A polymer nanofiber, its preparation method and application

Abstract

The present invention relates to the technical field of biomaterials, and discloses a polymer nanofiber, a preparation method thereof and an application thereof. The polymer nanofiber comprises the following raw materials for preparation: gelatin, a crosslinking agent, a nano reinforcing phase and a solvent; the nano reinforcing phase comprises nano-hydroxyapatite. The polymer nanofiber provided by the present invention uses gelatin as the main raw material and a substance including nano-hydroxyapatite as the nano reinforcing phase. The obtained polymer nanofiber has good biocompatibility, high cell activity, a large specific surface area, a pore structure, good structural stability and low cost, and has the ability to enhance the affinity between adjacent cells, strengthen the connection between cells, and further promote the formation of cell spheres by cells; the preparation method of the cell spheres provided by the present invention co-cultures the polymer nanofiber with cells, and then polymer nanofiber-reinforced cell spheres can be obtained. The operation is simple, the cost is low, it is easy to control, it is suitable for large-scale culture, and the cell spheres have a fast sphere formation speed and good structural stability.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a polymer nanofiber and its preparation method and application. Background Art

[0002] A spheroid is a three-dimensional cell aggregate structure that can better simulate the structure and function of in vivo tissues and has been widely used in fields such as tumor research, drug screening, tissue engineering, and toxicology.

[0003] The existing methods for preparing spheroids mainly include: (1) The hanging drop method: Drop the cell suspension in the form of droplets (usually 10 - 50 μL) on the lid of a culture plate, then invert the lid so that the droplets hang on the lid. Due to the action of gravity, the cells will gradually settle to the bottom of the droplets and aggregate with each other, eventually forming spheroids. The key to this method lies in the stability of the droplets and the control of the evaporation of the culture medium; (2) The ultra-low attachment culture method: Use a specially treated culture plate or culture dish with an ultra-low attachment surface, which can prevent cells from adhering to the wall, thereby promoting the aggregation of cells in a suspended state to form spheroids. The key to this method lies in optimizing the cell density and culture time to ensure that the size and morphology of the spheroids meet the experimental requirements; (3) The rotary culture method: Place the cell suspension in a rotary bottle and keep the cells in a suspended state and gradually aggregate to form spheroids by continuous stirring; (4) The microcarrier culture method: Use microcarriers (such as gelatin, agarose, or polymer microspheres) as scaffolds for cell attachment to promote the aggregation of cells to form spheroids. Microcarriers usually have a large specific surface area and can support the high-density growth of cells; (5) The air-liquid interface method: Inoculate cells on a porous membrane and culture them under air-liquid interface conditions to promote the aggregation of cells to form spheroids. The key to this method lies in the selection of the porous membrane and the optimization of the culture conditions.

[0004] In the above methods, the hanging drop method is easy to operate, low in cost, and the formed cell spheres are of uniform size, but it is only suitable for small-scale experiments and high-throughput screening; the ultra-low attachment culture method is easy to operate, suitable for large-scale culture, and has a high cell sphere formation efficiency, but the cell spheres are of uneven size and the culture medium needs to be replaced regularly to maintain cell viability; the rotary culture method can generate a large number of cell spheres, and the size of the cell spheres can be controlled by adjusting the stirring speed, but this method requires the use of special equipment (such as rotary flasks and stirrers), and the shear force generated during the stirring process may affect cell viability; the microcarrier culture method is suitable for large-scale culture, and the size and morphology of the cell spheres can be regulated by the properties of the microcarriers, but the microcarriers may affect the purity and function of the cell spheres, and the microcarrier materials and cell seeding density also need to be optimized; the air-liquid interface method can simulate the physiological environment of some tissues (such as the respiratory tract, skin) and promote the natural formation of cell spheres, but this method is complex to operate, requires special equipment, and the experimental conditions need to be optimized to ensure cell viability. In addition, the following challenges also exist in the preparation of cell spheres: for cell spheres with a larger diameter, inside the cell sphere, as the distance from the surface increases, the concentrations of oxygen and nutrients gradually decrease, and the metabolic activities of cells are restricted, thus affecting the survival and function of cells. For example, existing studies have shown that cell spheres with a diameter greater than 500 μm will have premature cell death in the central region of the cell sphere due to the difficulty of oxygen and nutrient diffusion.

[0005] Therefore, it is of great significance to develop a method for preparing cell spheres that is simple to operate, low in cost, easy to control, suitable for large-scale culture, and can ensure the normal diffusion of oxygen and nutrients inside the cells, for promoting the application of cell spheres in the fields of tumor research, drug screening, tissue engineering, and toxicology. Summary of the Invention

[0006] The present invention aims to at least solve one of the above technical problems existing in the prior art. For this reason, one of the purposes of the present invention is to provide a polymer nanofiber.

[0007] Another purpose of the present invention is to provide a method for preparing this polymer nanofiber.

[0008] A third purpose of the present invention is to provide a method for preparing cell spheres.

[0009] A fourth purpose of the present invention is to provide a cell sphere.

[0010] A fifth purpose of the present invention is to provide the application of this cell sphere.

[0011] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0012] The first aspect of the present invention provides a polymer nanofiber, comprising the following raw materials for preparation: gelatin, a crosslinking agent, a nano-reinforcing phase, and a solvent; the nano-reinforcing phase includes nano-hydroxyapatite (nHAP).

[0013] In some embodiments of the present invention, the diameter of the polymer nanofiber is 550 - 650 nm.

[0014] In some specific embodiments of the present invention, the diameter of the polymer nanofiber is 550 - 600 nm.

