Polymer nanofiber as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a polymer nanofiber as well as a preparation method and application thereof. The polymer nanofiber is prepared from the following raw materials: gelatin, a cross-linking agent, a nano reinforcing phase and a solvent, and the nano reinforced phase comprises nano hydroxyapatite. The polymer nanofiber provided by the invention takes gelatin as a main raw material and takes a substance comprising nano-hydroxyapatite as a nano reinforced phase, and the obtained polymer nanofiber has the advantages of good biocompatibility, high cell activity, large specific surface area, pore structure, good structural stability and low cost, and has the effects of enhancing the affinity between adjacent cells and improving the biocompatibility of the cells. The connection between the cells is enhanced, so that the cell sphere forming capability of the cells is promoted; according to the preparation method of the cell sphere, the polymer nanofiber reinforced cell sphere can be obtained by co-culturing the polymer nanofiber and the cell, the operation is simple, the cost is low, the control is easy, the method is suitable for large-scale culture, the cell sphere forming speed is high, and the structural stability is good.

Description

Technical Field

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

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

[0003] Existing methods for preparing cell spheres mainly include: (1) hanging drop method: drop the cell suspension in the form of droplets (usually 10-50µL) on the lid of the culture plate, and then invert the lid to make the droplets hang on the lid. Due to the effect of gravity, the cells will gradually settle to the bottom of the droplets and aggregate with each other to eventually form cell spheres. The key to this method is the stability of the droplets and the control of evaporation of the culture medium; (2) ultra-low adsorption culture method: use specially treated culture plates or culture dishes, whose surfaces have ultra-low adsorption, which can prevent cells from adhering to the wall, thereby causing cells to aggregate with each other in a suspended state to form cell spheres. The key to this method is to optimize the cell density and culture medium. (3) Rotational culture method: Place the cell suspension in a rotating bottle and keep the cells in suspension by continuous stirring so that they gradually aggregate to form cell spheres; (4) Microcarrier culture method: Use microcarriers (such as gelatin, agarose or polymer microspheres) as scaffolds for cell attachment to promote cell aggregation to form cell spheres. Microcarriers usually have a large specific surface area and can support high-density cell growth; (5) Air-liquid interface method: Inoculate cells on a porous membrane and culture them under air-liquid interface conditions to promote cell aggregation to form cell spheres. The key to this method lies in the selection of porous membranes and the optimization of culture conditions.

[0004] Among the above methods, the hanging drop method is easy to operate, low in cost, and the cell spheres formed are of uniform size, but it is only suitable for small-scale experiments and high-throughput screening; the ultra-low adsorption culture method is easy to operate, suitable for large-scale culture, and has high efficiency in forming cell spheres, but the size of the cell spheres is uneven, and the culture medium needs to be replaced regularly to maintain cell activity; the rotation 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 rotating bottles and stirrers), and the shear force generated during the stirring process may affect cell activity; 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 need to be optimized; the gas-liquid interface method can simulate the physiological environment of certain tissues (such as the respiratory tract and skin) and promote the natural formation of cell spheres, but this method is complicated to operate, requires special equipment, and requires optimization of experimental conditions to ensure cell activity. In addition, the preparation of cell spheroids faces the following challenges: for cell spheroids with larger diameters, the concentrations of oxygen and nutrients inside the cell spheroids gradually decrease as the distance from the surface increases, and the metabolic activity of the cells is restricted, thereby affecting the survival and function of the cells. For example, previous studies have shown that cell spheroids with a diameter greater than 500 μm will have difficulty in the diffusion of oxygen and nutrients, leading to premature death of cells in the central area of ​​the cell spheroids.

[0005] Therefore, developing a method for preparing cell spheroids 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 is of great significance for promoting the application of cell spheroids in tumor research, drug screening, tissue engineering, toxicology and other fields. Summary of the invention

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

[0007] The second object of the present invention is to provide a method for preparing the polymer nanofiber.

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

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

[0010] A fifth object of the present invention is to provide applications of the cell sphere.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides a polymer nanofiber, comprising the following preparation materials: gelatin, a cross-linking agent, a nano-reinforced phase and a solvent; the nano-reinforced phase comprises nano-hydroxyapatite (nHAP).

[0012] In some embodiments of the present invention, the polymer nanofibers have a diameter of 550-650 nm.

[0013] In some specific embodiments of the present invention, the diameter of the polymer nanofiber is 550-600 nm.

[0014] In some embodiments of the present invention, the molecular weight of the gelatin is 50-100 kDa.

