Method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosa mesenchymal stem cells and product

Abstract

The invention discloses a method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosa mesenchymal stem cells and a product, and belongs to the technical field of biological tissue engineering. Gelatin sponge particles and olfactory mucosa mesenchymal stem cells are mixed and inoculated into a 3D suspension shaking table, and the material and the cells form the 3D gelatin sponge cell microspheres under the action of an extracellular matrix and electrochemical adsorption force. The prepared cellularized biological material has the characteristics of high permeability, low cytotoxicity and good biocompatibility, is wide in preparation raw material source, low in cost and simple in process flow, and has a wide prospect in preparation of cell culture, extracellular vesicle extraction, exosome extraction and other applications.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and relates to a method and product for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells. Background Art

[0002] As a new tool for tissue repair in the field of bioengineering, stem cells have made many advances in the study of various disease models. However, for diseases with large amounts of local normal tissue loss, such as cerebral hemorrhage, skull defects, and spinal cord injuries, cells in a liquid suspension state are not easy to stay in the lesion site and continue to play a role. Therefore, the rise of 3D cell culture technology aims to solve this problem. Moreover, 3D cell culture also has good applications in the preparation of cell-derived supernatants, extracellular vesicles, and exosomes.

[0003] There are many known 3D cell culture schemes. Among them, pure cell type stem cell spheres have a large number of cells. As the sphere grows, the cells in the center of the sphere are prone to necrosis due to the lack of nutrient exchange, and it is not easy to control the cell size in the process. Another method uses stem cell microcarrier technology to form 3D microspheres by combining biological microcarriers with cells. This method has good stability, there are gaps between cells, there are channels for nutrient exchange, and the cell viability is high. This scheme has been successfully commercialized, but its dedicated microcarriers are expensive, which is not conducive to cell extraction and large-scale preparation of 3D microspheres.

[0004] The main component of gelatin sponge is gelatin, which is white or slightly yellow, light, soft and porous. It is mainly used for hemostasis and is widely used in surgical operations, especially brain and spinal cord operations. Due to its expansion characteristics, gelatin sponge particles are currently mainly used in interventional surgery to play the role of embolization of blood vessels. At present, gelatin sponges have been used to plant various types of mesenchymal stem cells to study their biocompatibility and application, but this method obtains few deep cells in the sponge, while the cells on the surface of the sponge are too dense. The uneven characteristics of cells on the gelatin sponge lead to reduced cell activity. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method and product for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells. The present invention belongs to the technology of stem cell microcarriers, using low-cost gelatin sponge particles with good biomaterials as microcarriers, and combining them with olfactory mucosal mesenchymal stem cells to prepare 3D cell microspheres. Cells and gelatin sponges can be cross-linked with each other, with uniform density, which can better maintain the activity of stem cells. The preparation method has a wide source of raw materials, low cost, simple process flow, and the prepared 3D microspheres have high permeability, low cytotoxicity and good biocompatibility. And the 3D microspheres have broad prospects for the extraction of derivatives such as extracellular vesicles and exosomes after preparation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells comprises the following steps:

[0008] ① Weigh the gelatin sponge particles, mix them evenly with the culture medium, and inoculate them into a culture bottle. Place the culture bottle on a universal shaker in a humidified incubator for culture;

[0009] The gentle shaking of the shaker allows the gelatin sponge particles to fully contact the surrounding culture medium, which is more conducive to the subsequent cell inoculation and the uniform distribution of cells on its surface and pores.

[0010] ② Collect OM-MSC single cell suspension and adjust the cell suspension density;

[0011] The density of the cell suspension is adjusted to a suitable range (such as the specific density value in the present invention) to ensure that after being inoculated into the culture bottle containing the gelatin sponge particles, a reasonable binding ratio can be formed between the cells and the carrier, so that the cells in the microspheres will not be sparse due to too few cells, affecting their functions, nor will the local nutrient competition be too fierce due to too many cells, affecting the cell activity, thereby ensuring that the cells in the prepared 3D microspheres are evenly distributed and have good activity.

[0012] ③ Inoculate the cells into the culture flask containing gelatin sponge particles in step ①;

[0013] This step is to inoculate the processed and density-adjusted olfactory mucosal mesenchymal stem cells onto the pre-treated gelatin sponge particles, so that the cells can adhere to the surface and pores of the gelatin sponge particles. The porous and loose structural characteristics of the gelatin sponge particles are used to provide physical support for the cells and a channel for exchanging nutrients with the outside world, thereby starting the process of cell growth on a specific carrier and gradually forming 3D microspheres. This is a key connecting step in the entire preparation process and is directly related to whether the subsequent 3D microspheres can be successfully constructed and whether the cells can grow well in them.

[0014] ④ Add 5-10 mL of culture medium to the culture bottle every day and culture continuously on a universal shaker.

[0015] Preferably, the culture medium is composed of DMEM / F12 culture medium and 10 v / v% fetal bovine serum. The culture medium formula can provide comprehensive and appropriate nutrients for olfactory mucosal mesenchymal stem cells, including various amino acids, vitamins, inorganic salts, and growth factors, etc., to meet the growth, proliferation, and metabolic requirements of cells during in vitro culture.