[0015] In some embodiments of the present invention, the molecular weight of the gelatin is 50 - 100 kDa.

[0016] In some embodiments of the present invention, the mass ratio of the gelatin to the crosslinking agent is (20 - 50):1.

[0017] In some embodiments of the present invention, the mass ratio of the gelatin to the nano-reinforcing phase is (10 - 60):1.

[0018] In some embodiments of the present invention, the solid-liquid ratio of the gelatin to the solvent is 1 g:(5 - 30) mL.

[0019] In some embodiments of the present invention, the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0020] In some specific embodiments of the present invention, the mass ratio of the gelatin to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is (25 - 50):1.

[0021] In some specific embodiments of the present invention, the mass ratio of the gelatin to N-hydroxysuccinimide is (20 - 30):1.

[0022] In the present invention, the dosage of the crosslinking agent is strictly controlled to ensure the sufficiency and stability of the crosslinking reaction, while avoiding the solution becoming too viscous due to excessive addition of the crosslinking agent, which affects the stretching and formation of the fibers.

[0023] In some embodiments of the present invention, the solvent includes a buffer solution and an alcohol solvent; the volume ratio of the buffer solution to the alcohol solvent is 1:(0.5 - 5).

[0024] In some specific embodiments of the present invention, the volume ratio of the buffer solution to the alcohol solvent is 1:(0.5 - 4).

[0025] In some embodiments of the present invention, the buffer solution includes PBS buffer solution.

[0026] In some embodiments of the present invention, the alcohol solvent includes at least one of ethanol and isopropanol.

[0027] The inventive concept of the present invention is as follows: In natural bone, the mass ratio of hydroxyapatite (HAP) to collagen is 7:3, and the hydroxyapatite in natural bone is in nanoscale. Therefore, nano-hydroxyapatite is used as the nano-reinforcing phase to provide corresponding biological activity. Gelatin is a hydrolysis product of collagen and contains multiple biologically active functional groups. Different from collagen, gelatin can be dissolved in water. Compared with collagen, gelatin is easier to shape, has a lower cost, has good biocompatibility, and has nearly zero immunogenicity. Therefore, the present invention uses gelatin and nano-hydroxyapatite as the main raw materials to prepare a polymer nanofiber with the characteristics of both raw materials.

[0028] The second aspect of the present invention provides a method for preparing the polymer nanofiber described in the first aspect of the present invention, including the following steps:

[0029] S1. Mix gelatin with a solvent to obtain a gelatin solution;

[0030] S2. Add a crosslinking agent to the gelatin solution and carry out a crosslinking reaction to obtain a crosslinked gelatin solution;

[0031] S3. Add a nano-reinforcing phase to the crosslinked gelatin solution, disperse it to obtain a spinning solution, and perform electrospinning to obtain the polymer nanofiber.

[0032] In some embodiments of the present invention, in step S1, the pH of the gelatin solution is 3.5 - 4.5.

[0033] In some specific embodiments of the present invention, in step S1, the pH of the gelatin solution is 3.8 - 4.2.

[0034] In some embodiments of the present invention, in step S1, it includes adding a pH regulator to adjust the pH of the gelatin solution; the concentration of the pH regulator is 0.1 - 10 mol / L.

[0035] In some specific embodiments of the present invention, the pH regulator includes acetic acid.

[0036] In some embodiments of the present invention, in step S1, it includes first stirring and mixing a buffer solution and an alcohol solvent, and the rotation speed of the stirring and mixing is 100 - 300 rpm, and the time is 5 - 15 min.

[0037] In some embodiments of the present invention, in step S1, the process of mixing gelatin with the solvent is assisted by water bath heating, and the temperature of the water bath heating is 40 - 50 °C.

[0038] In some embodiments of the present invention, in step S1, during the process of mixing the gelatin with the solvent, stirring is assisted, and the rotation speed of the stirring is 200 - 500 rpm.

[0039] In some embodiments of the present invention, in step S2, the temperature of the cross-linking reaction is 25 - 35 °C, and the time is 10 - 30 min.

[0040] In some embodiments of the present invention, in step S2, during the process of the cross-linking reaction, stirring is assisted, and the rotation speed of the stirring is 100 - 300 rpm.

[0041] In some embodiments of the present invention, in step S3, the temperature of the dispersion is 25 - 35 °C, and the time is 10 - 20 min.

[0042] In some embodiments of the present invention, in step S3, during the process of the dispersion, stirring is assisted, and the rotation speed of the stirring is 200 - 500 rpm.

[0043] In some embodiments of the present invention, in step S3, the process parameters of the electrospinning include at least one of the following parameters:

[0044] 1) The ambient temperature is 20 - 30 °C;

[0045] 2) The relative humidity is 40% - 50%;

[0046] 3) The injection speed is 0.10 - 0.15 mm / min;

[0047] 4) The positive voltage is 15 - 25 kV, and the negative voltage is -2 to -5 kV;

[0048] 5) The spinning distance is 15 - 20 cm;

[0049] 6) The rotation speed of the collection device is 80 - 120 rpm.

[0050] In the present invention, the process parameters of electrospinning need to be strictly controlled for the following reasons: ① When the environmental temperature and humidity change, it is easy to cause the nanofibers to be beaded; ② The spinning distance needs to be set comprehensively considering the fluidity of the spinning solution and the efficiency of electrospinning to avoid the fiber structure being beaded or broken. When the spinning distance is too close, the solvent cannot be fully volatilized, and beaded nanofibers will appear; ③ When the concentration of the gelatin solution increases, the fiber width will increase, and too high a concentration may directly lead to spinning failure; ④ Too fast a pushing speed is likely to cause fiber breakage or unevenness, while too slow a speed is likely to cause an increase in fiber diameter or the appearance of breakpoints; ⑤ For the setting of positive and negative voltages, it is necessary to ensure that the voltage is stable and meets the electrostatic field strength required for spinning. A higher voltage helps to stretch the solution to form fibers, but too high a voltage may lead to arc discharge or solution splashing; ⑥ The rotation speed of the collection device affects the uniformity of nanofiber collection. If set improperly, it will cause fiber crossing or aggregation. By controlling each parameter in the present invention, the stability of the spinning solution and the mechanical properties of the fibers can be effectively improved.