[0015] In some embodiments of the present invention, the mass ratio of gelatin to cross-linking agent is (20-50): 1.

[0016] In some embodiments of the present invention, the mass ratio of gelatin to nano-reinforced phase is (10-60): 1.

[0017] In some embodiments of the present invention, the solid-liquid ratio of the gelatin and the solvent is 1 g: (5-30) mL.

[0018] In some embodiments of the present invention, the cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0019] In some specific embodiments of the present invention, the mass ratio of gelatin to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is (25-50): 1.

[0020] In some specific embodiments of the present invention, the mass ratio of gelatin to N-hydroxysuccinimide is (20-30): 1.

[0021] In the present invention, the amount of the crosslinking agent is strictly controlled to ensure the sufficiency and stability of the crosslinking reaction while avoiding excessive addition of the crosslinking agent, which causes the solution to become too viscous and affects the stretching and formation of the fiber.

[0022] 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).

[0023] In some specific embodiments of the present invention, the volume ratio of the buffer solution to the alcohol solvent is 1: (0.5-4).

[0024] In some embodiments of the invention, the buffer comprises PBS buffer.

[0025] In some embodiments of the present invention, the alcohol solvent includes at least one of ethanol and isopropanol.

[0026] The inventive concept of the present invention is: in natural bones, the mass ratio of hydroxyapatite (HAP) to collagen is 7:3, and the hydroxyapatite in natural bones is nano-sized, so nano-hydroxyapatite is used as a nano-reinforcement phase to provide corresponding biological activity; gelatin is a hydrolysis product of collagen, containing multiple functional groups with biological activity. Unlike collagen, gelatin can be dissolved in water. Compared with collagen, gelatin is easier to shape, has lower cost, and has good biocompatibility and near-zero immunogenicity. Therefore, the present invention uses gelatin and nano-hydroxyapatite as main raw materials to prepare a polymer nanofiber having the characteristics of both raw materials.

[0027] The second aspect of the present invention provides a method for preparing the polymer nanofibers according to the first aspect of the present invention, 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.

[0028] In some embodiments of the present invention, in step S1, the pH of the gelatin solution is 3.5-4.5.

[0029] In some specific embodiments of the present invention, in step S1, the pH of the gelatin solution is 3.8-4.2.

[0030] In some embodiments of the present invention, step S1 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.

[0031] In some specific embodiments of the present invention, the pH adjuster includes acetic acid.

[0032] In some embodiments of the present invention, step S1 includes first stirring and mixing the buffer solution and the alcohol solvent, wherein the stirring and mixing is performed at a rotation speed of 100-300 rpm for 5-15 min.

[0033] In some embodiments of the present invention, in step S1, the process of mixing gelatin and solvent is assisted by water bath heating, and the water bath heating temperature is 40-50°C.

[0034] In some embodiments of the present invention, in step S1, the process of mixing the gelatin and the solvent is assisted by stirring, and the stirring speed is 200-500 rpm.

[0035] 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.

[0036] In some embodiments of the present invention, in step S2, the cross-linking reaction process is assisted by stirring, and the stirring speed is 100-300 rpm.

[0037] In some embodiments of the present invention, in step S3, the dispersion temperature is 25-35° C. and the dispersion time is 10-20 min.

[0038] In some embodiments of the present invention, in step S3, the dispersing process is assisted by stirring, and the stirring speed is 200-500 rpm.

[0039] In some embodiments of the present invention, 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 speed of the collection device is 80-120rpm.

[0040] In the present invention, it is necessary to strictly control the process parameters of electrospinning for the following reasons: ① When the ambient temperature and humidity change, the nanofibers are likely to be beaded; ② The spinning distance needs to be set based on the fluidity of the spinning solution and the efficiency of electrospinning to avoid the fiber being beaded or broken. When the spinning distance is too close, the solvent cannot fully volatilize, and beaded nanofibers will appear; ③ When the concentration of the gelatin solution increases, the fiber will become wider, and too high a concentration may directly lead to spinning failure; ④ Too fast a push speed may easily lead to fiber breakage or unevenness, while too slow may easily lead to an increase in fiber diameter or breakpoints; ⑤ The setting of positive and negative voltages needs 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 cause arc discharge or solution splashing; ⑥ The speed of the collecting device will affect the uniformity of nanofiber collection, and improper setting may cause fiber crossover or aggregation. The present invention can effectively improve the stability of the spinning solution and the mechanical properties of the fiber by controlling each parameter.

