A thermosensitive cell microcarrier and its preparation method, and a method for culturing cells

Abstract

The present invention provides a kind of thermosensitive cell microcarrier and preparation method thereof, and the method for culturing cells. The preparation method provided by the present invention, using glass microspheres as basic materials to carry out surface hydroxylation, then aminosilane coupling agent is used to carry out amino modification, then the N-carboxylic acid anhydride (NCA) of glutamic acid modified by propargyl alcohol is polymerized on the surface of glass microspheres, and finally click chemistry reaction is carried out with azido oligoethylene glycol to obtain a surface-covered thermosensitive polymer glass microsphere with good cell compatibility. The thermosensitive cell microcarrier prepared by the present invention has extremely low cytotoxicity and good biosafety; moreover, it has thermosensitive performance, can achieve efficient cell adhesion and detachment, and effectively detaches cells in a low temperature environment; when it is used as a carrier to culture cells, three-dimensional space is provided to cell growth, and compared with a two-dimensional plane, cell amplification can be achieved, the cost of cell culture is reduced, and the effect of protecting cell surface proteins is possessed.

Description

Technical Field

[0001] The present invention relates to the field of new biomedical materials, and in particular to a temperature-sensitive cell microcarrier and a preparation method thereof, as well as a method for culturing cells. Background Art

[0002] Regenerative medicine and cell therapy, emerging therapeutic approaches, both require large numbers of cells. However, the current cost of expanding cell culture is extremely high. Existing two-dimensional culture models are no longer sufficient, leading to the development of three-dimensional cell culture models (see Fronk A and E Vargis. Methods for Culturing Retinal Pigment Epithelial Cells: a Review of Current Protocols and Future Recommendations. Journal of Tissue Engineering, 7:1-23). ​​Among existing three-dimensional cell culture models, GE's polystyrene microspheres are the mainstream culture carrier. Their advantage lies in their ability to combine the high surface area of ​​the porous material with a density close to that of water, enabling expanded culture. However, cells adhere to the microspheres, making them difficult to detach, and trypsin detachment can damage the cell's protein structure, making this culture method costly and complex. Therefore, leveraging the hydrophilic and hydrophobic temperature responsiveness of thermosensitive materials to allow cells to detach at low temperatures and avoid trypsin is a viable approach. The resulting cell surface structure is protected from trypsin damage, and the recyclable nature of the thermosensitive material allows this culture carrier to be reused. Moreover, during cell culture, temperature-sensitive materials must have low toxicity.

[0003] Currently, most commonly used thermosensitive materials use poly (N-isopropylacrylamide) (PNIPAM), but PNIPAM has a certain degree of cytotoxicity, and the larger the molecular weight, the greater the cytotoxicity. In theory, cell detachment can be achieved by utilizing the hydrophilic and hydrophobic temperature responsiveness of thermosensitive materials. However, in actual applications, not all thermosensitive materials can achieve good cell adhesion and detachment after being combined with matrix microspheres, and some also have high cytotoxicity, which cannot achieve the ideal practical application effect. Therefore, the development of cell carriers still faces great challenges. Summary of the Invention

[0004] In view of this, the present invention provides a thermosensitive cell microcarrier, a method for preparing the same, and a method for culturing cells. The thermosensitive cell microcarrier provided by the present invention has extremely low cytotoxicity and can achieve efficient cell adhesion and detachment. When used as a carrier to culture cells, it provides a three-dimensional space for cell growth, which, compared to a two-dimensional plane, can achieve cell expansion, reduce cell culture costs, and protect cell surface proteins.

[0005] The present invention provides a thermosensitive cell microcarrier comprising glass microspheres and a thermosensitive polymer grafted onto the surface of the glass microspheres; the structure of the thermosensitive cell microcarrier is shown in formula (L):

[0006]

[0007] in:

[0008] represents glass microspheres;

[0009] m and n are the degree of polymerization, m is 2 to 8, and n is 30 to 120;

[0010] j and k are the molar percentages of the corresponding structural units, j+k=1, 0%≤j / (j+k)≤10%, 90%≤k / (j+k)≤100%.

[0011] Preferably, the mass fraction of the thermosensitive polymer in the thermosensitive cell microcarrier is ≥1%.

[0012] The present invention also provides a method for preparing the temperature-sensitive cell microcarrier described in the above technical solution, comprising the following steps:

[0013] A) performing surface hydroxylation treatment on glass microspheres to obtain surface hydroxylated microspheres; treating the surface hydroxylated microspheres with an aminosilane coupling agent to obtain amino-treated glass microspheres;

[0014] B) glutamic acid reacts with propargyl alcohol to generate propargyl alcohol-modified glutamic acid represented by formula (1); the propargyl alcohol-modified glutamic acid represented by formula (1) reacts with bis(trichloromethyl) carbonate to generate propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2);

[0015]

[0016] C) reacting the amino-modified glass microspheres obtained in step A) with the propargyl alcohol-modified glutamic acid N-carboxylic anhydride of formula (2) obtained in step B) to obtain glass microspheres with surface grafted polyglutamic acid;

[0017] D) reacting the glass microspheres with the surface grafted polyglutamic acid with polyethylene glycol azide to obtain glass microsphere microcarriers with the surface modified polyglutamic acid derivatives;

[0018] There is no order restriction for step A) and step B).

[0019] Preferably, in step D), the degree of polymerization of the azide polyethylene glycol is 2 to 8;

[0020] In step A), the aminosilane coupling agent is KH550.