[0051] The third aspect of the present invention provides a method for preparing cell spheres, including preparing a nanofiber solution by using a culture medium to dissolve the polymer nanofibers described in the first aspect of the present invention, and co-culturing with cells to obtain the cell spheres.

[0052] In some embodiments of the present invention, the concentration of the nanofiber solution is 1 - 5 mg / mL; the dosage of the nanofiber solution is 100 - 600 cells / μL.

[0053] In some specific embodiments of the present invention, the concentration of the nanofiber solution is 1 - 3 mg / mL.

[0054] In some embodiments of the present invention, the composition of the culture medium includes 400 - 500 mL of DMEM / F12 basal medium, 20 - 100 mL of fetal bovine serum, and 2 - 10 mL of penicillin-streptomycin.

[0055] In some embodiments of the present invention, the time of co-culture is 1 - 30 d.

[0056] In some specific embodiments of the present invention, the time of co-culture is 1 - 20 d.

[0057] In some embodiments of the present invention, the cells are selected from at least one of stem cells, cancer cells, cardiomyocytes, nerve cells, hepatocytes, fibroblasts, endothelial cells, and epithelial cells derived from stem cells, as well as cardiomyocytes, nerve cells, hepatocytes, fibroblasts, endothelial cells, and epithelial cells derived from organisms.

[0058] In some specific embodiments of the present invention, the cells include bone marrow mesenchymal stem cells (BMSCs).

[0059] The fourth aspect of the present invention provides a cell sphere, which is prepared by using the method for preparing a cell sphere described in the third aspect of the present invention.

[0060] In the present invention, the obtained cell sphere is a polymer nanofiber-reinforced cell sphere. Since the polymer nanofiber is a gelatin nanofiber containing a nano-reinforcing phase, and the gelatin contains an RGD sequence (arginine-glycine-asparagine acid), this sequence can bind well to integrins on the cell surface, thereby upregulating the expression of junction proteins on the cell membrane surface. The accumulation of junction proteins on the cell membrane surface can enhance the affinity between adjacent cells, strengthen the connection between cells, and thus form a cell sphere. In addition, the gelatin nanofiber has a high specific surface area and structural stability, which can provide more support and structural framework, so as to better maintain the stability of the cell sphere. When nano-hydroxyapatite is used as the nano-reinforcing phase, the good biological activity of nano-hydroxyapatite itself enhances the cell activity of the polymer nanofiber to cells, facilitates the aggregation of cells and polymer nanofibers, and the addition of nano-hydroxyapatite causes the polymer nanofiber to generate pores, thereby promoting the diffusion of oxygen and nutrients into the interior of the cell sphere, so that the cells in the central region of the cell sphere are not easily prematurely dead due to the lack of oxygen and nutrients, which is conducive to the long-term culture of the cell sphere.

[0061] The fifth aspect of the present invention provides the application of the cell sphere described in the fourth aspect of the present invention in tumor research, drug screening, tissue engineering, and toxicology research.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0063] 1) The polymer nanofiber provided by the present invention uses gelatin as the main raw material and a substance including nano-hydroxyapatite as the nano-reinforcing phase. The obtained polymer nanofiber has the characteristics of good biocompatibility, high cell activity, large specific surface area, porous structure, good structural stability, and low cost, and can enhance the affinity between adjacent cells, strengthen the connection between cells, and promote the formation of cell spheres by cells;

[0064] 2) The method for preparing the polymer nanofiber provided by the present invention has simple steps and mild process conditions. By controlling the process parameters of electrospinning, the stability of the spinning solution and the mechanical properties of the fibers can be improved, and uniform and good-looking polymer nanofibers can be obtained;

[0065] 3) The method for preparing the cell sphere provided by the present invention co-cultures the polymer nanofiber with cells, and then a polymer nanofiber-reinforced cell sphere can be obtained. The operation is simple, the cost is low, it is easy to control, and it is suitable for large-scale culture;

[0066] 4) The cell spheres provided by the present invention have a fast spheroidization speed and good structural stability. Oxygen and nutrients can diffuse into the interior of the cell spheres through the pores of the polymer nanofibers. The cells in the central region of the cell spheres are not prone to premature death, and the addition of the polymer nanofibers does not affect the reproduction of the cells, which can meet the application requirements of cell spheres in tumor research, drug screening, tissue engineering, toxicology research, etc. Description of the Drawings

[0067] Figure 1 It is the infrared spectrogram of the gelatin solution, nano-hydroxyapatite, spinning solution and polymer nanofibers in Example 1;

[0068] Figure 2 It is the laser confocal microscope image of the polymer nanofibers in Example 1;

[0069] Figure 3 It is the morphological observation of the adherent culture of the fourth-generation BMSCs in Application Comparative Example 1;

[0070] Figure 4 It is the DIC microscope image of the formation process of BMSCs cell spheres in Application Comparative Example 1;

[0071] Figure 5 It is the DIC microscope image of the BMSCs cell spheres on the 5th day after plating in Application Comparative Example 1;