[0041] The third aspect of the present invention provides a method for preparing a cell sphere, comprising using a culture medium to prepare the polymer nanofibers described in the first aspect of the present invention into a nanofiber solution, and co-culturing the solution with cells to obtain the cell sphere.

[0042] 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.

[0043] In some specific embodiments of the present invention, the concentration of the nanofiber solution is 1-3 mg / mL.

[0044] In some embodiments of the present invention, the culture medium comprises 400-500 mL DMEM / F12 basal medium, 20-100 mL fetal bovine serum and 2-10 mL penicillin-streptomycin.

[0045] In some embodiments of the present invention, the co-cultivation period is 1-30 days.

[0046] In some specific embodiments of the present invention, the co-cultivation time is 1-20 days.

[0047] In some embodiments of the present invention, the cells are selected from stem cells, cancer cells, stem cell-derived cardiomyocytes, nerve cells, hepatocytes, fibroblasts, endothelial cells and epithelial cells, and at least one of organism-derived cardiomyocytes, nerve cells, hepatocytes, fibroblasts, endothelial cells and epithelial cells.

[0048] In some specific embodiments of the present invention, the cells include bone marrow mesenchymal stem cells (BMSCs).

[0049] The fourth aspect of the present invention provides a cell sphere, comprising a cell sphere prepared by the preparation method of the cell sphere described in the third aspect of the present invention.

[0050] In the present invention, the obtained cell ball is a polymer nanofiber reinforced cell ball. Because the polymer nanofiber is a gelatin nanofiber containing a nano-enhanced phase, the gelatin contains an RGD sequence (arginine-glycine-asparagine), which can be well combined with the integrin on the cell surface, thereby upregulating the expression of the cell membrane surface connecting protein. The accumulation of the connecting protein on the cell membrane surface can enhance the affinity between adjacent cells, strengthen the connection between cells, and then form a cell ball. In addition, the gelatin nanofiber has a high specific surface area and structural stability, and can provide more support and structural framework, so as to better maintain the stability of the cell ball. When nano-hydroxyapatite is used as the nano-enhanced phase, the good biological activity of the nano-hydroxyapatite itself enhances the cell activity of the polymer nanofiber to the cell, which is beneficial to the aggregation of cells and polymer nanofibers, and the addition of nano-hydroxyapatite prompts the polymer nanofiber to produce pores, thereby promoting the diffusion of oxygen and nutrients into the interior of the cell ball, so that the cells in the central area of ​​the cell ball are not easy to die prematurely due to the lack of oxygen and nutrients, which is beneficial to the long-term culture of the cell ball.

[0051] The fifth aspect of the present invention provides the use of the cell spheroid described in the fourth aspect of the present invention in tumor research, drug screening, tissue engineering, and toxicology research.

