Microspheres with high diffusive permeability and their use in three-dimensional cell culture

Abstract

The application discloses a kind of microspheres of high diffusion permeability and its application in three-dimensional cell culture, and its synthesis method includes the following steps: step (1), preparation is obtained dispersed phase precursor liquid;Step (2), preparation is obtained continuous phase;Step (3), dispersed phase precursor liquid is slowly dropped to continuous phase under magnetic stirring, and the droplet particle size is controlled by adjusting stirring speed;Step (4), the emulsion obtained is irradiated under ultraviolet light to induce photopolymerization;Step (5), after polymerization is completed, continuous phase is removed by centrifugation, and cyclohexane is added to demulsification to remove residual organic phase;Step (6), the microspheres obtained are washed with ultrapure water, and are screened by screen mesh to obtain the microspheres of required particle size;Step (7), microspheres are sterilized and purified using sterile PBS.The microspheres of the application have excellent diffusion permeability and biomimetic porous structure, and have great application potential in in vitro cell culture and three-dimensional tissue model.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a microsphere with high diffusion permeability and application thereof in three-dimensional cell culture. BACKGROUND

[0002] Three-dimensional cell culture technology (TDCC) is a new in vitro cell culture technology, which can not only retain the material basis of the in vivo cell microenvironment, but also reflect the intuitiveness and controllability of cell culture. It refers to the co-culture of a carrier with a three-dimensional structure of different materials and various different types of cells in vitro, so that the cells can adhere, migrate and grow in the three-dimensional space structure of the carrier to form a three-dimensional cell-carrier composite. The scaffold for three-dimensional cell culture mainly includes hydrogel, fiber and porous structure. The porous microsphere scaffold has attracted widespread attention from the scientific research and industrial communities due to its high surface area, efficient cell expansion and injectability.

[0003] Notably, the pore size of the three-dimensional cell culture microsphere particles directly affects cell adhesion and proliferation. When the pore size is too small, the cells cannot enter the interior of the microsphere particles, i.e., they only adhere to the surface, the effective adhesion area is insufficient, and the cells are prone to fall off during the culture process. Generally speaking, the microsphere carrier with staggered distribution of large and small pores is more conducive to the diffusion of gas and nutrients, thereby promoting cell adhesion and proliferation.

[0004] The three-dimensional cell culture carrier obtained by traditional freeze-drying is in the form of a block, which cannot be applied to large-scale bioreactors for suspension culture. Moreover, the low heat transfer efficiency leads to high energy consumption in the preparation and processing process, thereby bringing greater cost to actual production. The three-dimensional culture microsphere particles obtained by microfluidic emulsification technology or atomizing the droplets into liquid nitrogen and then vacuum freeze-drying have a complicated carrier microsphere process, which is difficult to scale up. The pore size is difficult to control, thereby affecting the uniformity of the feeding ratio between the subsequent cells and the microsphere particles. SUMMARY

[0005] The application provides a microsphere with high diffusion permeability and application thereof in three-dimensional cell culture. The microsphere is synthesized by utilizing the high polymer miscibility effect and the reverse microemulsion method, and has great application potential in in vitro cell culture and three-dimensional tissue models.

[0006] To achieve the above object, the application adopts the following technical solutions:

[0007] In a first aspect, the application provides a microsphere with high diffusion permeability, and a synthesis method of the microsphere includes the following steps:

[0008] Step (1), preparation of the dispersed phase precursor solution: a mixed solution of DMSO and water was used as the dispersed phase solvent, and NIPAM, BIS and a photoinitiator Darocur 1173 were added to prepare the dispersed phase precursor solution;

[0009] Step (2), preparation of the continuous phase: Span 80 was added to methyl silicone oil to prepare the continuous phase;

[0010] Step (3), the dispersed phase precursor solution was slowly added to the continuous phase under magnetic stirring, and the droplet size was controlled by adjusting the stirring speed;

[0011] Step (4), the obtained emulsion was irradiated under 365 nm ultraviolet light to induce photopolymerization;

[0012] Step (5), after the polymerization was completed, the continuous phase was removed by centrifugation, and cyclohexane was added to break the emulsion to remove the residual organic phase;

[0013] Step (6), the obtained microspheres were washed with ultrapure water, and sieved through a screen to obtain microspheres with a desired particle size;

[0014] Step (7), the microspheres were sterilized and purified using sterile PBS.

[0015] In the above technical solution, in step (1), DMSO: water: NIPAM: BIS: Darocur 1173 = 12 mL: 8 mL: 4 g: 100 mg: 20 μL.

[0016] In the above technical solution, in step (2), methyl silicone oil: Span 80 = 100 mL: 500 μL.

[0017] In the above technical solution, in step (3), the droplet size is 50-500 μm.

[0018] In the above technical solution, in step (6), the microspheres obtained by sieving have a particle size of 100-500 μm.