[0016] Preferably, the gelatin sponge particles have a particle size of 700 to 1000 μm. The selection of this particle size range is based on comprehensive consideration of factors such as cell attachment, nutrient exchange, and the overall size and performance of subsequent 3D microspheres. Particles of this size have a suitable pore structure and specific surface area, which is conducive to the uniform distribution of cells and the exchange of substances with the external environment.

[0017] Preferably, the shaking table in step ① rotates at a constant speed of 40 rpm for 12 hours. This speed and time setting can provide a moderate dynamic environment for the combination of gelatin sponge particles and cells and the initial attachment of cells on their surface in the initial culture stage, and will not damage cells or carrier structures due to too fast a speed, nor will it cause problems such as insufficient mixing and uneven cell attachment due to too slow a speed. The duration of 12 hours has been verified by multiple experiments, and can enable cells to complete the initial attachment process better, laying a good foundation for subsequent continuous culture.

[0018] Preferably, the method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells comprises the following steps:

[0019] ① Weigh 30 mg of gelatin sponge particles, mix evenly with 30 mL of culture medium, and inoculate into a culture bottle. Place the culture bottle on a shaker at 40 rpm and culture in a humidified incubator for 12 hours;

[0020] ② The extracted OM-MSCs were digested with trypsin and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the precipitate was collected and separated into a single cell suspension. The cell density of the cell suspension was adjusted to 4×10 7 / mL;

[0021] ③ Inoculate the cells into the culture flask containing gelatin sponge particles in step ①;

[0022] ④Add 5-10 mL of culture medium to the conical flask every day and culture continuously for 7 days.

[0023] Preferably, the parameters of the universal shaker in step ④ are set to rotate at 30rpm for 5 minutes and stop for 25 minutes on the first day, and circulate for 24 hours; after the second day, culture is cultured at a uniform speed of 40rpm. The intermittent low-speed rotation mode on the first day can not only give cells a certain dynamic stimulation, promote their better attachment and adaptation to the new environment, but also avoid cell shedding or damage due to continuous mechanical force. After the first day of adaptation, 40rpm uniform rotation culture is adopted from the second day. At this time, the cells are relatively stably attached, and the uniform rotation can more effectively maintain the uniformity of the environment in which the cells are located, ensure the continuous and stable growth, proliferation and cell-to-cell interaction of the cells, and promote the gradual development and maturation of 3D microspheres during the 7-day culture process, forming 3D microspheres with stable structure and good cell activity, so that they have better application potential.

[0024] Furthermore, the present invention relates to 3D cell microspheres prepared by the method.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention produces a 3D microsphere containing stem cells, which combines the therapeutic potential of OM-MSCs and the scaffold function of gelatin sponge particles. Compared with the existing technology, the preparation method has a wide source of raw materials, low cost, and a simple process flow. The prepared 3D microspheres have high permeability, low cytotoxicity and good biocompatibility. Compared with the traditional method of directly planting cells on gelatin sponges, this method can better maintain cell viability. After preparation, 3D microspheres have broad prospects for the extraction of derivatives such as extracellular vesicles and exosomes. In practical applications, this cell carrier system may need to be customized and optimized according to specific clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Gelatin sponge particles.

[0028] Figure 2 : Gel sponge particles incubated in culture medium under light microscopy.

[0029] Figure 3 : Olfactory mucosa mesenchymal stem cells under light microscope.

[0030] Figure 4 : 3D microspheres cultured for 3 days.

[0031] Figure 5 : 3D microspheres cultured for 5 days under light microscope.

[0032] Figure 6 : CCK8 test results after 3D microsphere culture and 2D culture of OM-MSCs.

[0033] Figure 7: Comparison of live-dead staining results between 3D microsphere culture and direct seeding of OM-MSCs on gelatin sponge. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] In order to further illustrate the present invention, the following examples and drawings are used to describe the present invention in detail. The raw materials used in the following examples of the present invention are all commercially available products.

[0036] The following described sponge particles come from Jiangxi Xiangen Medical Technology Development Co., Ltd.

[0037] The olfactory mucosa mesenchymal stem cells are specifically obtained by a separation and culture method authorized by a Chinese patent entitled “A method for isolating and culturing human olfactory mucosa mesenchymal stem cells” and having application number CN201410228728.X.

[0038] Example 1

[0039] The method for incubating gelatin sponge particles is as follows:

[0040] ① Weigh 30 mg of gelatin sponge particles, see Figure 1 , mixed with 30 mL DMEM / F12 and 10% (v / v) fetal bovine serum medium, and inoculated into a triangular conical culture flask. Figure 2 ;

[0041] ② Place the triangular culture flask on a universal shaker at 37 degrees Celsius and 2% CO 2 in a humidified incubator;

[0042] ③ Set the shaker to a constant speed of 40 rpm and continue for 12 hours before use.

