molar concentration of the scaffold lysis buffer is 1–50 mmol / L. Even more preferably, the scaffold lysis buffer is a PBS solution of TECP with a molar concentration of 10 mmol / L.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] The present invention discloses a method for preparing a porous scaffold with a cell adhesion layer. By designing reactive groups in the hydrogel precursor and the cell adhesion layer material, the cell adhesion layer is successfully modified onto the surface of the porous hydrogel (including the inner walls of the pores), forming a porous hydrogel scaffold with cell adhesion properties. This porous scaffold with a cell adhesion layer combines cell proliferation and spheroid culture recovery functions, showing broad prospects in the fields of cell culture and tissue engineering. When applied to cell culture, it enables cells to adhere to the inner walls of the pores of the porous scaffold during cell seeding. After the cells proliferate to a predetermined number, the cell adhesion layer is lysed, allowing the cells to form three-dimensional cell microspheres within the pores of the porous scaffold. Following the formation of the cell microspheres, the scaffold matrix is ​​lysed, thereby achieving the collection of the cell microspheres. Attached Figure Description

[0039] Figure 1 This is a flowchart illustrating the fabrication process of the porous scaffold with a cell adhesion layer in Embodiment 1 of the present invention.

[0040] Figure 2 This is a SEM image of the porous scaffold with a cell adhesion layer prepared in Example 1 of the present invention;

[0041] Figure 3 This is a schematic diagram illustrating the chemical crosslinking and adhesion layer modification principle of the porous scaffold with a cell adhesion layer in Embodiment 1 of the present invention;

[0042] Figure 4 This is a schematic diagram illustrating the process of culturing and recovering cell spheroids using the porous scaffold prepared in Example 1 in Example 2 of the present invention;

[0043] Figure 5 SEM image of cell spheroids (GFP-HUVEC cells) prepared in Example 2 of this invention. Detailed Implementation

[0044] To make the present invention easier to understand, it will be described in further detail below with reference to the accompanying drawings and embodiments:

[0045] Example 1

[0046] 1. Preparation of cell adhesion layer modification materials:

[0047] 1) Preparation of thiolated dextran:

[0048] Dissolve 2g of carboxymethyl dextran in 10mL of deionized water;

[0049] Add 0.42g cysteamine, 1.58g EDC·HCl and 0.94g NHS sequentially and dissolve completely;

[0050] The reaction was stirred at room temperature for 24 hours.

[0051] The reaction was carried out by dialysis with deionized water for 2 days, followed by freeze-drying to obtain thiolated dextran.

[0052] 2) Preparation of RGD-grafted dextran:

[0053] Dissolve 1 g of the above lyophilized product, thiolated dextran, in 5 mL of deionized water;

[0054] Add 0.77g RGD peptide, 0.79g EDC·HCl and 0.47g NHS sequentially and dissolve thoroughly;

[0055] The reaction was stirred at room temperature for 24 hours.

[0056] After dialysis with deionized water for 2 days, the dextran modified with thiol and RGD peptides was obtained by freeze-drying, which is the cell adhesion layer modification material.

[0057] 2. Preparation of porous scaffolds with cell adhesion layers, such as... Figure 1 As shown:

[0058] 1) Dissolve 0.4g of acryloyl hyaluronic acid (-COOH is 1mmol, polymer molecular weight is 100,000-200,000, average molecular weight of structural unit is 403, degree of substitution is 30%) in 10ml of deionized water to prepare a hydrogel precursor solution.

[0059] 2) Dissolve 0.11g cystamine dihydrochloride, 0.19g EDC·HCl and 0.12g NHS in 10ml deionized water to prepare a crosslinking agent solution;

[0060] 3) Add 2g of pore-forming agent dextran microspheres (diameter 300-400μm) into the mold (internal dimensions 5x5x5mm);

[0061] 4) Mix the hydrogel precursor solution and crosslinking agent solution evenly, and slowly add the mixed solution into the mold until it just covers the densely packed dextran microspheres. Let it stand at 4°C for 72 hours to solidify.

[0062] 5) Remove the cured hydrogel from the mold and soak it in a 5% dextranase solution for 1 hour to decompose the pore-forming agent in the hydrogel;

[0063] 6) Rinse with running water to allow the decomposed porogen to flow out of the hydrogel. After the decomposed porogen is completely removed, a non-adhesive porous hydrogel is obtained.

[0064] 7) Immerse the above non-adhesive porous hydrogel in the above-prepared solution of thiol and RGD grafted dextran (solvent is water, mass fraction is 5%), adjust the pH to about 8, and soak for 4 hours;

[0065] 8) After soaking, remove the porous hydrogel and rinse it with running water. Then freeze it at -20℃ for 8 hours and finally freeze it in a freeze dryer to obtain a porous scaffold with a cell adhesion layer.