[0052] Compared with the prior art, the present invention has the following beneficial effects: 1) The polymer nanofibers provided by the present invention use gelatin as the main raw material and a substance including nano-hydroxyapatite as a nano-reinforcement phase. The obtained polymer nanofibers have the characteristics of good biocompatibility, high cell activity, large specific surface area, porous structure, good structural stability, low cost, etc., and can enhance the affinity between adjacent cells, strengthen the connection between cells, and promote cells to form cell spheres; 2) The preparation method of polymer nanofibers 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 to obtain uniform polymer nanofibers with good appearance. 3) The preparation method of the cell sphere provided by the present invention is to co-culture the polymer nanofibers with cells to obtain the polymer nanofiber-reinforced cell spheres, which is simple to operate, low in cost, easy to control, and suitable for large-scale culture; 4) The cell spheroids provided by the present invention have fast spheroidization speed and good structural stability. Oxygen and nutrients can diffuse into the interior of the cell spheroids through the pores of the polymer nanofibers. The cells in the central area of ​​the cell spheroids are not prone to premature death. The addition of polymer nanofibers will not affect the reproduction of cells, and can meet the application requirements of cell spheroids in tumor research, drug screening, tissue engineering, toxicology research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is the infrared spectra of the gelatin solution, nano-hydroxyapatite, spinning solution and polymer nanofibers in Example 1; Figure 2 This is a laser confocal microscope image of the polymer nanofiber in Example 1; Figure 3 For the morphological observation of the fourth generation BMSCs in adherent culture in comparative example 1; Figure 4 It is a DIC microscope image of the BMSCs cell sphere formation process in comparative example 1; Figure 5 This is a DIC microscopic image of the BMSCs spheres on the 5th day after plating in Comparative Example 1; Figure 6 The cytoskeleton structure comparison diagram of BMSCs before and after sphering in comparative example 1; Figure 7 The skeleton structure of the BMSCs cell sphere in Comparative Example 1 is used; Figure 8 This is a DIC microscopic image of the process of enhancing BMSCs sphere formation by using gelatin molecules in Comparative Example 2; Fig. 9 The polymer nanofiber-enhanced BMSCs sphere morphology on the 3rd day after plating in Application Example 1; Fig.10 The DIC micrographs of the polymer nanofiber-enhanced BMSCs cell sphere formation process on days 6-14 in Application Example 1 when the cell number is 1000-5000; Fig.11 This is a DIC microscopic image of the polymer nanofiber-reinforced BMSCs cell sphere on the third day when the cell number is 6000 in Application Example 1; Fig.12 This is an image of the failure of polymer nanofibers and BMSCs to form a ball; Fig.13 The results of the cell live-death experiment in Experimental Example 1; Fig.14 The results of the live-dead staining experiment of the cell spheres on the 20th day after cell plating in Application Example 1; Fig.15 The cell proliferation of polymer nanofiber-enhanced BMSCs cell spheres in Experimental Example 2; Fig.16 Schematic diagram of the principle of polymer nanofiber-enhanced BMSCs sphering. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0055] Example 1: In this example, a polymer nanofiber is prepared in the following steps: S11, adding 40 mL of PBS buffer and 60 mL of ethanol into a beaker, stirring and mixing at 200 rpm for 10 min to obtain a mixed solvent; 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 350 rpm in a 45°C water bath; S13, adding 0.5 mol / L acetic acid solution dropwise to the solution obtained in step S12, adjusting the pH of the solution to 4.0, and obtaining a gelatin solution; S21, adding 150 μL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 0.2 g of N-hydroxysuccinimide to the gelatin solution obtained in step S13, stirring at 200 rpm at 30° C. for 20 min to obtain a cross-linked gelatin solution; S31, adding 0.3 g of nano-hydroxyapatite to the cross-linked gelatin solution obtained in step S21, stirring and dispersing at 200 rpm for 15 min at 30° C. to obtain a uniform spinning solution; S32. The spinning solution obtained in step S31 is loaded into a syringe, and electrospinning is performed 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, and collection device rotation speed 100 rpm to obtain polymer nanofibers.

[0056] The gelatin solution, nano-hydroxyapatite, spinning solution and polymer nanofibers in Example 1 were tested by infrared spectroscopy. Figure 1 is the infrared spectra of the gelatin solution, nano-hydroxyapatite, spinning solution and polymer nanofibers in Example 1. Figure 1 It can be seen that in gelatin solution, 1300cm -1 The band at 1250-1400 cm-1 is mainly attributed to the swinging vibration of the proline side chain, indicating the presence of type I gelatin. -1 The obvious peaks in the range are all related to type I gelatin. In addition, the characteristic bands include the amide A band (3300-3500cm -1 ) and amide B band (1680-1780cm -1The amide A band is the strongest absorption band in the amide bond, mainly generated by NH stretching vibration, and the amide B band is mainly generated by C=O stretching vibration and NH bending vibration. In polymer nanofibers, the amide I band (1600-1700cm -1 ) is mainly generated by C=O stretching vibration, amide II band (1500-1550cm -1 ) is mainly generated by NH bending vibration and CN stretching vibration. The appearance of amide I band and amide II band indicates that the polymer nanofibers mainly adopt α-helical structure. Other spectral peaks include the stretching vibration of hydroxyl (-OH) (4000-3200cm -1 )、carbonate bending vibration(1540-1400cm -1 and 1530-1320cm -1 ) and phosphate bands (900-1200cm -1 ); in spinning solution, 1300cm -1 The shift of the gelatin band at 1000 nm can confirm the formation of chemical bonds between the carboxyl ions in gelatin and nanohydroxyapatite, which involves Ca 2+ R-COO with gelatin molecule - The formation of covalent bonds and cross-linking may lead to a shortening of the distance between nano-hydroxyapatite and gelatin nanofibers, promoting more Ca 2+ R-COO with gelatin molecule - Combine.