[0019] In a second aspect, the application provides the use of the above-mentioned microspheres in three-dimensional cell culture.

[0020] In the above technical solution, the cells are suspension cells.

[0021] The microspheres of the application have excellent diffusion permeability and biomimetic porous structure, which not only have great application potential in in vitro cell culture and three-dimensional tissue models, but also provide a more flexible carrier platform for drug delivery, immune cell therapy and tissue engineering. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a flow chart of the synthesis of microspheres of the present application;

[0023] Figure 2 is a basic characterization of the microspheres of Comparative Example 1 of the present application;

[0024] Figure 3 is a basic characterization of the microspheres of Example 1 of the present application;

[0025] Figure 4 is a flow chart of the co-incubation experiment of microspheres with spleen cells of the present application;

[0026] Figure 5 is a graph of the results of the co-incubation experiment of microspheres with spleen cells of the present application. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples. The present application can be implemented in many different forms, and should not be understood as being limited to the examples set forth herein. On the contrary, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the idea of the present application to those skilled in the art, which will be limited only by the claims.

[0028] The present application provides a microsphere with high diffusive permeability, the synthesis method of which comprises the following steps:

[0029] Step (1), preparation of the dispersed phase precursor solution: a mixed solution of DMSO and water is used as the dispersed phase solvent, NIPAM, BIS and a photoinitiator Darocur 1173 are added to prepare the dispersed phase precursor solution;

[0030] Step (2), preparation of the continuous phase: Span 80 is added to methyl silicone oil to prepare the continuous phase;

[0031] Step (3), the dispersed phase precursor solution is slowly added to the continuous phase under magnetic stirring, and the droplet size is controlled by adjusting the stirring speed;

[0032] Step (4), the obtained emulsion is irradiated under 365 nm ultraviolet light to induce photopolymerization;

[0033] Step (5), after the polymerization is completed, the continuous phase is removed by centrifugation, and cyclohexane is added to break the emulsion to remove the residual organic phase;

[0034] Step (6), the obtained microspheres are washed with ultrapure water, and sieved through a screen to obtain microspheres with the desired particle size;

[0035] Step (7), the microspheres are sterilized and purified using sterile PBS.

[0036] Preferably, Span 80 is a good surfactant for the system of the present application, and other surfactants cannot achieve the effect of the present application.

[0037] Preferably, the droplet size is controlled by adjusting the stirring speed. The suitable particle size is about 50-500 μm, and then a screen can be used to screen the suitable particle size according to the need.

[0038] The present application also provides the use of the above microspheres in three-dimensional cell culture. Preferably, the cells are suspension cells.

[0039] Example 1: Synthesis method of microspheres

[0040] The method comprises the following steps:

[0041] (1) Preparation of the dispersed phase precursor solution: 12 mL of a mixed solution of DMSO (dimethyl sulfoxide) and 8 mL of water is used as the dispersed phase solvent, 4 g of NIPAM (N-isopropyl acrylamide), 100 mg of BIS (methylene acrylamide) and 20 μL of a photoinitiator Darocur 1173 are added to prepare the dispersed phase precursor solution.

[0042] (2) Preparation of the continuous phase: 500 μL of Span 80 is added to 100 mL of methyl silicone oil to prepare the continuous phase.

[0043] (3) The dispersed phase precursor solution is slowly added to the continuous phase under magnetic stirring, and the stirring speed is adjusted to control the droplet size to be about 100 μm.

[0044] (4) The obtained emulsion is irradiated under 365 nm ultraviolet light for 200 s to induce photopolymerization.

[0045] (5) After the polymerization is completed, the continuous phase is removed by centrifugation, and 50 mL of cyclohexane is added to break the emulsion to remove the residual organic phase.

[0046] (6) The obtained microspheres are washed with ultrapure water, and screened by a screen to obtain microspheres with a particle size of 100-500 μm.

[0047] (7) The microspheres are sterilized and purified using sterile PBS.

[0048] Comparative Example 1: Synthesis method of microspheres

[0049] The method comprises the following steps:

[0050] (1) 2 g monomer N-isopropyl acrylamide, 1.6 mL monomer sodium vinyl sulfonate, 1 mL crosslinking agent N, N-methylene bisacrylamide and 1 mL photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone were added to a mixed solution of 10 mL glycerol and 10 mL water as the dispersion phase solvent to prepare a dispersion phase precursor solution.

[0051] (2) 100 mL methyl silicone oil and 1 mL surfactant Span 80 were mixed to prepare a continuous phase.

[0052] (3) The dispersion phase precursor solution was slowly dropped into the continuous phase, the droplet size was controlled by adjusting the rotation speed of the continuous phase magnetic sub, and after the particle size reached about 100 μm, it was cured in a UV curing box (160 W) for 200 seconds.