[0072] Figure 6 It is the comparison diagram of the cytoskeleton structure of BMSCs before and after spheroidization in Application Comparative Example 1;

[0073] Figure 7 It is the cytoskeleton structure of the BMSCs cell spheres in Application Comparative Example 1;

[0074] Figure 8 It is the DIC microscope image of the process of enhancing the formation of BMSCs cell spheres by gelatin molecules in Application Comparative Example 2;

[0075] Figure 9 It is the morphology of the polymer nanofiber-reinforced BMSCs cell spheres on the 3rd day after plating in Application Example 1;

[0076] Figure 10 It is the DIC microscope image of the formation process of the polymer nanofiber-reinforced BMSCs cell spheres on the 6th - 14th day when the cell number is 1000 - 5000 in Application Example 1;

[0077] Figure 11 It is the DIC microscope image of the polymer nanofiber-reinforced BMSCs cell spheres on the 3rd day when the cell number is 6000 in Application Example 1;

[0078] Figure 12Image of the failure of polymer nanofibers and BMSCs to form spheres;

[0079] Figure 13 Results of the cell viability and cytotoxicity assay in Experimental Example 1;

[0080] Figure 14 Results of the cell viability and cytotoxicity staining assay of cell spheres on the 20th day after cell seeding in Application Example 1;

[0081] Figure 15 Cell proliferation of polymer nanofiber-reinforced BMSCs cell spheres in Experimental Example 2;

[0082] Figure 16 Schematic diagram of the principle of polymer nanofiber-reinforced BMSCs spheroid formation. Detailed implementation manners

[0083] The content of the present invention will be further described in detail through specific embodiments below. The raw materials, reagents or devices used in the embodiments and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0084] Example 1, in this example, a polymer nanofiber is prepared as follows:

[0085] S11. Add 40 mL of PBS buffer solution and 60 mL of ethanol to a beaker, stir and mix at a speed of 200 rpm for 10 min to obtain a mixed solvent;

[0086] S12. Weigh 5.5 g of gelatin powder (molecular weight 80 kDa), slowly add it to the mixed solvent obtained in step S11, and stir and dissolve it at a speed of 350 rpm under the condition of water bath heating at 45 °C;

[0087] S13. Slowly add 0.5 mol / L acetic acid solution to the solution obtained in step S12, and adjust the pH of the solution to 4.0 to obtain a gelatin solution;

[0088] S21. Add 150 μL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.2 g of N-hydroxysuccinimide to the gelatin solution obtained in step S13, and stir and react at a speed of 200 rpm at 30 °C for 20 min to obtain a cross-linked gelatin solution;

[0089] S31. Add 0.3 g of nano-hydroxyapatite to the cross-linked gelatin solution obtained in step S21, and stir and disperse it at a speed of 200 rpm at 30 °C for 15 min to obtain a uniform spinning solution;

[0090] S32. Load the spinning solution obtained in step S31 into a syringe and perform electrospinning under the following process parameters: ambient temperature 25°C, relative humidity 45%, injection speed 0.10 mm / min, positive voltage 20 kV, negative voltage -3 kV, spinning distance 18 cm, rotational speed of the collection device 100 rpm to obtain polymer nanofibers.

[0091] Perform infrared spectroscopy tests on the gelatin solution, nano-hydroxyapatite, spinning solution, and polymer nanofibers in Example 1. Figure 1 For the infrared spectra of the gelatin solution, nano-hydroxyapatite, spinning solution, and polymer nanofibers in Example 1, it can be seen from Figure 1 that in the gelatin solution, the band at 1300 cm -1 is mainly attributed to the wagging vibration of the proline side chain, indicating the presence of type I gelatin. Other obvious spectral peaks in the range of 1250 - 1400 cm -1 are all related to type I gelatin. In addition, the characteristic spectral bands include amide A band (3300 - 3500 cm -1 ) and amide B band (1680 - 1780 cm -1 ). The amide A band is the strongest absorption band in the amide bond, mainly generated by the N-H stretching vibration, and the amide B band is mainly generated by the combined action of C=O stretching vibration and N-H bending vibration; in the polymer nanofibers, the amide I band (1600 - 1700 cm -1 ) is mainly generated by the C=O stretching vibration, and the amide II band (1500 - 1550 cm -1 ) is mainly generated by the N-H bending vibration and C-N stretching vibration. The appearance of the amide I band and amide II band indicates that the polymer nanofibers mainly adopt an α-helical structure. Other spectral peaks include the stretching vibration of hydroxyl (-OH) (4000 - 3200 cm -1 ), carbonate bending vibration (1540 - 1400 cm -1 and 1530 - 1320 cm -1 ) and phosphate band (900 - 1200 cm -1 ); in the spinning solution, the shift of the gelatin band at 1300 cm -1 can confirm the formation of a chemical bond between the carboxyl ions in gelatin and nano-hydroxyapatite. This bonding involves the formation of a covalent bond between Ca 2+ and the R-COO - of the gelatin molecule. Crosslinking may lead to a shortening of the distance between nano-hydroxyapatite and gelatin nanofibers, promoting more Ca 2+ to combine with the R-COO - of the gelatin molecule.

[0092] After dyeing the polymer nanofibers prepared in Example 1 with Rhodamine B, laser confocal microscopy characterization was carried out. Figure 2 Figure 226 is the laser confocal microscopy image of the polymer nanofibers in Example 1, where Figure 2 Figure 227(a) is the laser confocal microscopy image with a magnification of 1000 times, Figure 2 Figure 228(b) is the laser confocal microscopy image with a magnification of 2000 times. As can be seen from Figure 2 it, the polymer nanofibers prepared in Example 1 have a nanoscale size with a diameter of about 590 nm, so they have the characteristic of a high specific surface area and can show extremely strong activity. Existing studies have shown that the diameter of nanofibers affects the migration rate of cells, and different cells have different responses to the diameter of nanofibers. In the study on BMSCs, BMSCs showed the fastest migration rate on poly(L-lactic acid) fibers with a diameter of 600 nm. Therefore, it is speculated that the polymer nanofibers prepared in Example 1 are also beneficial to promoting cell spheroid formation.