[0021] Preferably, in step A), the surface hydroxylated microspheres are treated with an aminosilane coupling agent at a temperature of 60 to 120° C. for 4 to 24 hours.

[0022] Preferably, in step B), the reaction conditions of glutamic acid and propargyl alcohol are: temperature 0-8° C., time 8-12 hours.

[0023] Preferably, in step B), the reaction conditions of the propargyl alcohol-modified glutamic acid represented by formula (1) and bis(trichloromethyl) carbonate are: temperature 25-55° C., time 0.5-3 hours.

[0024] Preferably, in step C), the reaction conditions are: temperature 25-40° C., time 3-7 days;

[0025] In step D), the reaction conditions are: temperature 30-50° C., and time 3-7 days.

[0026] Preferably, in step A):

[0027] The surface hydroxylation treatment process is as follows: soaking the glass microspheres in a mixed solution to hydroxylate the surface of the glass microspheres, and then separating the solid and the liquid to obtain surface hydroxylated microspheres;

[0028] The mixed solution is a mixture of sulfuric acid solution and hydrogen peroxide solution, wherein the molar ratio of H2SO4 to hydrogen peroxide is 7:3; the soaking conditions are: temperature 40-80°C, time 1-4 hours;

[0029] The process of treating the surface hydroxylated microspheres with an aminosilane coupling agent comprises: mixing the surface hydroxylated microspheres, a solvent and the aminosilane coupling agent to react to obtain amino glass microspheres;

[0030] Step C) specifically comprises: dispersing the amino-modified glass microspheres in an anhydrous organic solvent, adding the propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2), and stirring the mixture under a protective atmosphere to obtain glass microspheres with surface grafted polyglutamic acid;

[0031] Step D) specifically comprises: mixing glass microspheres with surface grafted polyglutamic acid with an organic solvent, bubbling with an inert gas for a period of time, adding copper sulfate pentahydrate and polyethylene glycol azide, continuing to bubble for a period of time, then adding sodium ascorbate, and performing a click chemistry reaction under an inert gas atmosphere to obtain glass microspheres with surface modified polyglutamic acid derivatives.

[0032] The present invention also provides a method for culturing cells, wherein the culture carrier used is the temperature-sensitive cell microcarrier described in the above technical solution or the temperature-sensitive cell microcarrier prepared by the preparation method described in the above technical solution.

[0033] The preparation method provided by the present invention uses glass microspheres as the base material for surface hydroxylation, then uses an aminosilane coupling agent for amino modification, then triggers the polymerization of propargyl alcohol-modified glutamic acid N-carboxylic anhydride (NCA) on the surface of the glass microspheres, and finally performs a click chemistry reaction with azide oligoethylene glycol to obtain a surface-covered layer of thermosensitive polymer glass microspheres with good cell compatibility. The thermosensitive cell microcarrier prepared by the present invention has extremely low cytotoxicity and good biosafety; moreover, it has thermosensitive properties, can achieve efficient cell adhesion and detachment, and effectively detach cells in a low-temperature environment; when it is used as a carrier to culture cells, it provides a three-dimensional space for cell growth, which can achieve cell expansion and reduce the cost of cell culture compared to a two-dimensional plane, while also having the effect of protecting cell surface proteins; in addition, the obtained thermosensitive glass microsphere carrier has a reusable function; in addition, the above-mentioned preparation method is simple, the conditions are mild, and it is easy to operate. The raw material source is low-toxic or non-toxic and low-priced, which is conducive to large-scale production and application.

[0034] The test results show that the thermosensitive glass microsphere carrier prepared by the present invention can successfully adhere to cells and at the same time can achieve a cell detachment rate of more than 78%, showing efficient cell detachment; the cell survival rate reaches more than 91.9%, showing extremely low cytotoxicity and extremely high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0036] Figure 1 This is a SEM image of the temperature-sensitive glass microsphere carrier obtained in Example 4 of the present invention;

[0037] Figure 2 This is a fluorescence microscope image of cells cultured on the thermosensitive glass microsphere carrier obtained in Example 4 of the present invention;

[0038] Figure 3 Graph showing the temperature-sensitive desorption curves of the temperature-sensitive glass microsphere carriers obtained in Examples 1 to 4 of the present invention. DETAILED DESCRIPTION

[0039] The present invention provides a thermosensitive cell microcarrier comprising glass microspheres and a thermosensitive polymer grafted onto the surface of the glass microspheres; the structure of the thermosensitive cell microcarrier is shown in formula (L):

[0040]

[0041] in:

[0042] represents glass microspheres;

[0043] m and n are the degree of polymerization; m is 2 to 8, specifically 2, 3, 4, 5, 6, 7, 8, more preferably 2, 3, 4, 6, 8. n is 30 to 120, specifically 30, 40, 50, 60, 70, 80, 90, 100, 110, 120.

[0044] j and k are the molar percentages of the corresponding structural units (specifically the molar percentages of the total amount of the structural units corresponding to j and k), j+k=1 (i.e., 100%), 0%≤j / (j+k)≤10% (can also be directly expressed as 0%≤j≤10%), and can specifically be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; 90%≤k / (j+k)≤100% (can also be directly expressed as 90%≤k≤100%), and can specifically be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0045] In the present invention, the thermosensitive polymer is evenly distributed on the surface of the glass microsphere to form a thermosensitive layer. The mass fraction of the thermosensitive polymer in the thermosensitive cell microcarrier is preferably ≥1%, more preferably 1% to 2%.

