Cell scaffold for promoting proliferation of corneal limbal stem cells
The cell scaffold composed of quaternized chitosan and sodium hyaluronate cross-linked with magnetic nanoparticles solves the problem that the two-dimensional culture method cannot simulate the in vivo microenvironment, promotes the proliferation and functional differentiation of corneal limbal stem cells, and provides stable growth support.
Patent Information
- Application Number
- CN202510909346.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-10-17
AI Technical Summary
Existing two-dimensional culture methods cannot fully simulate the in vivo microenvironment of corneal limbal stem cells, resulting in changes in cell morphology and function, and there are ethical and safety issues. The three-dimensional culture system lacks standardization, and the existing three-dimensional culture environment is significantly different from the in vivo physiological environment.
A mixture of quaternized chitosan and sodium hyaluronate is cross-linked with magnetic nanoparticles under an external magnetic field to form a directionally arranged cell scaffold. Through glutaraldehyde cross-linking and freeze-drying, the extracellular matrix structure in the body is simulated to provide a growth environment closer to the physiological state.
Promote the proliferation of limbal stem cells, improve cell attachment and functional differentiation, provide a good growth microenvironment, avoid immune response and cytotoxicity, and maintain mechanical stability and structural integrity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a cell scaffold for promoting proliferation of limbal stem cells. BACKGROUND
[0002] Limbal stem cells (LSCs) are the stem cells of the corneal epithelium, mainly responsible for the renewal and repair of the corneal epithelium. They play a key role in maintaining corneal transparency and normal function. However, limbal stem cell deficiency (LSCD) is one of the main causes of corneal diseases and visual impairment, severely affecting the quality of life of patients. At present, corneal transplantation is the main method for treating severe corneal diseases, but due to the shortage of donor corneas, many patients cannot obtain suitable corneas for transplantation in time. Therefore, the development of technologies and products that can effectively expand limbal stem cells is of great significance to meet clinical needs.
[0003] Traditional two-dimensional culture methods can achieve partial expansion of limbal stem cells, but there are many problems. For example, cells in a two-dimensional environment cannot completely simulate the three-dimensional microenvironment in vivo, leading to changes in cell morphology and function, and accelerated cell differentiation. In addition, the commonly used murine feeder layer cells (such as NIH-3T3) in two-dimensional culture have ethical and safety problems, such as immune rejection and potential viral infection risk. Three-dimensional cell culture technology provides a new idea for the in vitro expansion of limbal stem cells, but there is currently a lack of standardized three-dimensional culture system, and the differences in scaffold materials and culture conditions used in different studies are also large. In addition, there are still significant differences between the existing three-dimensional culture environment and the in vivo physiological environment, and how to construct a three-dimensional culture system closer to the in vivo microenvironment is a difficulty in current research. SUMMARY
[0004] The purpose of the present application is to provide a cell scaffold that constructs a three-dimensional culture system closer to the in vivo microenvironment and promotes the proliferation of limbal stem cells.
[0005] The present application provides a cell scaffold for promoting proliferation of limbal stem cells, which is prepared by the following method: Mix 1-2% w / v quaternized chitosan solution and 0.5-1% w / v sodium hyaluronate aqueous solution according to a volume ratio of 0.5-2:1, and slowly add a pre-dispersed magnetic nanoparticle suspension to obtain a cross-linking liquid; pour the cross-linking liquid into a mold, then apply a uniform external magnetic field to the mold containing the cross-linking liquid; add 0.1-0.5% w / v glutaraldehyde solution dropwise to the cross-linking liquid to perform cross-linking reaction, and obtain a cross-linked material; freeze-dry the cross-linked material to obtain a cell scaffold; the final concentration of magnetic nanoparticles in the cross-linking liquid is 0.1-1 mg / mL.
[0006] Preferably, the preparation method of the quaternary ammonium chitosan solution is as follows: chitosan is dissolved in 1-2% v / v acetic acid solution, then glycidyltrimethylammonium chloride is added, and the reaction is carried out at 50-60 DEG C for 6-10 hours; after the reaction is completed, the precipitate is removed by filtration, and then the concentration is adjusted to 1-2% w / v with distilled water to obtain the quaternary ammonium chitosan solution; the molar ratio of the glycidyltrimethylammonium chloride to chitosan is 2-3:1.
[0007] Preferably, the volume ratio of the chitosan solution to the sodium hyaluronate solution is 1-1.5:1.
[0008] Preferably, the magnetic nanoparticles are iron oxides.