[0057] The polymer nanofibers prepared in Example 1 were stained with rhodamine B and then characterized by laser confocal microscopy. Figure 2 is a laser confocal microscope image of the polymer nanofiber in Example 1, wherein: Figure 2 (a) is a laser confocal microscope image with a magnification of 1000 times. Figure 2 (b) is a laser confocal microscope image with a magnification of 2000 times. Figure 2 It can be seen that the polymer nanofibers prepared in Example 1 have a nanometer size and a diameter of about 590nm, so they have the characteristics of high specific surface area and can show extremely strong activity. Studies have shown that the diameter of nanofibers affects the migration rate of cells, and different cells respond differently to the diameter of nanofibers. In the study of BMSCs, BMSCs showed the fastest migration rate on poly (L-lactic acid) fibers with a diameter of 600nm, so it is speculated that the polymer nanofibers prepared in Example 1 are also beneficial to promote cell sphering.

[0058] Comparative Example 1: Isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs): (1) Preparation of culture medium: Mix 450 mL of DMEM / F12 basal culture medium, 50 mL of fetal bovine serum, and 5 mL of penicillin-streptomycin. Filter the resulting mixture through a 220 μm filter to obtain culture medium, which is then sealed and stored at 4°C. Reagents related to cell culture and cell passaging need to be placed in a water bath at 37°C before each use.

[0059] (2) Cell extraction: prepare scalpels, surgical scissors, tweezers, pipettes, syringes and culture dishes, place them on a clean bench, and sterilize them under ultraviolet light for 30 min. 10 min before the experiment, kill the rats by dislocating the neck, and soak the rats in 75wt% alcohol for 5-10 min. Take out the soaked rats, squeeze out the excess alcohol from their mouths, and then fix the rats' front feet with a needle. Peel off the rats' skin, cut them around, remove the excess meat, cut the femur and tibia at the joints, and soak them in a PBS solution containing 10wt%-15wt% double antibody (penicillin-streptomycin). Cut the head of the bone to expose the red bone marrow. Use a syringe filled with 5 mL of culture medium to flush the bone marrow into a new culture dish that has been sterilized by ultraviolet light. Use a pipette to add 5 mL of culture medium to the culture dish. Place the culture dish containing the collected rat bone marrow in a cell culture incubator at 37°C and 5% CO2 for culture.

[0060] (3) Cell separation and purification: Taking advantage of the BMSCs’ ability to adhere to the wall, bone marrow mesenchymal stem cells were separated and purified by attaching to the surface of the culture dish. Within 48 hours after cell extraction, the culture dish was placed in an incubator to promote the cell’s adherent growth and avoid cell medium replacement. This helped the cells to fully adhere to the surface of the culture dish 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 using 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. The dish was then placed in a cell culture incubator at 37°C and 5% CO2 for further culture. When the cell density reached more than 80% (usually 4-6 days), the cells could be subcultured.

[0061] (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.

[0062] 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.

[0063] In the subcultured BMSCs in this application comparison, the primary BMSCs were oval and spindle-shaped, and some of the cells were in an adherent state. After a few days of culture, the cells showed synaptic morphological changes and gradually began to fuse. Compared with the primary cells, the second-generation BMSCs appeared larger cells and extended from triangles to polygons. The proliferation rate of the second-generation BMSCs was accelerated, and some cells had 1-2 nucleoli. Although it was found that the cells were poorly integrated with the surrounding environment in terms of shape and distribution, the proliferation rate was significantly increased compared with the original cells. The third subculture of BMSCs was easy to carry out, and the cells had a strong proliferation ability at this time. The proliferation rate of the fourth-generation BMSCs continued to accelerate, and the cells were polygonal and long spindle-shaped. This application comparison selected the fourth-generation BMSCs to participate in the preparation of cell spheres. Figure 3 In order to observe the morphology of the fourth generation BMSCs adherent culture in comparative example 1, Figure 3 It can be seen that the fourth-generation BMSCs in adherent culture showed a long spindle shape.

[0064] In this application comparative example, the fourth-generation BMSCs were taken, an appropriate amount of culture medium was added for resuspending, and a cell suspension was obtained. The cell suspension was counted, and different numbers of cells (1000, 2000, 3000, 4000, 5000 and 6000) were added to each well of an ultra-low adsorption U-shaped 96-well plate. Ten parallel samples were made for each group, and the growth status of the cells at different time points was observed using a DIC microscope (20x objective, 50μm scale).