[0046] The present invention also provides a method for preparing the temperature-sensitive cell microcarrier described in the above technical solution, comprising the following steps:

[0047] A) performing surface hydroxylation treatment on glass microspheres to obtain surface hydroxylated microspheres; treating the surface hydroxylated microspheres with an aminosilane coupling agent to obtain amino-treated glass microspheres;

[0048] B) glutamic acid reacts with propargyl alcohol to generate propargyl alcohol-modified glutamic acid represented by formula (1); the propargyl alcohol-modified glutamic acid represented by formula (1) reacts with bis(trichloromethyl) carbonate to generate propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2);

[0049]

[0050] C) reacting the amino-modified glass microspheres obtained in step A) with the propargyl alcohol-modified glutamic acid N-carboxylic anhydride of formula (2) obtained in step B) to obtain glass microspheres with surface grafted polyglutamic acid;

[0051] D) reacting the glass microspheres with the surface grafted polyglutamic acid with polyethylene glycol azide to obtain glass microsphere microcarriers with the surface modified polyglutamic acid derivatives;

[0052] There is no order restriction for step A) and step B).

[0053] The preparation method provided by the present invention uses glass microspheres as the base material for surface hydroxylation, then uses an aminosilane coupling agent for amino modification, then triggers the polymerization of propargyl alcohol-modified glutamic acid N-carboxylic anhydride (NCA) on the surface of the glass microspheres, and finally undergoes a click chemistry reaction with azide oligoethylene glycol to obtain a temperature-sensitive polymer glass microsphere with good cell compatibility covered on the surface.

[0054] About step A) :

[0055] A) performing surface hydroxylation treatment on glass microspheres to obtain surface hydroxylated microspheres; and treating the surface hydroxylated microspheres with an aminosilane coupling agent to obtain aminoated glass microspheres.

[0056] According to the present invention, the glass microspheres are first subjected to surface hydroxylation treatment to obtain surface hydroxylated microspheres.

[0057] In the present invention, the process of performing the surface hydroxylation treatment is preferably: immersing the glass microspheres in a mixed solution to hydroxylate the surfaces of the glass microspheres, and then performing solid-liquid separation to obtain surface hydroxylated microspheres.

[0058] in:

[0059] The glass microspheres are used as the basic material for preparing cell microcarriers, and their particle size is preferably 100 to 300 μm, specifically 100 μm, 150 μm, 200 μm, 250 μm, and 300 μm.

[0060] The mixed solution is preferably a mixture of sulfuric acid solution and hydrogen peroxide solution, wherein the molar ratio of H2SO4 to hydrogen peroxide is preferably 7:3. There is no particular limitation on the amount of the mixed solution and the glass microspheres, as long as the glass microspheres are completely immersed in the mixed solution.

[0061] The immersion temperature is preferably 40-80°C, specifically 40°C, 50°C, 60°C, 70°C, or 80°C. The immersion time is preferably 1-4 hours, specifically 1 hour, 2 hours, 3 hours, or 4 hours. After the immersion treatment, the surface of the glass microspheres undergoes hydroxylation.

[0062] The solid-liquid separation method is not particularly limited and can be any conventional separation method known to those skilled in the art, such as filtering or removing the microspheres, etc. After solid-liquid separation, surface hydroxylated glass microspheres are obtained.

[0063] According to the present invention, after surface hydroxylated microspheres are obtained, the surface hydroxylated microspheres are treated with an aminosilane coupling agent to obtain amino glass microspheres.

[0064] In the present invention, the aminosilane coupling agent is preferably KH550. Using the aminosilane coupling agent, the surface of the glass microspheres is bound to amino chains with a certain chain structure, which is beneficial for subsequent preparation and improves product performance. The reaction route for converting surface hydroxylated microspheres into amino-containing glass microspheres using the aminosilane coupling agent is as follows:

[0065]

[0066] In the present invention, the process of treating the surface hydroxylated microspheres with an aminosilane coupling agent preferably comprises: mixing and reacting the surface hydroxylated microspheres, a solvent and the aminosilane coupling agent to obtain amino-treated glass microspheres.

[0067] in:

[0068] The solvent is preferably toluene and / or ethanol. The ratio of the glass microspheres (i.e., the initial glass microspheres before hydroxylation) to the solvent is preferably 1 g:(10-100) mL, specifically 1 g:10 mL, 1 g:20 mL, 1 g:30 mL, 1 g:40 mL, 1 g:50 mL, 1 g:60 mL, 1 g:70 mL, 1 g:80 mL, 1 g:90 mL, or 1 g:100 mL.

[0069] The type of the aminosilane coupling agent is as described above and will not be repeated here. The amount ratio of the glass microspheres (i.e., the initial glass microspheres before hydroxylation treatment) to the aminosilane coupling agent is preferably 1g:amL, a≥0.4; more preferably 1g:(0.4-1.4)mL, specifically 1g:0.4mL, 1g:0.5mL, 1g:0.6mL, 1g:0.7mL, 1g:0.8mL, 1g:0.9mL, 1g:1.0mL, 1g:1.1mL, 1g:1.2mL, 1g:1.3mL, 1g:1.4mL.

[0070] The reaction temperature is preferably 60-120°C, specifically 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C, and more preferably 80°C. The reaction time is preferably 4-24 hours, specifically 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours. The reaction is preferably carried out under stirring. The stirring rate is preferably 1000-2500 rpm, specifically 1000 rpm, 1500 rpm, 2000 rpm, or 2500 rpm.

