Porous gel microspheres capable of being cryopreserved as well as preparation method and application of porous gel microspheres

Abstract

The invention provides a porous gel microsphere capable of being cryopreserved as well as a preparation method and application thereof. The method comprises the following steps: preparing a water-phase solution and an oil-phase solution into gel-state micro-droplets, wherein the water-phase solution contains methacrylic acid gelatin synthesized by low-jelly-strength pigskin gelatin; soaking the gel-state micro-droplets in a liquid containing a photoinitiator, and freezing after full permeation, so that water in the gel-state micro-droplets forms ice crystals; and performing ultraviolet irradiation to form gel-state micro-droplets of ice crystals, and performing cross-linking curing to obtain the porous gel microspheres capable of being cryopreserved. The porous gel microspheres support preloading cells, get rid of the time urgency of current preparation and current use, and can be taken in different time and space for cell printing and injection manufacturing; for a patient, one-time extraction, long-term cryopreservation and taking as required can be realized, so that repeated trauma is avoided.

Description

Technical Field

[0001] The present invention relates to the field of regenerative medicine, in particular to the field of tissue engineering and biomanufacturing using gel microspheres as raw materials, and specifically to a porous gel microsphere that can be cryopreserved, and a preparation method and application thereof. Background Art

[0002] The development of tissue engineering and regenerative medicine technology has provided new solutions for the repair and regeneration of tissues and organs. Gel microsphere particles are a new type of material. Gel microsphere particles are composed of tightly packed submillimeter gel spheres. The gap space between gel microspheres has a higher porosity than traditional hydrogels, which is conducive to cell physiological activities, including migration, proliferation and differentiation. In clinical applications, it is often necessary to inject biomaterials. The submicron size and weak physical interaction of gel microsphere particles give them significant shear-thinning behavior, which can be easily injected through a fine needle. After injection, the friction between the gel microspheres quickly induces the structure to self-repair. This physical form gives shear-thinning and self-repairing properties to materials that are difficult to extrude, and can be extruded through a needle and maintain shape after extrusion. In addition, gel microsphere particles can also be used as ink for bio-3D printing. As an advanced technology in the field of biomanufacturing, bio-3D printing can manufacture biomimetic functional structures with cells, active molecules and biomaterials as basic units, and reconstruct the complexity of tissues and organs to a greater extent. Gel microsphere particle ink combined with bio-3D printing can construct customized tissue engineering scaffolds. Therefore, gel microsphere particles have broad application prospects in the field of clinical injection and tissue engineering applications.

[0003] The porosity of traditional gel microsphere ink is related to the packing density, which is about 15% when densely packed and about 25% when loosely packed. Too loose packing will lead to poor extrusion characteristics, making it impossible to print high-fidelity structures, and the bonding between microspheres is also weak. Some technologies modify the surface of gel microspheres to provide dynamic bonding for loosely packed gel microspheres, further reducing the stacking density threshold required for printing, thereby increasing the porosity. However, although the porosity of gel microsphere ink is improved compared with traditional block hydrogels, it still has certain limitations in biology, including difficulty in stretching cells in microspheres and insufficient nutrient transport. More interconnected micropores and larger void spaces are not only conducive to the transport of nutrients, thereby increasing cell viability, but also can promote cell stretching without waiting for hydrogel degradation.

[0004] A variety of strategies have been used to create smaller-scale pore features inside gel microspheres. Some technologies prepare freeze-dried gel microspheres (also called microcarriers) for stem cell expansion, but freeze-dried gel microspheres cannot be preloaded with cells and can only be used as microcarriers to inoculate cells, with limited inoculation efficiency. Some other technologies use aqueous two-phase precursor hydrogels to prepare gel microspheres, and obtain interconnected pores by sacrificing one of the phases. This method can preload cells at a higher density and obtain better pore connectivity, but is limited by the material system. Currently, only a limited number of biocompatible material systems can form spontaneous liquid-liquid phase separation. In addition, there is currently no work showing that aqueous two-phase cell-loaded microspheres support storage and can only be used immediately after manufacturing. In summary, it is still challenging to achieve high-porosity cell-loaded porous gel microspheres that can be accessed in one step. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a porous gel microsphere that can be cryopreserved and a preparation method and application thereof. The porous gel microsphere has little restriction on materials, supports pre-loaded cells, gets rid of the time urgency of current preparation and current use, and can be used in different time and space.