[0009] Preferably, the particle size of the magnetic nanoparticles is 10-100 nm.
[0010] Preferably, the preparation method of the magnetic nanoparticle suspension is as follows: the magnetic nanoparticles are mixed with PBS buffer to have a concentration of 0.3-0.7 mg / mL, and then ultrasonic treatment is carried out at 60-120 W for 10-20 min.
[0011] Preferably, the final concentration of the glutaraldehyde solution in the crosslinking solution is 0.05-0.1% w / v.
[0012] Preferably, the temperature of the crosslinking reaction is 25-37 DEG C, and the duration of the crosslinking reaction is 12-24 hours.
[0013] Preferably, the strength of the magnetic field is 100-300 mT, and the direction of the magnetic field is perpendicular to the surface of the mold.
[0014] Preferably, the temperature of the freeze-drying is -60 DEG C to -40 DEG C, and the cooling rate is 1-5 DEG C / min.
[0015] The application further provides the use of the cell scaffold in the above technical solution in the in-vitro expansion of limbal stem cells.
[0016] The application has the following advantages: The cell scaffold provided by the application is made of chitosan and sodium hyaluronate, which has good biocompatibility and bioactivity, can simulate the characteristics of the extracellular matrix, provides necessary physical and chemical support for stem cells, and does not cause immune response or cytotoxicity. The addition of magnetic nanoparticles further enhances the performance of the cell scaffold. The directional arrangement of the cell scaffold under the action of a magnetic field can provide the cells with a structure support closer to the in-vivo microenvironment, and promote the attachment and proliferation of the cells. The cell scaffold provided by the application can provide a good growth microenvironment for limbal stem cells, and helps to solve the problem that limbal stem cells easily lose activity and function in in-vitro culture.
[0017] The present application applies a uniform external magnetic field during preparation, and the magnetic nanoparticles can form a directional arrangement inside the cell scaffold. The directional arrangement can simulate the fiber structure of the extracellular matrix in vivo, provide a growth environment closer to the physiological state for the corneal limbal stem cells, and promote the directional growth and functional differentiation of the cells. Through glutaraldehyde cross-linking and freeze-drying treatment, the cell scaffold has good mechanical stability and structural integrity, and can maintain its performance for a long time. DETAILED DESCRIPTION
[0018] In order to further illustrate the present application, the schemes provided by the present application are described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.
[0019] Unless otherwise specified, the substances used in the examples of the present application are all conventional commercially available products.
[0020] Example 1 The chitosan was dissolved in a 2% v / v acetic acid solution, then glycidyltrimethylammonium chloride (GTMAC) with a molar amount of 2 times that of the chitosan was added, and the reaction was carried out at 60°C for 8 h, then the precipitate was removed by filtration, and the concentration was adjusted to 1% v / v with distilled water.
[0021] The sodium hyaluronate was dissolved in deionized water to prepare a 1% w / v sodium hyaluronate solution.
[0022] The ferroferric oxide nanoparticles with a particle size of 40 nm were mixed with a PBS buffer to make the concentration 1.2 mg / mL, and then ultrasonically treated at 60 W for 10 min to obtain a pre-dispersed magnetic nanoparticle suspension for standby.
[0023] The chitosan solution and the sodium hyaluronate solution were mixed in a volume ratio of 1:1, then the pre-dispersed magnetic nanoparticle suspension was slowly added to make the final concentration of the magnetic nanoparticle suspension 1 mg / mL, to obtain a cross-linking liquid; after uniform mixing, the cross-linking liquid was poured into a mold, an external magnetic field with a strength of 200 mT was applied outside the mold by using an electromagnet, and the direction of the magnetic field was perpendicular to the plane of the mold, and the magnetic field was removed after the reaction was completed.
[0024] The 0.2% w / v glutaraldehyde solution was added dropwise to the mold to start the cross-linking reaction; when the concentration of the glutaraldehyde solution in the cross-linking liquid was 0.05% w / v, the dropwise addition was stopped; the cross-linking was carried out at room temperature for 18 h to obtain a cross-linked material.
[0025] The cross-linked material was subjected to freeze-drying treatment: the temperature was lowered at a rate of 5°C / min to-55°C until the cross-linked material was fixed and shaped to obtain a cell scaffold.
[0026] Example 2 Chitosan was dissolved in 1% v / v acetic acid solution, then glycidyltrimethylammonium chloride (GTMAC) was added in an amount of 2 times the molar amount of chitosan, and the mixture was reacted at 50°C for 8 h, after which the precipitate was removed by filtration, and the concentration was adjusted to 1% v / v with distilled water.