[0093] Application of Comparative Example 1: Isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs):

[0094] (1) Preparation of the culture medium: Mix 450 mL of DMEM / F12 basal medium, 50 mL of fetal bovine serum, and 5 mL of penicillin-streptomycin. The resulting mixture was filtered through a 220 μm filter to obtain the culture medium, which was sealed and stored at 4 °C. Reagents related to cell culture and cell passage need to be water-bathed at 37 °C before each use.

[0095] (2) Cell extraction: Prepare a scalpel, surgical scissors, forceps, pipette, syringe, and culture dish, place them on the ultra-clean workbench, and sterilize them with ultraviolet light for 30 min; decapitate the rat 10 min before the experiment and soak the rat in 75 wt% alcohol for 5 - 10 min; take out the soaked rat, squeeze out the excess alcohol from the mouth, then fix the front paw of the rat with a needle, peel off the skin of the rat, cut it around, remove the excess meat, cut off the femur and tibia at the joint, soak the two in PBS solution containing 10 wt% - 15 wt% double antibody (penicillin-streptomycin), cut open the head of the bone to expose the red bone marrow, and use a syringe filled with 5 mL of culture medium to flush out the bone marrow into a new culture dish that has been sterilized by ultraviolet light. Additionally, use a pipette to add 5 mL of culture medium to the culture dish. Place the culture dish containing rat bone marrow in an incubator at 37 °C and 5% CO 2 for cell culture.

[0096] (3) Cell separation and purification: Taking advantage of the characteristics of BMSCs adherent growth, bone marrow mesenchymal stem cells were separated and purified by attachment to the culture dish surface. Within 48 hours after cell extraction, the culture dish was placed in the incubator to promote cell adhesion growth and avoid cell medium replacement. This helped the cells to fully adhere to the culture dish surface and improved the efficiency of separation and purification. After 48 hours of culture, the cell medium was replaced every two days, and the growth status and number of cells were closely observed. The specific operation of cell medium replacement was as follows: first, the old culture medium was poured out, and then 10 mL of PBS buffer was added to the culture dish with a pipette for rinsing. The PBS buffer in the culture dish was poured out, and then 10 mL of new culture medium was added to the culture dish, and the dish was kept at 37°C and 5% CO. 2 Continue culturing in a cell culture incubator until the cell density reaches more than 80% (usually takes 4-6 days), then cell subculture can be performed.

[0097] (4) Cell subculture: Before cell subculture, pour out the old culture medium, then add 10 mL of PBS buffer to the culture dish for rinsing, pour out the PBS buffer in the culture dish, then add 3 mL of trypsin to the culture dish, digest in the cell culture incubator for 2 min, take out and add 2 times the amount of culture medium used for trypsin to terminate the digestion; place the mixed solution in a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, pour out the supernatant to obtain the cell pellet, resuspend the cell pellet with new culture medium to obtain a cell suspension, transfer the cell suspension to the culture dish, add 20 mL of culture medium for cell culture, and pay attention to ensure that the culture medium is spread evenly on the bottom of the culture dish during the culture process. When the cells are cultured to the fourth generation, the sphere formation experiment can be started.

[0098] In in vitro cell culture, there are usually three types of cell morphology: fibroblast-like morphology, epithelial cell-like morphology, and lymphoblastoid morphology. Cell morphology is affected by the microenvironment, biophysical and morphological signals. Studies have shown that the morphology of cells cultured in vitro often changes from the original spherical shape to a spindle-like or elongated shape. In in vitro adherent culture, BMSCs are usually spindle-shaped or star-shaped, with a large nucleus and a small amount of cytoplasm, and have abundant plasmids and endoplasmic reticulum. The spindle-shaped or star-shaped morphology provides more surface area, which is conducive to interaction with the surrounding environment, receiving signals and regulating cell fate. The large nucleus and a small amount of cytoplasm provide it with sufficient intracellular space for storing genetic material and performing cell functions. This morphology is suitable for its pluripotent stem cell characteristics, enabling it to remain stable in different environments and exert its differentiation potential.

[0099] In the BMSCs cultured by passage in the comparative example of this application, the primary BMSCs were oval and spindle-shaped. At this time, some cells were in an adherent state. After culturing for several days, the cells showed synaptic-like morphological changes and gradually began to fuse. Compared with the primary cells, the second-generation BMSCs were larger cells and extended from triangular to polygonal. The proliferation rate of the second-generation BMSCs increased, and some cells had 1-2 nucleoli. Although it was found that the integration degree of the cells with the surrounding environment in terms of shape and distribution was very low, compared with the original cells, the proliferation rate increased significantly. The third passage of BMSCs was easy to perform, and the cells had a strong proliferation ability at this time. The proliferation rate of the fourth-generation BMSCs continued to increase, and the cells presented a polygonal and long spindle shape. The fourth-generation BMSCs were selected in the comparative example of this application to participate in the preparation of cell spheres. Figure 3 For the morphological observation of the fourth-generation BMSCs in adherent culture in Comparative Example 1 of the application, Figure 3 it can be seen that the fourth-generation BMSCs in adherent culture presented a long spindle shape.