[0065] The cell sphere formation process of different numbers of BMSCs was recorded from day 1 to day 9 starting from the day of cell plating. Figure 4 The DIC micrograph of the BMSCs sphere formation process in Comparative Example 1 is shown in FIG. Figure 4 It can be seen that in the early stage of plating, cells gather together to form a monolayer of cell groups, and a more obvious cell ball structure can be formed after 4-5 days of plating, but the stability of this cell ball structure is poor. Obvious scattering begins to appear on the 9th day after plating, and as the number of cells increases, the scattering phenomenon becomes more obvious. When the number of cells is 1000-3000, it is more conducive to the formation and stability of cell balls. In addition, cell balls with better quality should be displayed as translucent spheres under a bright field microscope, with clear external boundaries and a slightly darker core. In the figure, only a few cell balls can meet this standard, and irregularly shaped or loose cell aggregates represent spheroids of poor quality. It can be seen that the cell ball structure formed only by cells is of poor quality and difficult to exist stably.

[0066] Figure 5 This is a DIC microscope image of the BMSCs cell sphere on the 5th day after plating in Comparative Example 1, wherein: Figure 5 (a) shows a cell sphere formed by 1000 BMSCs. Figure 5 (b) shows a cell sphere formed by 2000 BMSCs. Figure 5 (c) shows a cell sphere formed by 3000 BMSCs. Figure 5 (d) shows a cell sphere formed by 4000 BMSCs. Figure 5 (e) in the figure shows a cell sphere formed by 5000 BMSCs. Figure 5 (f) shows a cell sphere formed by 6000 BMSCs. Figure 5It can be seen that on the 5th day after plating, the diameters of the cell spheres formed by 1000, 2000, 3000, 4000, 5000 and 6000 BMSCs were 108.03μm, 112.24μm, 143.10μm, 154.33μm, 180.99μm and 205.54μm, respectively, that is, as the number of cells increases, the diameter of the cell sphere increases, but the number of cells scattered around also increases. Studies have shown that the distance between most cells in the body and the nearest capillaries does not exceed 100-200μm. Beyond this distance, it is difficult for cells to obtain sufficient oxygen and nutrients. Similarly, the oxygen and nutrients of the cell sphere depend on diffusion supply. If the radius of the cell sphere exceeds 200μm, it is difficult for cells inside the cell sphere to obtain oxygen and nutrients through diffusion, and necrosis is likely to occur inside the cell sphere, and the vitality of its core cells will be lost.

[0067] 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). It presents a filamentous structure in cells and participates 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 culture, 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, which can bind to these extracellular matrices. This integrin-mediated adhesion of cells to extracellular matrix determines the position of cells. At the same time, integrins and corresponding ligands connect to form a complex. This complex can directly connect to F-actin with the assistance of other proteins (focal adhesion protein and talin). Therefore, integrins directly connect F-actin and the surrounding environment of cells. It is in this way that the adhesion of cells to extracellular matrix can guide the formation of actin stress fibers, thereby affecting the overall organizational structure of the cytoskeleton. Figure 6 This is a comparison diagram of the cytoskeleton structure of BMSCs before and after sphering in Comparative Example 1, wherein: Figure 6 (a) is the staining observation of the nucleus and cytoskeleton of BMSCs before sphering. Figure 6 (b) is the staining observation of the nucleus and cytoskeleton of BMSCs after sphering. Figure 6 It is known that changes in the state of cell aggregation will affect the structure of the cytoskeleton (F-actin). Studies have shown that changes in the structure of the cytoskeleton have a significant impact on the differentiation direction of stem cells. Therefore, it is speculated that in the sphere culture of BMSCs, significant changes in the cytoskeleton structure may also have an important impact on the direction of cell differentiation.

[0068] Figure 7 In order to apply the skeleton structure of the BMSCs cell sphere in Comparative Example 1, Figure 7It can be seen that F-actin has a complex network structure. The reason is that F-actin has polarity. When the cell moves, one end aggregates and elongates, located on the cell membrane side, and the other end depolymerizes and shortens, located inside the cell. The F-actin network is regulated by related binding proteins and is a highly ordered structure rather than a uniform structure. Generally speaking, the more stable and compact the cell sphere structure is, the more difficult it is for the fluorescent marker to enter the cell sphere, and the more difficult it is for the cell sphere to be stained.