[0027] Sodium hyaluronate was dissolved in deionized water to prepare a 1% w / v sodium hyaluronate solution.
[0028] Ferroferric oxide nanoparticles with a particle size of 40 nm were mixed with PBS buffer to a concentration of 1.2 mg / mL, and were treated with ultrasound at 80 W for 10 min to obtain a pre-dispersed magnetic nanoparticle suspension for standby use.
[0029] The chitosan solution and the sodium hyaluronate solution were mixed in a volume ratio of 1:1, and then the pre-dispersed magnetic nanoparticle suspension was slowly added to obtain a final concentration of 0.6 mg / mL of the magnetic nanoparticle suspension, thereby obtaining a crosslinking solution; after uniform mixing, the crosslinking solution was poured into a mold, an external magnetic field with a strength of 200 mT was applied to the outside of the mold by using an electromagnet, and the direction of the magnetic field was perpendicular to the plane of the mold, and the magnetic field was removed after the reaction was completed.
[0030] A 0.5% w / v glutaraldehyde solution was added dropwise to the mold to start the crosslinking reaction; the addition was stopped when the concentration of the glutaraldehyde solution in the crosslinking solution reached 0.1% w / v; and the crosslinking was carried out at room temperature for 18 h to obtain a crosslinked material.
[0031] The crosslinked material was subjected to freeze-drying treatment: the temperature was lowered to -60°C at a rate of 3°C / min until the crosslinked material was fixed and shaped, thereby obtaining a cell scaffold.
[0032] Example 3 Chitosan was dissolved in 2% v / v acetic acid solution, then glycidyltrimethylammonium chloride (GTMAC) was added in an amount of 3 times the molar amount of chitosan, and the mixture was reacted at 50°C for 8 h, after which the precipitate was removed by filtration, and the concentration was adjusted to 1% v / v with distilled water. Sodium hyaluronate was dissolved in deionized water to prepare a 1% w / v sodium hyaluronate solution.
[0033] Ferroferric oxide nanoparticles with a particle size of 40 nm were mixed with PBS buffer to a concentration of 1 mg / mL, and were treated with ultrasound at 60 W for 10 min to obtain a pre-dispersed magnetic nanoparticle suspension for standby use.
[0034] The chitosan solution and the sodium hyaluronate solution were mixed in a volume ratio of 1.5:1, and then the pre-dispersed magnetic nanoparticle suspension was slowly added to obtain a cross-linking solution with a final concentration of 0.3 mg / mL of the magnetic nanoparticles; after uniform mixing, the cross-linking solution was poured into a mold, and an external magnetic field with a strength of 200 mT was applied to the mold by an electromagnet, with the magnetic field direction being perpendicular to the plane of the mold, until the reaction was completed and the magnetic field was removed.
[0035] The cross-linking reaction was started by adding a 0.2% w / v glutaraldehyde solution dropwise to the mold; the addition was stopped when the concentration of the glutaraldehyde solution in the cross-linking solution was 0.05% w / v; and the cross-linking was performed at room temperature for 18 h to obtain a cross-linked material.
[0036] The cross-linked material was subjected to freeze-drying treatment: the temperature was lowered to -55°C at a rate of 5°C / min until the cross-linked material was fixed and shaped to obtain a cell scaffold.
[0037] Comparative Example 1 The difference from Example 1 is that no magnetic nanoparticles were added and no magnetic field was applied.
[0038] The chitosan was dissolved in a 2% v / v acetic acid solution, and then glycidyltrimethylammonium chloride (GTMAC) in an amount of 2 times the molar amount of the chitosan was added, and the mixture was reacted at 50°C for 8 h, followed by filtration to remove the precipitate and adjustment of the concentration to 1% v / v with distilled water.
[0039] The sodium hyaluronate was dissolved in deionized water to prepare a 1% w / v sodium hyaluronate solution.
[0040] The chitosan solution and the sodium hyaluronate solution were mixed in a volume ratio of 1:1 to obtain a cross-linking solution; after uniform mixing, the cross-linking solution was poured into a mold, and a cross-linking reaction was started by adding a 0.2% w / v glutaraldehyde solution dropwise to the mold; the addition was stopped when the concentration of the glutaraldehyde solution in the cross-linking solution was 0.05% w / v; and the cross-linking was performed at room temperature for 18 h to obtain a cross-linked material.
[0041] The cross-linked material was subjected to freeze-drying treatment: the temperature was lowered to -55°C at a rate of 5°C / min until the cross-linked material was fixed and shaped to obtain a cell scaffold.