[0100] In the comparative example of this application, the fourth-generation BMSCs were taken, resuspended with an appropriate amount of culture medium to obtain a cell suspension. By counting the cell suspension, different numbers of cells (1000, 2000, 3000, 4000, 5000, and 6000) were added to each well in a 96-well ultra-low attachment U-shaped plate, and 10 parallel samples were made for each group. The growth state of the cells at different time points was observed using a DIC microscope (20× objective lens, scale bar 50 μm).

[0101] The process of cell sphere formation of different numbers of BMSCs on the 1st - 9th day was recorded starting from the day when the cells were plated. Figure 4 For the DIC microscope image of the process of BMSCs cell sphere formation in Comparative Example 1 of the application, Figure 4 it can be seen that in the initial stage of plating, the cells aggregated together to form a monolayer cell population, and a relatively obvious cell sphere structure could be formed only after 4 - 5 days of plating. However, the stability of this cell sphere structure was poor, and obvious scattering began to occur on the 9th day after plating. Moreover, with the increase in the number of cells, the scattering phenomenon became more obvious. When the number of cells was 1000 - 3000, it was more conducive to the formation and stability of cell spheres. In addition, a cell sphere with better quality should appear as a semi-transparent sphere under a bright-field microscope, with a clear outer boundary and a slightly darker core. In the figure, only a few cell spheres could meet this standard, and cell aggregates with irregular shapes or being loose represented poor-quality spheroids. It can be seen that the cell spheres formed only by cells had poor quality and were difficult to exist stably.

[0102] Figure 5 For the DIC microscope image of the BMSCs cell spheres on the 5th day after plating in Comparative Example 1 of the application, among which, Figure 5Among them, (a) is a cell sphere formed by 1000 BMSCs, Figure 5 among them, (b) is a cell sphere formed by 2000 BMSCs, Figure 5 among them, (c) is a cell sphere formed by 3000 BMSCs, Figure 5 among them, (d) is a cell sphere formed by 4000 BMSCs, Figure 5 among them, (e) is a cell sphere formed by 5000 BMSCs, Figure 5 among them, (f) is a cell sphere formed by 6000 BMSCs. It can be seen from Figure 5 that on the 5th day after plating, the diameters of the cell spheres formed by 1000, 2000, 3000, 4000, 5000, and 6000 BMSCs are 108.03 μm, 112.24 μm, 143.10 μm, 154.33 μm, 180.99 μm, and 205.54 μm respectively. That is, as the cell number increases, the diameter of the cell sphere increases, but the more scattered cells there are around. Research shows that the distance of most cells in the body from the nearest capillary will not exceed 100 - 200 μm. Beyond this distance, it is difficult for cells to obtain sufficient oxygen and nutrients. Similarly, the oxygen and nutrients of the cell sphere rely on diffusion supply. If the radius of the cell sphere exceeds 200 μm, the cells inside the cell sphere will be difficult to obtain oxygen and nutrients through diffusion, and necrosis is likely to occur inside the cell sphere, and the viability of its core cells will be lost.

[0103] Rhodamine-labeled phalloidin was used to label F-actin in cells. F-actin is a polymer formed by the polymerization of monomeric actin (G-actin), presenting a filamentous structure inside the cell and participating in multiple biological processes such as maintaining cell morphology and cell movement. The adherent growth of cells is often due to the fact that cells can secrete extracellular matrix during the culture process, and these extracellular matrices can adhere to the surface of the support. At the same time, there are some adhesion molecules on the cell surface, such as integrins, etc., which can bind to these extracellular matrices. This adhesion of cells mediated by integrins to the extracellular matrix determines the position of the cells. At the same time, integrins and the corresponding ligands connect to form a complex, and this complex can directly connect to F-actin with the assistance of other proteins (talin and vinculin). Therefore, integrins directly connect F-actin and the cell's surrounding environment. It is through this way that the adhesion of cells to the extracellular matrix can guide the formation of actin stress fibers, thereby affecting the overall organizational structure of the cytoskeleton. Figure 6 It is a comparison diagram of the cytoskeleton structure of BMSCs before and after spheroid formation in Application Comparative Example 1. Among them, Figure 6 in (a) is an observation diagram of the nucleus and cytoskeleton staining of BMSCs before spheroid formation, Figure 6(b) in it is the observation diagram of the nucleus and cytoskeleton staining after BMSCs form spheres. It can be seen from Figure 6 that the change in the cell aggregation state will affect the structure of the cytoskeleton (F-actin). Research shows that the structural changes of the cytoskeleton have a significant impact on the differentiation direction of stem cells. Therefore, it is speculated that in the sphere formation culture of BMSCs, the obvious change in the cytoskeleton structure may also have an important impact on the cell differentiation direction.

[0104] Figure 7 For the skeleton structure of the BMSCs cell spheres in Application Comparative Example 1, it can be seen from Figure 7 that F-actin has a complex network structure. The reason is that F-actin has polarity. When the cell moves, one segment aggregates and elongates, located on the cell membrane side, and the other segment depolymerizes and shortens, located inside the cell. And the F-actin network is regulated by related binding proteins and is a highly ordered structure, rather than a uniform and unified structure. Usually, the more stable and tight the cell sphere structure is, the more difficult it is for the fluorescent marker to enter the inside of the cell sphere, and the more difficult it is for the inside of the cell sphere to be stained.