[0071] After the above reaction, it is preferred to further perform washing. The washing is preferably performed with deionized water. After the above treatment, amino-treated glass microspheres are obtained.

[0072] About step B) :

[0073] B) glutamic acid reacts with propargyl alcohol to generate propargyl alcohol-modified glutamic acid represented by formula (1); the propargyl alcohol-modified glutamic acid represented by formula (1) reacts with bis(trichloromethyl) carbonate to generate propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2).

[0074] According to the present invention, glutamic acid is first reacted with propargyl alcohol to produce propargyl alcohol-modified glutamic acid shown in formula (1). The reaction scheme is as follows:

[0075]

[0076] In the present invention, the usage ratio of glutamic acid to propargyl alcohol is preferably 20g:(30-60)mL, specifically 20g:30mL, 20g:35mL, 20g:40mL, 20g:45mL, 20g:50mL, 20g:55mL, and 20g:60mL.

[0077] In the present invention, the reaction is preferably carried out under the action of concentrated sulfuric acid catalyst. In the present invention, the usage ratio of glutamic acid to concentrated sulfuric acid is preferably 20g:(8-16)mL, specifically 20g:8mL, 20g:9mL, 20g:10mL, 20g:11mL, 20g:12mL, 20g:13mL, 20g:14mL, 20g:15mL, 20g:16mL.

[0078] In the present invention, the reaction temperature is preferably 0-8° C., specifically 0° C., 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., or 8° C. The reaction time is preferably 8-12 h, specifically 8 h, 9 h, 10 h, 11 h, or 12 h.

[0079] After the reaction, a neutralization treatment is preferably performed to neutralize the sulfuric acid introduced in the previous step. The neutralization treatment is preferably performed using a saturated sodium bicarbonate solution. The volume ratio of the saturated sodium bicarbonate solution to concentrated sulfuric acid is preferably 1: (0.8-1).

[0080] In the present invention, after the neutralization treatment, filtration and recrystallization are preferably performed. The solvent used for the recrystallization is preferably a methanol-water solution. The volume ratio of methanol to water in the methanol-water solution is preferably 1:(0.3-0.7). After recrystallization, the solvent is removed by filtration to obtain a white powder, which is the propargyl alcohol-modified glutamic acid (GAP) represented by formula (1).

[0081] In the present invention, the above reaction process preferably specifically comprises: mixing glutamic acid and propargyl alcohol, adding concentrated sulfuric acid while stirring in an ice bath, and reacting overnight to obtain a reaction solution; mixing the reaction solution with a saturated sodium bicarbonate solution for neutralization, then filtering off the solution, recrystallizing in a methanol aqueous solution, and then filtering off the solvent to obtain a white powder, i.e., propargyl alcohol-modified glutamic acid (GAP) represented by formula (1).

[0082] According to the present invention, after obtaining the propargyl alcohol-modified glutamic acid represented by formula (1), the propargyl alcohol-modified glutamic acid represented by formula (1) is reacted with bis(trichloromethyl) carbonate to produce the propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2). The reaction scheme is as follows:

[0083]

[0084] In the present invention, the mass ratio of glutamic acid to bis(trichloromethyl) carbonate is preferably 20:(6-15), specifically 20:6, 20:7, 20:8, 20:9, 20:10, 20:11, 20:12, 20:13, 20:14, or 20:15.

[0085] In the present invention, the reaction is preferably carried out in a solvent medium. The solvent is preferably tetrahydrofuran (THF). The ratio of glutamic acid to solvent is preferably 20 g: (150-400) mL.

[0086] In the present invention, the reaction temperature is preferably 25-55° C., specifically 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., or 55° C. The reaction time is preferably 0.5-3 h, specifically 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, or 3 h.

[0087] In the present invention, after the above reaction, the following post-treatment is preferably further performed: removing the solvent by rotary evaporation. After the above treatment, the propargyl alcohol-modified glutamic acid N-carboxylic anhydride (GAP-NCA) represented by formula (2) is obtained.

[0088] In the present invention, there is no order restriction for the above steps A) and B).

[0089] Regarding step C) :

[0090] C) reacting the amino-modified glass microspheres obtained in step A) with the propargyl alcohol-modified glutamic acid N-carboxylic anhydride of formula (2) obtained in step B) to obtain glass microspheres with surface grafted polyglutamic acid.

[0091] The reaction scheme of this step is as follows:

[0092]

[0093] In the present invention, the mass ratio of the amino glass microspheres to the propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2) is preferably 0.1:(0.1-2), specifically 0.1:0.1, 0.1:0.2, 0.1:0.3, 0.1:0.4, 0.1:0.5, 0.1:0.6, 0.1:0.7, 0.1:0.9, 0.1:1.0, 0.1:1.1, 0.1:1.2, 0.1:1.3, 0.1:1.4, 0.1:1.5, 0.1:1.6, 0.1:1.7, 0.1:1.8, 0.1:1.9, and 0.1:2.0.

[0094] In the present invention, the reaction is preferably carried out in an anhydrous organic solvent medium. The anhydrous organic solvent is preferably at least one of N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran, and chloroform. In the present invention, the amount ratio of the amino glass microspheres to the anhydrous organic solvent is preferably 100 mg: (6-10) mL, specifically 100 mg: 6 mL, 100 mg: 7 mL, 100 mg: 8 mL, 100 mg: 9 mL, and 100 mg: 10 mL.