[0043] Example 2

[0044] The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells is as follows:

[0045] ① Collect OM-MSCs and culture them, see Figure 3 ;

[0046] ② The extracted OM-MSCs were digested with 0.25% (mass percentage) trypsin, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, the precipitate was collected and separated into a single cell suspension;

[0047] ③ Determine the number of cells by cell counting and adjust to the required cell density of 4×10 7 / mL, for spare use;

[0048] ④ Inoculate 1 mL of the cells at the above density into a triangular pyramidal culture flask containing gelatin sponge particles and culture continuously; the parameters of the universal shaker are set to rotate at 30 rpm for 5 minutes and stop for 25 minutes on the first day, with a cycle of 24 hours; after the second day, culture is carried out with a constant rotation speed of 40 rpm.

[0049] ⑤On the third day, a sphere visible to the naked eye was formed. Figure 4 ;

[0050] ⑥ Add 5-10mL of culture medium to the conical flask every day and extract 1mL of sample for testing. The spheres can be seen to be enlarged. Figure 5 , cultured continuously for 7 days to obtain gelatin sponge 3D microspheres.

[0051] Example 3

[0052] The experimental method for the effect of 3D microsphere culture on the proliferation ability of OM-MSCs is as follows:

[0053] ① The collected OM-MSCs were equally divided and cultured in 3D microspheres and 2D according to the above method, with 5 samples in each group;

[0054] ② After 1 day of culture, collect the cell precipitates from the culture medium of the above two methods and set aside;

[0055] ③ Place the two groups of cell pellets in a centrifuge tube and add 90% culture medium and 10% CCK8 reagent stock solution;

[0056] ④ Place the centrifuge tube at 37 degrees Celsius and 2% CO 2 in a humidified incubator for 30 minutes;

[0057] ⑤ Take the supernatant of the above centrifuge tube, detect the OD value of each group in an ELISA instrument, and make statistics. Figure 6 .

[0058] The results showed that 3D microsphere culture promoted the proliferation ability of OM-MSCs.

[0059] Example 4

[0060] Comparison of cell activity between direct inoculation of OM-MSCs on gelatin sponge and 3D microspheres, the steps are as follows:

[0061] ① Cut 30 mg of the gelatin sponge cube, mix it evenly with DMEM / F12 and 10% (v / v) fetal bovine serum culture medium, and inoculate it into a six-well plate;

[0062] ② OM-MSCs cells were evenly planted on the surface of gelatin sponge blocks and incubated at 37 degrees Celsius and 2% CO 2 Culture in a humidified incubator;

[0063] ③ After 7 days of continuous culture, the grown gelatin sponge 3D microspheres and gelatin sponge blocks were obtained respectively.

[0064] ④ Gently wash the samples obtained above with PBS for 3 times and then add live-dead staining reagent according to the manufacturer's protocol;

[0065] After incubation, the cell viability was observed under a microscope. Figure 7 The results showed that the cell viability of the 3D microsphere method was significantly higher than that of the direct inoculation method with gelatin sponge.

[0066] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells, characterized in that: The following steps are involved: ① Weigh the gelatin sponge particles, mix them evenly with the culture medium, and inoculate them into a culture bottle. Place the culture bottle on a universal shaker in a humidified incubator for culture; ② Collect OM-MSC single cell suspension and adjust the cell suspension density; ③ Inoculate the cells into the culture flask containing gelatin sponge particles in step ①; ④ Add 5-10 mL of culture medium to the culture bottle every day and culture continuously on a universal shaker.

2. The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells according to claim 1, characterized in that: The culture medium consists of DMEM / F12 culture medium and 10 v / v% fetal bovine serum.

3. The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells according to claim 1, characterized in that: The particle size of the gelatin sponge particles is 700-1000 μm.

4. The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells according to claim 1, characterized in that: Step ① The shaking table rotates at a constant speed of 40 rpm for 12 hours.

5. The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells according to claim 1, characterized in that: The following steps are involved: ① Weigh 30 mg of gelatin sponge particles, mix evenly with 30 mL of culture medium, and inoculate into a culture bottle. Place the culture bottle on a shaker at 40 rpm and culture in a humidified incubator for 12 hours; ② The extracted OM-MSCs were digested with trypsin and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and the precipitate was collected and separated into a single cell suspension. The cell density of the cell suspension was adjusted to 4×10 7 / mL; ③Inoculate 1 mL of cells into the culture flask containing gelatin sponge particles in step ①; ④Add 5-10 mL of culture medium to the conical flask every day and culture continuously for 7 days.

6. The method for preparing 3D microspheres by combining gelatin sponge particles with olfactory mucosal mesenchymal stem cells according to claim 1, characterized in that: In step ④, the parameters of the universal shaker are set to rotate at 30 rpm for 5 minutes and stop for 25 minutes on the first day, with a cycle of 24 hours; after the second day, the culture is cultured with a constant rotation speed of 40 rpm.

7. 3D cell microspheres prepared by the method according to any one of claims 1 to 6.

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