[0053] (4) The mixed solution was centrifuged to remove the upper continuous phase, 50 mL cyclohexane was added to break the emulsion and remove the upper organic phase, a large amount of ultrapure water was added for washing, and a sieve was used for screening to obtain white microspheres MS.

[0054] (5) 40 mg of dopamine hydrochloride was dissolved in 10 mL of pH=8.5 200 mM Tris-HCl buffer, 2 mL of microspheres MS was added and reacted for 70 minutes, and then sieved to obtain polydopamine functionalized microspheres PDA-MS.

[0055] (6) After the PDA-MS was washed with ultrapure water for several times, 75% ethanol was added for soaking for 12 hours, and then a UV curing box was used for irradiation for 10 minutes, 1000 rpm centrifugation for 1 minute, and then the supernatant was removed in an ultraclean table to complete the sterilization of the microspheres, thereby preparing the microspheres of Comparative Example 1.

[0056] Basic characterization of the microspheres

[0057] As shown in Figure 2 , the basic characterization of the microspheres prepared in Comparative Example 1 of the application was carried out, the microspheres were freeze-dried using liquid nitrogen and a freeze dryer and adhered to the conductive glue on the sample table, and it was found by scanning electron microscopy that the microspheres had a surface film structure and an open structure inside.

[0058] As shown in Figure 3 , the basic characterization of the microspheres prepared in Example 1 of the application was carried out, the microspheres synthesized in the DMSO and water system also had a highly loose and porous structure, which was different from the microspheres synthesized in water and glycerol in Comparative Example 1, and did not have a special film structure on the surface, which could meet the needs of different biological applications.

[0059] The novel microspheres of this invention remove the outer membrane structure, exhibiting a more open porous system compared to the membrane-bound microspheres of Comparative Example 1. This structural characteristic makes them suitable for the three-dimensional infiltration and culture of suspended cells (such as CAR-T cells and spleen cells), as the suspended cells are not restricted by the outer membrane and can freely enter the interior of the microspheres and distribute themselves within the porous network, thus fully realizing their biological functions. In contrast, the membrane-bound microspheres of Comparative Example 1 are restricted by the outer membrane, making it difficult for suspended cells to enter their interior, and are only suitable for the adhesion and growth of adherent cells.

[0060] Microsphere and suspended cell co-incubation experiment

[0061] Mouse spleen cells were extracted and co-cultured with microspheres prepared in Example 1 for 8 hours. The culture medium was then removed and the cells were washed twice with PBS. Live cells were then stained with Calcein-AM assay solution, and the infiltration of suspended spleen cells was observed using CLSM.

[0062] like Figure 4 The diagram shown is the experimental flowchart. Figure 5 The image shown depicts the experimental results, revealing that the microspheres support the infiltration of suspended spleen cells. Figure 5 a is the bright-field image from the microscope. Figure 5 b is a dark-field / live-cell staining image under a microscope. Figure 5 c is a CLSM observation image after incubation. The microspheres of this invention have excellent diffusion and permeability, and can be applied to the three-dimensional infiltration and culture of suspended cells.

[0063] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Microspheres of high diffusive permeability, characterized in that: The synthesis method of the microspheres comprises the following steps: Step (1), preparation of the dispersed phase precursor solution: a mixed solution of dimethyl sulfoxide (DMSO) and water is used as the dispersed phase solvent, N-isopropyl acrylamide (NIPAM), methylenebisacrylamide (BIS) and a photoinitiator Darocur 1173 are added to prepare the dispersed phase precursor solution; Step (2), preparation of the continuous phase: Span 80 is added to methyl silicone oil to prepare the continuous phase; Step (3), the dispersed phase precursor solution is slowly added to the continuous phase under magnetic stirring, and the droplet size is controlled by adjusting the stirring speed; Step (4), the obtained emulsion is irradiated under 365 nm ultraviolet light to induce photopolymerization; Step (5), after the polymerization is completed, the continuous phase is removed by centrifugation, and cyclohexane is added to break the emulsion to remove the residual organic phase; Step (6), the obtained microspheres are washed with ultrapure water, and are screened through a screen to obtain microspheres with a desired particle size; Step (7), the microspheres are sterilized and purified using sterile PBS; In step (1), dimethyl sulfoxide (DMSO): water: N-isopropyl acrylamide (NIPAM): methylenebisacrylamide (BIS): Darocur 1173 = 12 mL: 8 mL: 4 g: 100 mg: 20 μL; In step (2), methyl silicone oil: Span 80 = 100 mL: 500 μL; In step (3), the droplet size is 50-500 μm.

2. The microspheres of claim 1, wherein: In step (6), the particle size of the microspheres obtained by screening through a screen is 100-500 μm.

3. Use of the microspheres of any one of claims 1-2 in three-dimensional cell culture, which use is not for the diagnosis and treatment of diseases.

4. The use according to claim 3, wherein the cells are suspension cells.

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