[0095] In the present invention, the reaction is preferably carried out under a protective atmosphere. The present invention has no particular limitation on the type of gas providing the protective atmosphere, and any conventional protective gas in the art, such as nitrogen or argon, can be used.

[0096] In the present invention, the reaction temperature is preferably 25-40°C, specifically 25°C, 30°C, 35°C, or 40°C. The reaction time is preferably 3-7 days, specifically 3 days, 4 days, 5 days, 6 days, or 7 days. In the present invention, stirring is preferably performed during the reaction. The stirring rate is preferably 200-1000 rpm. Through the above reaction, glass microspheres with surface-grafted polyglutamic acid are generated in the system.

[0097] In the present invention, the step C) preferably specifically comprises: dispersing the amino glass microspheres in an anhydrous organic solvent, adding the propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2), and stirring the reaction under a protective atmosphere to obtain glass microspheres with surface grafted polyglutamic acid.

[0098] In the present invention, after the above reaction, the following post-treatments are preferably performed: solid-liquid separation, washing, and drying. The solid-liquid separation method is not particularly limited and can be conventional methods known to those skilled in the art, such as filtration. Washing is preferably performed with an organic solvent; the type of organic solvent used is preferably the same as the anhydrous organic solvent medium used in the above reaction process. Drying is preferably performed under vacuum. The drying temperature is preferably 30-80°C. After the above post-treatments, glass microspheres with polyglutamic acid grafted on their surfaces are obtained.

[0099] Regarding step D) :

[0100] D) reacting the glass microspheres with surface grafted polyglutamic acid with polyethylene glycol azide to obtain glass microsphere microcarriers with surface modified polyglutamic acid derivatives.

[0101] The reaction scheme of this step is as follows:

[0102]

[0103] In the present invention, the degree of polymerization m of the azido polyethylene glycol (N3-PEG) is preferably 2 to 8. If an azido polyethylene glycol with a too high degree of polymerization or an azidoethanol with m of 1 is used, the final product will not be able to effectively adhere to cells. The present invention controls the above-mentioned degree of polymerization to achieve efficient cell adhesion. The degree of polymerization m can specifically be 2, 3, 4, 5, 6, 7, or 8, and more preferably 2, 3, 4, 6, or 8. Moreover, the polyethylene glycol main chain in the above-mentioned azido polyethylene glycol is a pure polyethylene glycol. If the main chain contains other substituents or is grafted with other molecular chains, the effect of the product will be deteriorated or even ineffective. The present invention has no special restrictions on the source of the azido polyethylene glycol, and it can be a commercial product or prepared according to a conventional preparation method well known to those skilled in the art.

[0104] In the present invention, the usage ratio of the glass microspheres with surface grafted polyglutamic acid to azide polyethylene glycol is preferably 100 mg: (10-100) μL, specifically 100 mg: 10 μL, 100 mg: 20 μL, 100 mg: 30 μL, 100 mg: 40 μL, 100 mg: 50 μL, 100 mg: 60 μL, 100 mg: 70 μL, 100 mg: 80 μL, 100 mg: 90 μL, 100 mg: 100 μL, and more preferably 100 mg: 50 μL.

[0105] In the present invention, the reaction is preferably carried out under a protective atmosphere. The present invention has no particular limitation on the type of gas providing the protective atmosphere, and any conventional protective gas in the art, such as nitrogen or argon, can be used.

[0106] In the present invention, the reaction is preferably carried out in the presence of a catalyst. The catalyst is preferably copper sulfate pentahydrate and sodium ascorbate. The mass of the copper sulfate pentahydrate is preferably 10% to 30% of the mass of the glass microspheres with surface grafted polyglutamic acid, specifically 10%, 15%, 20%, 25%, 30%, and more preferably 20%. The mass of the sodium ascorbate is preferably 20% to 60% of the mass of the glass microspheres with surface grafted polyglutamic acid, specifically 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and more preferably 40%.

[0107] In the present invention, the reaction is preferably carried out in an organic solvent medium. The organic solvent is preferably at least one of dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). In the present invention, the ratio of the glass microspheres with surface grafted polyglutamic acid to the organic solvent is preferably 100 mg:(10-30) mL, specifically 100 mg:10 mL, 100 mg:20 mL, or 100 mg:30 mL.

[0108] In the present invention, the reaction temperature is preferably 30°C to 50°C, specifically 30°C, 35°C, 40°C, 45°C, or 50°C. The reaction time is preferably 3 to 7 days, specifically 3 days, 4 days, 5 days, 6 days, or 7 days. Under the above conditions, a click chemistry reaction occurs, generating glass microspheres surface-modified with a polyglutamic acid derivative.

[0109] In the present invention, the step D) preferably specifically comprises: mixing glass microspheres with surface grafted polyglutamic acid with an organic solvent, bubbling with an inert gas for a period of time, adding copper sulfate pentahydrate and polyethylene glycol azide, continuing to bubble for a period of time, then adding sodium ascorbate, and performing a click chemistry reaction under an inert gas atmosphere to obtain glass microspheres with surface modified polyglutamic acid derivatives. The first bubbling time is preferably 10 to 50 minutes, specifically 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and more preferably 30 minutes; the second bubbling time (i.e., the continued bubbling) is preferably 3 to 30 minutes, specifically 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, and more preferably 10 minutes.