[0069] Application Comparative Example 2: In this application comparative example, gelatin solution is used to participate in the preparation of cell spheres to obtain a gelatin molecule enhanced BMSCs cell sphere. The preparation steps are as follows: (1) Weigh gelatin powder (molecular weight 80 kDa) and add it to culture medium to prepare a 1 mg / mL gelatin solution; (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, count the cell suspension, add different numbers of cells (1000, 2000, 3000, 4000, 5000 and 6000) to each well of an ultra-low adsorption U-shaped well 96-well plate, then add 10 μL of 1 mg / mL gelatin solution to each well, add culture medium until the liquid volume in the well is 200 μL, mix well, and avoid the generation of bubbles in the well during the process. Make 10 parallel samples for each group, and use a DIC microscope (20x objective, scale 50 μm) to observe the growth status of the cells at different time points.

[0070] The cell sphere formation process of different numbers of BMSCs was recorded from day 1 to day 9 starting from the day of cell plating. Figure 8 The DIC micrograph of the process of enhancing BMSCs sphere formation by gelatin molecules in Comparative Example 2 is shown in FIG. Figure 8 It can be seen that compared with the application of comparative example 1, after the addition of gelatin molecules, the formation speed of BMSCs cell spheres is significantly accelerated, and a relatively obvious cell sphere structure can be formed about 2-3 days after plating, but the stability of the structure is poor, and obvious scattering begins on the 7th day after plating, and as the number of cells increases, the scattering phenomenon becomes more obvious. On the 9th day, all cell spheres have been scattered. Therefore, although the addition of gelatin molecules can accelerate the formation of BMSCs cell spheres and improve the morphology of cell spheres, it still cannot maintain the stability of the cell sphere structure. It is speculated that the cell adhesion effect brought by gelatin molecules is not enough to support the structure of the entire cell sphere.

[0071] Application Example 1: In this application example, the polymer nanofibers prepared in Example 1 are used to participate in the preparation of cell spheres to obtain polymer nanofiber-reinforced BMSCs cell spheres. The preparation steps are as follows: (1) The polymer nanofibers prepared in Example 1 are dissolved in 75% alcohol and collected in a centrifuge tube. The supernatant is then removed by centrifugation to collect the polymer nanofibers. The polymer nanofibers are resuspended in a culture medium, the supernatant is removed, and the polymer nanofibers are taken. This step can remove the alcohol solution mixed in the polymer nanofibers. The culture medium is added again to resuspend the polymer nanofibers, thereby obtaining a nanofiber solution that can be co-cultured with cells. (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, count the cell suspension, add different numbers of cells (1000, 2000, 3000, 4000, 5000 and 6000) to each well of an ultra-low adsorption U-shaped well 96-well plate, then add 10 μL of 1 mg / mL nanofiber solution to each well, add culture medium until the liquid volume in the well is 200 μL, mix well, and avoid the generation of bubbles in the well during the process. Make 10 parallel samples for each group, and use a DIC microscope (20x objective, 50 μm scale) to observe the growth status of the cells at different time points.

[0072] The cell sphere formation process of different numbers of BMSCs was recorded from day 1 to day 14 starting from the day of cell plating. Fig. 9 The polymer nanofibers enhanced the BMSCs cell sphere morphology on the third day after plating in Application Example 1. Fig. 9 It can be seen that compared with the comparative example 1, after adding the polymer nanofibers prepared in Example 1, the sphering speed of BMSCs was accelerated, and the cell spheroid structure was basically formed on the third day of culture.

[0073] Fig.10 The DIC micrographs of the polymer nanofiber-enhanced BMSCs sphere formation process on days 6-14 in Application Example 1 when the number of cells is 1000-5000. Fig.10 It can be seen that compared with the comparative examples 1 and 2, after adding the polymer nanofibers prepared in Example 1, the BMSCs cell sphere structure was more stable and no obvious scattering occurred within 6-14 days.

[0074] Fig.11 This is a DIC microscopic image of the polymer nanofiber-enhanced BMSCs cell sphere on the third day when the number of cells in Application Example 1 is 6000, where: Fig.11 (a) Fig.11 (b) and Fig.11 (c) is a DIC micrograph of three randomly selected cell spheres from 10 parallel samples. Fig.11 It can be seen that the diameters of the three cell spheroids are 276.46 μm, 152.78 μm and 177.75 μm, respectively.