[0105] In Application Comparative Example 2, this application comparative example uses a gelatin solution to participate in the preparation of cell spheres to obtain a gelatin molecule-enhanced BMSCs cell sphere. The preparation steps are as follows:

[0106] (1) Weigh gelatin powder (molecular weight 80 kDa), add it to the culture medium, and prepare a 1 mg / mL gelatin solution;

[0107] (2) Take the fourth-generation BMSCs cultured in Application Comparative Example 1, add an appropriate amount of culture medium to resuspend them to obtain a cell suspension. By counting the cell suspension, in an ultra-low attachment U-shaped well 96-well plate, add different numbers of cells (1000, 2000, 3000, 4000, 5000, and 6000) to each well, and then add 10 μL of the 1 mg / mL gelatin solution to each well. Add culture medium until the liquid volume in the well is 200 μL, and mix well, avoiding the generation of air bubbles in the well. Make 10 parallel samples for each group, and use a DIC microscope (20× objective lens, scale 50 μm) to observe the growth state of the cells at different time points.

[0108] Record the cell sphere formation process of different numbers of BMSCs on the 1st - 9th day starting from the day when the cells are plated. Figure 8 For the DIC microscope diagram of the formation process of the gelatin molecule-enhanced BMSCs cell spheres in Application Comparative Example 2, it can be seen from Figure 8It can be seen that, compared with Application Comparative Example 1, after adding gelatin molecules, the formation rate of BMSCs cell spheres becomes significantly faster. A relatively obvious cell sphere structure can be formed approximately 2 - 3 days after plating. However, the stability of this structure is poor, and obvious scattering begins to occur on the 7th day after plating. Moreover, as the number of cells increases, the scattering phenomenon becomes more obvious. By the 9th day, all cell spheres have scattered. Therefore, although the addition of gelatin molecules can accelerate the formation of BMSCs cell spheres and improve the morphology of the cell spheres, it still cannot maintain the stability of the cell sphere structure. It is speculated that this is related to the fact that the cell adhesion effect brought by gelatin molecules is insufficient to support the entire cell sphere structure.

[0109] Application Example 1. In this application example, the polymer nanofibers prepared in Example 1 were used to participate in the preparation of cell spheres, and a polymer nanofiber-reinforced BMSCs cell sphere was obtained. The preparation steps are as follows:

[0110] (1) Dissolve the polymer nanofibers prepared in Example 1 with 75% alcohol, collect them in a centrifuge tube, and then remove the supernatant by centrifugation to collect the polymer nanofibers; resuspend the polymer nanofibers with the culture medium, remove the supernatant, and take the polymer nanofibers. Through this step, the alcohol solution mixed in the polymer nanofibers can be removed; after adding the culture medium again to resuspend the polymer nanofibers, a nanofiber solution that can be co-cultured with cells is obtained;

[0111] (2) Take the fourth-generation BMSCs cultured in Application Comparative Example 1, add an appropriate amount of culture medium to resuspend them to obtain a cell suspension. By counting the cell suspension, in an ultra-low attachment U-shaped well 96-well plate, add different numbers of cells (1000, 2000, 3000, 4000, 5000, and 6000) to each well, and then add 10 μL of a 1 mg / mL nanofiber solution to each well. Add the culture medium until the liquid volume in the well is 200 μL, and mix well, avoiding the generation of air bubbles in the well. Make 10 parallel samples for each group, and use a DIC microscope (20× objective lens, scale 50 μm) to observe the growth state of the cells at different time points.

[0112] Record the cell sphere formation process of different numbers of BMSCs from the day of cell plating for the 1st - 14th days. Figure 9 This is the morphology of the polymer nanofiber-reinforced BMSCs cell sphere on the 3rd day after plating in Application Example 1. It can be Figure 9 seen that, compared with Application Comparative Example 1, after adding the polymer nanofibers prepared in Example 1, the BMSCs sphere formation speed is accelerated, and the cell sphere structure has basically been formed on the 3rd day of culture.

[0113] Figure 10DIC microscopy images of the formation process of polymer nanofiber-reinforced BMSCs cell spheres on the 6th - 14th day when the cell number in Application Example 1 was 1000 - 5000. It can be seen from Figure 10 that compared with Application Comparative Example 1 and Application Comparative Example 2, after adding the polymer nanofibers prepared in Example 1, the structures of BMSCs cell spheres were more stable and there was no obvious scattering within 6 - 14 days.

[0114] Figure 11 DIC microscopy images of polymer nanofiber-reinforced BMSCs cell spheres on the 3rd day when the cell number in Application Example 1 was 6000. Among them, (a) in Figure 11 , (b) in Figure 11 and (c) in Figure 11 are DIC microscopy images of 3 cell spheres randomly selected from 10 parallel samples. It can be seen from Figure 11 that the diameters of the 3 cell spheres are 276.46 μm, 152.78 μm and 177.75 μm respectively.

[0115] Figure 12 Images of the failure of polymer nanofibers to form spheres with BMSCs. Among them, (a) in Figure 12 is the DIC microscopy image of BMSCs cell spheres, and (b) in Figure 12 is the stained image of cell nuclei and cytoskeletons. Compared with cells, the sedimentation rate of polymer nanofibers is faster and it is easy to appear aggregated at the bottom of the well plate, thus unable to effectively aggregate with cells. As shown in Figure 12 , for this situation, after digesting cells with trypsin, centrifuging, and resuspending with medium to obtain a cell suspension, add the nanofiber solution that has been resuspended with medium. During this process, calculate the cell density in the mixed suspension clearly, and then aspirate the required suspension according to the required number of cells and place it in a microcentrifuge tube (0.2 mL), centrifuge at 1200 rpm for 5 min, then place the centrifuge tube in a cell culture incubator for cultivation, and slightly loosen the centrifuge tube cap to ensure the normal supply of 2 O 2 and CO₂ to ensure that polymer nanofibers and cells can effectively aggregate into spheres.