[0110] In the present invention, after the above reaction, the following post-treatment is preferably performed: solid-liquid separation, washing and drying. The method of solid-liquid separation is not particularly limited and can be a conventional method well known to those skilled in the art, such as filtration. The washing is preferably done with deionized water. The drying is preferably done under vacuum. The drying temperature is preferably 30 to 80°C. After the above post-treatment, glass microspheres with surface modified polyglutamic acid derivatives (G-PGAP-g-MEO m ), namely temperature-sensitive cell microcarriers.

[0111] The present invention also provides a temperature-sensitive cell microcarrier prepared by the preparation method described in the above technical solution.

[0112] The thermosensitive cell microcarrier prepared by the present invention comprises a glass microsphere having a thermosensitive polymer main chain grafted onto the surface of the glass microsphere and polyglutamic acid, the side groups of which are oligoethylene glycols, the amount of which can reach more than 90% of the amount of the polyglutamic acid structural units; and the thermosensitive polymer is evenly distributed on the surface of the glass microsphere to form a thermosensitive layer, the mass fraction of which can reach more than 1%.

[0113] The present invention also provides a method for culturing cells, wherein the culture carrier is the temperature-sensitive cell microcarrier described in the above technical solution.

[0114] The preparation method provided by the present invention uses glass microspheres as the base material for surface hydroxylation, then uses an aminosilane coupling agent for amino modification, then triggers the polymerization of propargyl alcohol-modified glutamic acid N-carboxylic anhydride (NCA) on the surface of the glass microspheres, and finally performs a click chemistry reaction with azide oligoethylene glycol to obtain a surface-covered layer of thermosensitive polymer glass microspheres with good cell compatibility. The thermosensitive cell microcarrier prepared by the present invention has extremely low cytotoxicity and good biosafety; moreover, it has thermosensitive properties, can achieve efficient cell adhesion and detachment, and effectively detach cells in a low-temperature environment; when it is used as a carrier to culture cells, it provides a three-dimensional space for cell growth, which can achieve cell expansion and reduce the cost of cell culture compared to a two-dimensional plane, while also having the effect of protecting cell surface proteins; in addition, the obtained thermosensitive glass microsphere carrier has a reusable function; in addition, the above-mentioned preparation method is simple, the conditions are mild, and it is easy to operate. The raw material source is low-toxic or non-toxic and low-priced, which is conducive to large-scale production and application.

[0115] The test results show that the thermosensitive glass microsphere carrier prepared by the present invention can successfully adhere to cells and at the same time can achieve a cell detachment rate of more than 78%, showing efficient cell detachment; the cell survival rate reaches more than 91.9%, showing extremely low cytotoxicity and extremely high biosafety.

[0116] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0117] Examples 1 to 4

[0118] A) Glass microspheres (approximately 200 μm in diameter) were immersed in a mixed solution (a mixture of sulfuric acid and hydrogen peroxide, with a molar ratio of H₂SO₄ to hydrogen peroxide of 7:3) and refluxed at 80°C for 1 hour to hydroxylate the microspheres. The microspheres were then removed and washed. The hydroxylated microspheres were then dispersed in toluene, and the aminosilane coupling agent KH550 was added. The mixture was stirred at 80°C overnight and then rinsed with deionized water to obtain the amino-treated microspheres.

[0119] According to the above process, four groups of experiments were carried out, namely Examples 1 to 4, and the experimental groups and samples were respectively denoted as a, b, c, and d (in subsequent steps, the products of each step are also represented by a, b, c, and d).

[0120] The information on the amount of each raw material used in the four groups of experiments is shown in Table 1:

[0121] Table 1: Experimental information for step A)

[0122]

[0123] B) 20 g of glutamic acid was added to 30 mL of propargyl alcohol, and 8 mL of concentrated sulfuric acid was added under stirring in an ice bath to react overnight. The mixture was then added to a saturated sodium bicarbonate solution (the volume ratio of the saturated sodium bicarbonate solution to the concentrated sulfuric acid was 1:0.9) for neutralization. The solution was then filtered off, and the mixture was recrystallized in a methanol-water solution. The solvent was then filtered off to obtain a white powder, i.e., propargyl alcohol-modified glutamic acid (GAP) represented by formula (1).

[0124] The obtained propargyl alcohol-modified glutamic acid represented by formula (1), 6 g of bis(trichloromethyl) carbonate and 200 mL of tetrahydrofuran solvent were mixed, reacted at 50° C. for 2 hours, and then post-treated to obtain propargyl alcohol-modified glutamic acid N-carboxylic anhydride (GAP-NCA) represented by formula (2).

[0125] C) 100 mg of each of amino-modified glass microspheres a to d were dispersed in 8 mL of anhydrous DMF, and 0.8 g of propargyl alcohol-modified glutamic acid N-carboxylic anhydride (GAP-NCA) represented by formula (2) was added. The mixture was stirred and reacted at 25°C under nitrogen for 3 days. The solution was then filtered off, the microspheres were washed with an organic solvent, and vacuum-dried to obtain glass microspheres with surface grafted polyglutamic acid (G-PGAP).

[0126] The experimental information of the four groups of experiments is shown in Table 2:

[0127] Table 2: Experimental information of step C)

[0128]

[0129] The grafting amounts in Table 2 were obtained through thermogravimetric analysis. It can be seen that the surface grafting densities of samples a to d are between 0.25% and 1.05%.