[0042] Comparative Example 2 The difference from Example 1 is that a non-quaternized chitosan solution was used.
[0043] The chitosan was dissolved in a 2% v / v acetic acid solution to prepare a 2% w / v chitosan solution.
[0044] The sodium hyaluronate was dissolved in deionized water to prepare a 1% w / v sodium hyaluronate solution.
[0045] Fe3O4 nanoparticles with a particle size of 40 nm were mixed with PBS buffer to a concentration of 1.2 mg / mL, and treated with 60 W ultrasonic for 10 min to obtain a pre-dispersed magnetic nanoparticle suspension for standby.
[0046] The chitosan solution and the sodium hyaluronate solution were mixed at a volume ratio of 1:1, and then the pre-dispersed magnetic nanoparticle suspension was slowly added to obtain a crosslinking liquid with a final concentration of 1 mg / mL of the magnetic nanoparticle suspension; after uniform mixing, the crosslinking liquid was poured into a mold, an external magnetic field with a strength of 200 mT was applied outside the mold by using an electromagnet, and the direction of the magnetic field was perpendicular to the plane of the mold, and the magnetic field was removed after the reaction was completed.
[0047] A 0.2% w / v glutaraldehyde solution was added dropwise to the mold to start the crosslinking reaction; the addition was stopped when the concentration of the glutaraldehyde solution in the crosslinking liquid was 0.05% w / v; and the crosslinking was performed at room temperature for 18 h to obtain a crosslinked material.
[0048] The crosslinked material was subjected to freeze-drying treatment: the temperature was lowered to -55°C at a rate of 5°C / min until the crosslinked material was fixed and shaped to obtain a cell scaffold.
[0049] Comparative Example 3 The difference from Example 1 is that hyaluronic acid is used instead of sodium hyaluronate.
[0050] Chitosan was dissolved in a 2% v / v acetic acid solution, and then glycidyltrimethylammonium chloride (GTMAC) with a molar amount of 2 times that of chitosan was added, and the mixture was reacted at 60°C for 8 h, and then the precipitate was removed by filtration, and the concentration was adjusted to 1% v / v with distilled water.
[0051] Hyaluronic acid was dissolved in deionized water at 45°C to prepare a 1% w / v hyaluronic acid solution.
[0052] Fe3O4 nanoparticles with a particle size of 40 nm were mixed with PBS buffer to a concentration of 1.2 mg / mL, and treated with 60 W ultrasonic for 10 min to obtain a pre-dispersed magnetic nanoparticle suspension for standby.
[0053] The chitosan solution and the sodium hyaluronate solution were mixed at a volume ratio of 1:1, and then the pre-dispersed magnetic nanoparticle suspension was slowly added to obtain a crosslinking liquid with a final concentration of 1 mg / mL of the magnetic nanoparticle suspension; after uniform mixing, the crosslinking liquid was poured into a mold, an external magnetic field with a strength of 200 mT was applied outside the mold by using an electromagnet, and the direction of the magnetic field was perpendicular to the plane of the mold, and the magnetic field was removed after the reaction was completed.
[0054] The cross-linking reaction was started by adding 0.2% w / v glutaraldehyde solution dropwise into the mold; when the concentration of the glutaraldehyde solution in the cross-linking liquid was 0.05% w / v, the dropwise addition was stopped; and the cross-linking was performed at room temperature for 18 h to obtain the cross-linked material.
[0055] The cross-linked material was subjected to freeze-drying treatment: the temperature was lowered to -55°C at a rate of 5°C / min until the cross-linked material was fixed and shaped to obtain the cell scaffold.
[0056] Test Example 1 The cell scaffolds prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were respectively taken as samples for testing.
[0057] Twenty-one T25 culture bottles were taken and divided into six groups, i.e., test groups and a control group, with three parallel samples in each group.
[0058] Group 1: One cell scaffold prepared in Example 1 was added to each bottle; Group 2: One cell scaffold prepared in Example 2 was added to each bottle; Group 3: One cell scaffold prepared in Example 3 was added to each bottle; Group 4: One cell scaffold prepared in Comparative Example 1 was added to each bottle; Group 5: One cell scaffold prepared in Comparative Example 2 was added to each bottle; Group 6: One cell scaffold prepared in Comparative Example 3 was added to each bottle; Control group: No cell scaffold was added.