[0116] Experimental Example 1: Take the cell spheres formed on the 4th day and the 10th day after cell seeding in the groups with 6000 cells in Application Example 1, Application Comparative Example 1 and Application Comparative Example 2 for cell viability and dead staining experiments:

[0117] Figure 13 Results of the cell viability and dead experiment in Experimental Example 1. Among them, green are live cells stained with calcein, and yellow are polymer nanofibers stained with rhodamine B. In cell viability and dead staining, the more complete and compact the structure of the cell sphere, the more difficult it is to be stained inside. It can be seen fromFigure 13 It can be seen that on the 4th day after cell seeding, the cell spheres obtained in Application Example 1, Application Comparative Example 1, and Application Comparative Example 2 all had complete cell sphere structures, and the cell spheres had good morphology; on the 10th day after cell seeding, the cell spheres in Application Comparative Example 1 and Application Comparative Example 2 no longer had complete cell sphere structures, and what was shown in the pictures were only the cell sphere fragments of the two, rather than complete cell sphere structures; while the cell spheres in Application Example 1 could still be successfully stained and still presented complete structures, and the results were consistent with the observation results of the DIC microscope.

[0118] Figure 14 The results of the cell viability and death staining experiment of the cell spheres on the 20th day after cell seeding in Application Example 1 are shown in Figure 14 It can be seen that the cell spheres on the 20th day after cell seeding in Application Example 1 could still be successfully stained, indicating that the polymer nanofibers provided by the present invention enhanced the ability of BMSCs to form cell spheres and maintained the stability of the sphere structure. However, at this time, the diameter of the cell spheres was only about 100 μm, and it was speculated that the cell spheres were already in a declining state at this time.

[0119] Experimental Example 2: The CCK-8 reagent was used to evaluate the cell proliferation ability of the polymer nanofiber-reinforced BMSCs cell spheres:

[0120] The experimental groups were the formation of cell spheres with 1000, 2000, 3000, 4000, 5000, and 6000 cells respectively. According to the principle of CCK-8, the faster the cell proliferation rate, the more formazan produced by cell reduction, the deeper the yellow color of the test system, and the higher the absorbance value at OD 450 nm. Figure 15 The cell proliferation of the polymer nanofiber-reinforced BMSCs cell spheres in Experimental Example 2 is shown in Figure 15 It can be seen that within the test time of 52 h, the absorbance of all groups maintained a good upward state, indicating good cell proliferation, that is, the addition of polymer nanofibers would not have an adverse effect on cell proliferation.

[0121] Figure 16 The schematic diagram of the principle of the polymer nanofiber-reinforced BMSCs sphere formation is shown in Figure 16 It can be seen that in the cell culture with the addition of polymer nanofibers, the formation of cell spheres is mediated by the binding of cells to polymer nanofibers. First, vitronectin and fibronectin in the serum added to the culture medium are adsorbed on the polymer nanofibers, and then the cells adhere to these nanofibers, thus forming spheroids. Therefore, the addition of polymer nanofibers improves the efficiency of cell sphere formation and enhances the cell viability.

Claims

1. A method for preparing cell spheres, characterized in that: The method comprises using culture medium to prepare polymer nanofibers into nanofiber solution, and co-culturing with cells to obtain the cell sphere; Wherein, the polymer nanofibers include the following preparation raw materials: gelatin, a cross-linking agent, a nano-reinforcement phase and a solvent; The nano-reinforced phase is nano-hydroxyapatite; The mass ratio of the gelatin to the nano-enhanced phase is (10-60): 1; The polymer nanofibers are prepared by a method comprising the following steps: S1, mixing gelatin with a solvent to obtain a gelatin solution; S2, adding a cross-linking agent to the gelatin solution to undergo a cross-linking reaction to obtain a cross-linked gelatin solution; S3, adding the nano-reinforcement phase to the cross-linked gelatin solution, dispersing to obtain a spinning solution, and electrospinning to obtain the polymer nanofibers.

2. The method for preparing cell spheres according to claim 1, characterized in that: The mass ratio of the gelatin to the cross-linking agent is (20-50): 1; And / or, the solid-to-liquid ratio of the gelatin and the solvent is 1g: (5-30)mL.

3. The method for preparing cell spheres according to claim 1, characterized in that: The cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

4. The method for preparing cell spheres according to claim 1, characterized in that: The solvent includes a buffer solution and an alcohol solvent; the volume ratio of the buffer solution to the alcohol solvent is 1: (0.5-5).

5. The method for preparing cell spheres according to claim 1, characterized in that: In preparing the polymer nanofibers, in step S1, the pH of the gelatin solution is 3.5-4.5; And / or, in step S2, the temperature of the cross-linking reaction is 25-35° C. and the time is 10-30 min; And / or, in step S3, the electrospinning process parameters include at least one of the following parameters: 1) The ambient temperature is 20-30℃; 2) Relative humidity is 40%-50%; 3) The injection speed is 0.10-0.15 mm / min; 4) Positive voltage is 15-25kV, negative voltage is -2 ~ -5kV; 5) Spinning distance is 15-20cm; 6) The collection device speed is 80-120rpm.

6. The method for preparing cell spheres according to claim 1, characterized in that: The concentration of the nanofiber solution is 1-5 mg / mL; the dosage of the nanofiber solution is 100-600 cells / μL.

7. A cell spheroid, characterized in that: The method comprises preparing the cell sphere by using the method for preparing the cell sphere according to any one of claims 1 to 6.

8. Use of the cell spheroid according to claim 7 in tumor research, drug screening, tissue engineering or toxicology research.

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