[0130] D) 100 mg of each of glass microspheres a to d with surface grafted polyglutamic acid was taken, 20 mL of dimethyl sulfoxide (DMSO) was added, and argon was bubbled for 30 minutes. Then, 20 mg of copper sulfate pentahydrate and 50 μL of oligoethylene glycol azide (degree of polymerization m = 4) were added. Bubbling was continued for 10 minutes, and 40 mg of sodium ascorbate was added. Click chemistry reaction was carried out at 40°C under argon atmosphere for 3 days. After filtering the solution, the product was rinsed with deionized water to obtain a thermosensitive glass microsphere carrier (G-PGAP-g-MEO grafted with oligoethylene glycol (degree of polymerization m = 4) m ).

[0131] The experimental information of the four groups of experiments is shown in Table 3:

[0132] Table 3: Experimental information for step D)

[0133]

[0134] Finally, four temperature-sensitive glass microsphere products (denoted as a, b, c, and d) were obtained, which were the products of Examples 1 to 4, respectively.

[0135] Test Example 1: Product Testing

[0136] 1. SEM characterization

[0137] The SEM image of the thermosensitive glass microsphere carrier obtained in Example 4 is shown in Figure 1 It can be seen that the particle size of the thermosensitive glass microsphere carrier product obtained in the present invention is about 200 μm, and the particles are well dispersed.

[0138] 2. Cell adsorption and desorption test

[0139] Cell culture:

[0140] Normally cultured cells expressing green fluorescent protein (GFP) were trypsinized and centrifuged for counting. 100 mg of thermosensitive glass microspheres and 1 million cells were placed into each well of a 24-well plate. The GFP cells were cultured in a medium containing 10% fetal bovine serum and incubated at 37°C in an incubator containing 5% carbon dioxide for 48 hours.

[0141] After 48 hours of culture, the 24-well plate was taken and the expression of GFP protein was observed under a microscope. Specifically, the green fluorescent protein signal was observed using a fluorescence microscope. Figure 2 (Scale bar 100 μm), it can be seen that the cells adhered successfully.

[0142] After the cells have successfully adhered, the culture plate was placed in a 4°C refrigerator for a certain period of time (2 minutes, 5 minutes, 10 minutes, and 30 minutes respectively). Then, it was gently blown and the supernatant was aspirated. The cells were counted and it was observed that the cells gradually detached at low temperature. At the same time, the thermosensitive glass microspheres were taken after 30 minutes and the undetached cells were digested with trypsin. The cell detachment rate was calculated using the following formula:

[0143] Cell thermosensitive detachment rate (%) = A / (A+B) × 100;

[0144] Among them, A is the number of cells removed by thermosensitive detachment, and B is the number of cells removed by trypsin digestion; each group of experiments was repeated three times, and the average value was taken as the test result.

[0145] The desorption rate results are shown in Figure 3 It can be seen that as time goes by, the desorption rate of the blank control group (i.e., the control group) does not change significantly, while the cell desorption effect of groups 1 to 4 is significantly improved, especially after 10 minutes. The desorption efficiency of each group after 30 minutes is shown in Table 4:

[0146] Table 4: Cell detachment rate

[0147] Culture carrier type Cell thermosensitive detachment rate (%) None (blank group) 2 Example 1 78 Example 2 81 Example 3 87 Example 4 95

[0148] 3. Cytotoxicity test

[0149] The cell counting reagent CCK-8 was used to evaluate the cytotoxicity of thermosensitive glass microspheres:

[0150] Within 24 hours before the experiment, cells in logarithmic growth phase were obtained, digested with trypsin, and diluted with culture medium. 1×10 4 Cells were seeded into a 96-well culture plate at a density of 50 mg. After adding 50 mg of thermosensitive glass microspheres, the cells were cultured in a 37°C incubator containing 5% CO2 until confluence reached 80-90%. After aspirating the cell culture medium, 100 μL of 10% CCK-8 medium was added to each well and the cells were incubated at 37°C for 1 hour. The absorbance of each well was then measured using a microplate reader at a wavelength of 450 nm. Cell viability was calculated using the following formula:

[0151] Cell survival rate (%) = (A sample / A control )×100;

[0152] Among them, Asample is the absorption of the cell sample well with thermosensitive glass microspheres added, A control is the absorption of the cell sample well without the addition of temperature-sensitive glass microspheres; each group of experiments was repeated three times, and the average value was taken as the test result.

[0153] The test results are shown in Table 5:

[0154] Table 5: Cell viability

[0155] Culture carrier type Cell survival rate (%) Example 1 96.1 Example 2 94.1 Example 3 95.0 Example 4 91.9

[0156] It can be seen from the test results in Table 5 that the use of the thermosensitive glass microspheres obtained in the present invention as culture carriers can achieve a cell survival rate of more than 91.9%, showing extremely low cytotoxicity and extremely high biosafety.

[0157] Example 5 was prepared according to the preparation process of Example 4, except that the degree of polymerization of the azide polyethylene glycol in step D) was adjusted to 2.

[0158] Comparative Example 1

[0159] The preparation process of Example 4 was followed, except that the degree of polymerization of the azido polyethylene glycol in step D) was adjusted to 1, i.e., azidoethanol was used.

[0160] Comparative Example 2

[0161] The preparation process of Example 4 was followed, except that the degree of polymerization of the azide polyethylene glycol in step D) was adjusted to 10.

[0162] Test Example 2: Product Testing

[0163] The products obtained in Example 5 and Comparative Examples 1-2 were tested according to the test method in Test Example 1. The results are shown in Table 6.