[0059] 5 mL of DMEM medium containing 10% FBS was added to each bottle, and then 500 μL of LSC cell solution with a concentration of 1×10 6 cells / mL was inoculated, and the culture was performed at 37°C under 5% CO2, the medium was replaced every 2 days during the culture, and the samples were taken at 0 d, 1 d, 3 d and 5 d, respectively, the cell concentration was calculated by using a hemocytometer, the cell proliferation rate was calculated, and the cell viability was determined by using a CCK-8 kit, and the results are shown in Tables 1 to 2.
[0060] Cell proliferation rate = (cell concentration after culture-cell concentration before culture) / cell concentration before culture×100% Table 1 Cell growth
[0061] From Table 1, compared with the control group, the cell proliferation rates of the first to third groups are significantly improved, which shows that the cell scaffold provided by the application is more conducive to simulating the in-vivo microenvironment and promoting the growth of LSCs cells; the cell proliferation rate of the fourth group is lower than that of the first group, which shows that the external magnetic field in the preparation of the cell scaffold can make the cell scaffold more simulate the fiber structure of the in-vivo extracellular matrix, which is conducive to cell adhesion and growth; the cell proliferation rate of the first group is higher than that of the fifth group, which shows that compared with unmodified chitosan, quaternary ammonium modified chitosan is more conducive to cell proliferation; the cell proliferation rate of the sixth group is also significantly lower than that of the first group, which shows that compared with hyaluronic acid, the effect of using sodium hyaluronate as raw material to prepare the cell scaffold is better.
[0062] Table 2 Cell viability
[0063] From Table 2, when the LSCs cells are cultured by using the cell scaffold provided by the application, compared with the control group without using the cell scaffold, the cell viability is significantly improved, which shows that the cell scaffold provided by the application has good biocompatibility and no cytotoxicity, which is conducive to supporting the growth and proliferation of LSCs cells.
[0064] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, not all the embodiments, and people can also obtain other embodiments according to the embodiment without creativity, which all belong to the protection scope of the application.
Claims
1. A cell scaffold for promoting the proliferation of limbal stem cells, characterized in that: The cell scaffold is prepared by the following method: Mix 1-2% w / v quaternized chitosan solution and 0.5-1% w / v sodium hyaluronate aqueous solution at a volume ratio of 0.5-2:1, and slowly add the pre-dispersed magnetic nanoparticle suspension to obtain a cross-linked solution; Pour the cross-linking liquid into the mold, and then apply a uniform external magnetic field to the mold containing the cross-linking liquid; adding 0.1-0.5% w / v glutaraldehyde solution dropwise to the cross-linking solution to carry out a cross-linking reaction to obtain a cross-linked material; The cross-linked material is freeze-dried to obtain a cell scaffold; The final concentration of the magnetic nanoparticles in the cross-linking solution is 0.1-1 mg / mL.
2. The cell scaffold according to claim 1, characterized in that The quaternized chitosan solution is prepared by dissolving chitosan in a 1-2% v / v acetic acid solution, then adding glycidyl trimethyl ammonium chloride, reacting at 50° C.-60° C. for 6-10 hours, filtering to remove the precipitate, and then adjusting the concentration to 1-2% w / v with distilled water to obtain the quaternized chitosan solution; the molar ratio of glycidyl trimethyl ammonium chloride to chitosan is 2-3:
1.
3. The cell scaffold according to claim 1, characterized in that The magnetic nanoparticles are iron oxide.
4. The cell scaffold according to claim 1, characterized in that The particle size of the magnetic nanoparticles is 10-100 nm.
5. The cell scaffold according to claim 1, characterized in that The preparation method of the magnetic nanoparticle suspension is as follows: mixing the magnetic nanoparticles with PBS buffer to a concentration of 0.3-1.2 mg / mL, and performing ultrasonic treatment at 60-120W for 10-20 minutes.
6. The cell scaffold according to claim 1, characterized in that The final concentration of the glutaraldehyde solution in the cross-linking solution is 0.05-0.1% w / v.
7. The cell scaffold according to claim 1, characterized in that The cross-linking reaction temperature is 25° C. to 37° C.; the cross-linking reaction time is 12 to 24 hours.
8. The cell scaffold according to claim 1, characterized in that The intensity of the magnetic field is 100-300 mT, and the direction of the magnetic field is perpendicular to the mold surface.
9. The cell scaffold according to claim 1, characterized in that The freeze-drying temperature is -60°C to -40°C, and the cooling rate is 1 to 5°C / min.
10. Use of the cell scaffold according to any one of claims 1 to 9 in in vitro expansion of limbal stem cells.