[0066] Scanning electron microscope images of the porous scaffold with cell adhesion layer prepared above are shown below. Figure 2 .Depend on Figure 2 As can be seen, the porous scaffold exhibits a honeycomb-like porous structure with relatively uniform pore sizes. The pores are interconnected, and small pores connect the pore surfaces. The pore size is approximately 400-500 μm.

[0067] The chemical cross-linking reaction and cell adhesion layer modification principle in the preparation process of the above porous scaffold are described in [reference needed]. Figure 3 .

[0068] Example 2: Cell seeding culture and spheroid harvesting

[0069] This embodiment uses endothelial cells (GFP-HUVEC) as an example, performing seeding culture and cell spheroid recovery on the porous scaffold prepared in Example 1. Figure 4 As shown, the process for recovering the cell seeding medium spheres is as follows:

[0070] 1) The porous scaffold (5x5x5mm) prepared in Example 1 was sterilized by irradiation and then placed in a 48-well plate;

[0071] 2) Add 80 μL of cell suspension (GFP-HUVEC, 1 x 10⁻⁶) to the porous scaffold in the well plate. 4 After the cell suspension has been completely absorbed, slowly add 300 μL of cell culture medium to the well plate.

[0072] 3) Continue culturing for 7-10 days, replacing the culture medium with fresh medium every 2 days;

[0073] 4) After the cell proliferation phase is completed, the porous scaffold is immersed in PBS solution containing 5% (w / w) dextranase and incubated at 37°C for 20 min to decompose the cell adhesion layer;

[0074] 5) The porous scaffold was then transferred into the cell culture medium and cultured for another 7 days until the cells in the pores of the porous scaffold aggregated into a spherical structure.

[0075] 6) After the cells aggregate into spheres, the porous scaffold is immersed in PBS solution containing 10 mmol / L TECP and incubated for 20 min to lyse the scaffold;

[0076] 7) Centrifuge and wash with PBS to recover the cell spheroids.

[0077] Microscopic images of the recovered cell spheroids are shown below. Figure 5 ,Depend on Figure 5It can be seen that the cell spheres prepared by the above method are relatively uniform in size, with a diameter of about 200 μm, and are approximately spherical and relatively dense.

Claims

1. A method for preparing a porous scaffold with a cell adhesion layer, characterized in that, Includes the following steps: (1) Pour an aqueous solution of hydrogel precursor, crosslinking agent and condensing agent into a mold containing pore-forming agent until the pore-forming agent is covered, and then solidify and demold to obtain hydrogel; (2) The obtained hydrogel is immersed in the pore-forming agent lysis solution to decompose the pore-forming agent, and then rinsed with water to obtain a porous hydrogel. (3) The porous hydrogel was immersed in a cell adhesion layer modification solution for surface modification, washed with water, and freeze-dried to obtain the porous scaffold with a cell adhesion layer. The hydrogel precursor is a polymer containing carboxyl groups and double bond functional groups; The pore-forming agent is cross-linked dextran microspheres; The pore-forming agent lysis solution is a dextranase solution; The cell adhesion layer modification solution is a dextran solution modified with thiol and RGD peptide; The crosslinking agent is cystamine dihydrochloride; The condensing agent is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The method for preparing a porous scaffold with a cell adhesion layer according to claim 1, characterized in that, The molar ratio of carboxyl groups to crosslinking agents in the hydrogel precursor is (1~10):

1.

3. The method for preparing a porous scaffold with a cell adhesion layer according to claim 1, characterized in that, The molar ratio of crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(1~4):(1~4).

4. The method for preparing a porous scaffold with a cell adhesion layer according to claim 1, characterized in that, The thiol- and RGD peptide-modified dextran was obtained by sequentially coupling carboxylated dextran with cysteine ​​and RGD peptide.

5. The method for preparing a porous scaffold with a cell adhesion layer according to claim 1, characterized in that, In step (3), when performing surface modification, the pH of the adhesion layer modification solution is 7-8 and the soaking time is greater than 2 hours.

6. A porous scaffold with a cell adhesion layer, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 5.

7. A method for culturing and recovering cell spheroids, characterized in that, Includes the following steps: a. Place the porous scaffold in the cell culture plate, add cell suspension and culture medium, and culture. b. After cell proliferation, add adhesion layer lysis buffer to the well plate to decompose the adhesion layer and cause the cells to detach from the porous scaffold and aggregate in the pores to form multicellular spheres; c. After the multicellular spheroids are formed, scaffold lysis buffer is added to the well plate. After the porous scaffold is lysed, the cell spheroids can be collected. The porous scaffold is the porous scaffold with a cell adhesion layer as described in claim 6.

8. The method for culturing and recovering cell spheroids according to claim 7, characterized in that, The adhesion layer lysis solution is a dextranase solution; The scaffold lysis buffer is a PBS solution of dithiothreitol, tri(2-chloroethyl) phosphate, or glutathione.

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