[0164] Table 6: Test results of various embodiments and comparative examples

[0165] Culture carrier type Cell detachment rate (%) Cell survival rate (%) Example 5 95 94 Comparative Example 1 5 90 Comparative Example 2 3 89

[0166] As can be seen from Table 6, combined with the test results in Tables 4-5, the thermosensitive glass microsphere carriers obtained in Examples 1-5 of the present invention can achieve a cell detachment rate of over 78%, demonstrating efficient cell detachment; the cell survival rate reaches over 91.9%, demonstrating extremely low cytotoxicity and high biosafety. Compared with Examples 1-5, the cell detachment rate and cell survival rate of Comparative Examples 1-2 are significantly reduced, demonstrating that the introduction of azido-polyethylene glycol and controlling the degree of polymerization of the azido-polyethylene glycol within a certain range in the present invention is beneficial for improving cell adhesion and detachment, as well as reducing cytotoxicity.

[0167] It should be noted that the definition of glass microspheres in the art is: "Glass microspheres (microspheres) are hollow, sealed, spherical, powder-like, ultra-lightweight filling materials with particle sizes ranging from 15 to 300 μm. Therefore, the definition of glass microspheres is common knowledge in the art and will not be elaborated here."

[0168] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including making and using any device or system, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal description of the claims, or if they include equivalent structural elements that are not substantially different from the literal description of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A thermosensitive cell microcarrier, characterized in that It comprises glass microspheres and a thermosensitive polymer grafted on the surface of the glass microspheres; the structure of the thermosensitive cell microcarrier is shown in formula (L): in: represents glass microspheres; m and n are the degree of polymerization, m is 2 to 8, and n is 30 to 120; j and k are the molar percentages of the corresponding structural units, j+k=1, 0%≤j / (j+k)≤10%, 90%≤k / (j+k)≤100%; The mass fraction of the thermosensitive polymer in the thermosensitive cell microcarrier is ≥1%.

2. A method for preparing the thermosensitive cell microcarrier according to claim 1, characterized in that: The following steps are involved: A) performing surface hydroxylation treatment on glass microspheres to obtain surface hydroxylated microspheres; treating the surface hydroxylated microspheres with an aminosilane coupling agent to obtain amino-treated glass microspheres; B) glutamic acid reacts with propargyl alcohol to generate propargyl alcohol-modified glutamic acid shown in formula (1); The propargyl alcohol-modified glutamic acid represented by formula (1) reacts with bis(trichloromethyl) carbonate to generate propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2); C) reacting the amino-modified glass microspheres obtained in step A) with the propargyl alcohol-modified glutamic acid N-carboxylic anhydride of formula (2) obtained in step B) to obtain glass microspheres with surface grafted polyglutamic acid; D) reacting the glass microspheres with the surface grafted polyglutamic acid with polyethylene glycol azide to obtain glass microsphere microcarriers with the surface modified polyglutamic acid derivatives; There is no order restriction for step A) and step B).

3. The preparation method according to claim 2, characterized in that In step D), the degree of polymerization of the azide polyethylene glycol is 2 to 8; In step A), the aminosilane coupling agent is KH550.

4. The preparation method according to claim 2, characterized in that In step A), the conditions for treating the surface hydroxylated microspheres with an aminosilane coupling agent are: a temperature of 60 to 120° C. and a time of 4 to 24 hours.

5. The preparation method according to claim 2, characterized in that In step B), the reaction conditions of glutamic acid and propargyl alcohol are: temperature 0-8° C., time 8-12 hours.

6. The preparation method according to claim 2, characterized in that In step B), the reaction conditions of the propargyl alcohol-modified glutamic acid represented by formula (1) and bis(trichloromethyl) carbonate are: temperature 25-55° C., time 0.5-3 hours.

7. The preparation method according to claim 2, characterized in that In step C), the reaction conditions are: temperature 25-40°C, time 3-7 days; In step D), the reaction conditions are: temperature 30-50° C., and time 3-7 days.

8. The preparation method according to claim 2, characterized in that In step A): The surface hydroxylation treatment process is as follows: soaking the glass microspheres in a mixed solution to hydroxylate the surface of the glass microspheres, and then separating the solid and the liquid to obtain surface hydroxylated microspheres; The mixed solution is a mixture of sulfuric acid solution and hydrogen peroxide solution, wherein the molar ratio of H2SO4 to hydrogen peroxide is 7:3; the soaking conditions are: temperature 40-80°C, time 1-4 hours; The process of treating the surface hydroxylated microspheres with an aminosilane coupling agent comprises: mixing the surface hydroxylated microspheres, a solvent and the aminosilane coupling agent to react to obtain amino glass microspheres; Step C) specifically comprises: dispersing the amino-modified glass microspheres in an anhydrous organic solvent, adding the propargyl alcohol-modified glutamic acid N-carboxylic anhydride represented by formula (2), and stirring the mixture under a protective atmosphere to obtain glass microspheres with surface grafted polyglutamic acid; Step D) specifically comprises: mixing glass microspheres with surface grafted polyglutamic acid with an organic solvent, bubbling with an inert gas for a period of time, adding copper sulfate pentahydrate and polyethylene glycol azide, continuing to bubble for a period of time, then adding sodium ascorbate, and performing a click chemistry reaction under an inert gas atmosphere to obtain glass microspheres with surface modified polyglutamic acid derivatives.

9. A method for culturing cells, characterized in that: The culture carrier used is the temperature-sensitive cell microcarrier according to claim 1 or the temperature-sensitive cell microcarrier prepared by the preparation method according to any one of claims 2 to 8.

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