[0075] Fig.12 This is an image of the failure of polymer nanofibers and BMSCs to form a ball, where Fig.12 (a) is a DIC microscope image of BMSCs cell spheres. Fig.12 (b) is a staining image of the cell nucleus and cytoskeleton. Compared with cells, the polymer nanofibers settle faster and tend to aggregate at the bottom of the well plate, thus failing to aggregate with cells effectively. Fig.12 As shown, in this case, after using trypsin to digest the cells, centrifuge, and resuspend them in culture medium to obtain a cell suspension, add the nanofiber solution that has been resuspended in culture medium. In this process, calculate the cell density in the mixed suspension, and then pipette the required suspension into a microcentrifuge tube (0.2mL) according to the required number of cells. Centrifuge at 1200rpm for 5min, then place the centrifuge tube in a cell culture incubator for culture, and slightly loosen the centrifuge tube cap to ensure the normal supply of O2 and CO2, so as to ensure that the polymer nanofibers and cells can effectively aggregate into spheres.

[0076] Experimental Example 1: In Application Example 1, Application Comparative Example 1 and Application Comparative Example 2, cell spheres formed on the 4th and 10th days after cell plating in the group of 6000 cells were subjected to cell live-death staining experiments: Fig.13 The results of the cell live-death experiment in Experimental Example 1, where green represents live cells stained with calcein, and yellow represents polymer nanofibers stained with rhodamine B. In the cell live-death staining, the more complete and compact the cell sphere is, the more difficult it is to stain its interior. Fig.13 It can be seen that on the 4th day after cell plating, the cell spheres obtained in Application Example 1, Application Comparative Example 1 and Application Comparative Example 2 all have complete cell sphere structures and good cell sphere morphology; on the 10th day after cell plating, the cell spheres in Application Comparative Example 1 and Application Comparative Example 2 no longer have complete cell sphere structures, and the pictures only show cell sphere fragments of the two, not complete cell sphere structures; while the cell spheres in Application Example 1 can still be successfully stained and still show a complete structure, and the results are consistent with the observation results of DIC microscopy.

[0077] Fig.14 The results of the live-dead staining experiment of the cell spheres on the 20th day after cell plating in Application Example 1 are as follows: Fig.14 It can be seen that the cell spheres in Application Example 1 can still be successfully stained on the 20th day after cell plating, indicating that the polymer nanofibers provided by the present invention enhance the ability of BMSCs to form cell spheres and maintain the stability of the sphere structure, but the diameter of the cell spheres is only about 100 μm at this time, and it is speculated that the cell spheres are already in a decaying state at this time.

[0078] Experimental Example 2: CCK-8 reagent was used to evaluate the cell proliferation ability of polymer nanofiber-enhanced BMSCs cell spheres: The experimental groups were 1000, 2000, 3000, 4000, 5000 and 6000 cells, respectively. According to the CCK-8 principle, the faster the cell proliferation rate, the more formazan is reduced by the cells, the darker the yellow color of the test system, and the higher the absorbance value at OD 450nm. Fig.15 The cell proliferation of polymer nanofiber-enhanced BMSCs cell spheres in Experimental Example 2 is shown in FIG. Fig.15 It can be seen that within the test time of 52 h, the absorbance of all groups maintained a good rising state, indicating that the cell proliferation was good, that is, the addition of polymer nanofibers did not have an adverse effect on cell proliferation.

[0079] Fig.16 Schematic diagram of the principle of polymer nanofiber-enhanced BMSCs sphering. Fig.16 It can be seen that in 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 to form spheres. Therefore, the addition of polymer nanofibers improves the efficiency of cell sphere formation and enhances cell viability.

Claims

1. A polymer nanofiber, characterized in that: The preparation method comprises the following raw materials: gelatin, a cross-linking agent, a nano-enhanced phase and a solvent; the nano-enhanced phase comprises nano-hydroxyapatite.

2. The polymer nanofiber 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 mass ratio of the gelatin to the nano-enhanced phase is (10-60): 1; And / or, the solid-to-liquid ratio of the gelatin and the solvent is 1g: (5-30)mL.

3. The polymer nanofiber according to claim 1 or 2, characterized in that: The cross-linking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

4. The polymer nanofiber according to claim 1 or 2, 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 the polymer nanofiber according to any one of claims 1 to 4, characterized in that: The following steps are involved: 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.

6. The method for preparing polymer nanofibers according to claim 5, characterized in that: 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.

7. A method for preparing cell spheres, characterized in that: The method comprises using culture medium to prepare the polymer nanofibers according to any one of claims 1 to 4 into a nanofiber solution, and co-culturing the solution with cells to obtain the cell spheres.

8. The method for preparing cell spheres according to claim 7, 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.

9. A cell spheroid, characterized in that: The method comprises adopting the cell sphere preparation method according to claim 7 or 8.

10. Use of the cell spheroid according to claim 9 in tumor research, drug screening, tissue engineering, and toxicology research.

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