[0006] To achieve the above object, the present invention provides a method for preparing porous gel microspheres that can be frozen, wherein the method comprises:

[0007] The aqueous phase solution and the oil phase solution are prepared into gel-state microdroplets; wherein the aqueous phase solution contains methacrylated gelatin (GelMA) synthesized from pig skin gelatin with low jelly strength;

[0008] The gel-state micro-droplets are soaked in a liquid containing a photoinitiator, and after being fully infiltrated, they are frozen so that the water inside the gel-state micro-droplets forms ice crystals;

[0009] Ultraviolet light is used to cross-link and solidify the gel-state microdroplets that form ice crystals, thereby obtaining porous gel microspheres that can be frozen.

[0010] According to a specific embodiment of the present invention, preferably, the freezing time is more than 90 minutes, and the freezing time affects the growth morphology of ice crystals in the hydrogel.

[0011] According to a specific embodiment of the present invention, preferably, the method comprises:

[0012] The aqueous phase solution and the oil phase solution are prepared into gel-state microdroplets; wherein the aqueous phase solution contains methacrylated gelatin synthesized from pig skin gelatin with low jelly strength and also contains cells to be preloaded;

[0013] The gel-state microdroplets are soaked in a liquid containing a photoinitiator, and after sufficient penetration, the gel-state microdroplets are transferred to a freezing solution and subjected to gradient freezing, so that the water inside the gel-state microdroplets forms ice crystals;

[0014] Ultraviolet light is used to cross-link and solidify the gel-state microdroplets that form ice crystals, thereby obtaining porous gel microspheres that can be frozen.

[0015] According to a specific embodiment of the present invention, the method for synthesizing methacrylated gelatin synthesized from pigskin gelatin with low jelly strength comprises: slowly adding methacrylic anhydride to a gelatin solution prepared from pigskin gelatin with a gel strength of 100-150 Bloomg, and stirring the solution sufficiently to allow the solution to react sufficiently. After the reaction is completed, unreacted methacrylic anhydride is neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances are removed by a dialysis bag (molecular weight cutoff ~12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000Da); the purified methacrylated gelatin solution is washed and dried to obtain the final methacrylated gelatin synthesized from pigskin gelatin with low jelly strength.

[0016] According to a specific embodiment of the present invention, preferably, the low temperature ranges from 0 to 10°C.

[0017] According to a specific embodiment of the present invention, preferably, when there are no cells to be preloaded in the aqueous solution, that is, when preparing cell-free porous gel microspheres, the cross-linking environment temperature is 4° C. and the time is 2 minutes.

[0018] According to a specific embodiment of the present invention, preferably, when the cell-laden hydrogel precursor solution (i.e., the aqueous phase solution) contains cells to be pre-loaded, i.e., when preparing cell-laden porous gel microspheres, the cross-linking environment temperature is room temperature and the time is 2 minutes. However, before taking out the porous gel microspheres after gradient freezing for cross-linking, they must always be kept below 4°C.

[0019] According to a specific embodiment of the present invention, preferably, the low jelly strength pigskin gelatin has a jelly strength of 100-150 Bloomg.

[0020] According to a specific embodiment of the present invention, preferably, based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin is 2-20 g / 100 mL, more preferably 5-12.5 g / 100 mL, and further preferably 8-12 g / 100 mL.

[0021] In the preparation method of the present invention, the material is optimized. The microspheres of GelMA generally used for tissue culture cannot produce pores when frozen (not dried and dehydrated), so the present invention adopts a variety of schemes to optimize the materials of GelMA microspheres so that pores appear after freezing (not dried and dehydrated), and finally obtain an optimized scheme, that is, GelMA synthesized with low jelly strength pigskin gelatin is used to prepare porous gel microspheres preloaded with cells in situ. In the present invention, methacrylic anhydride (MA) is added to low jelly strength pigskin gelatin, and a substitution reaction is performed to prepare the methacrylated gelatin. The microspheres prepared by this method can form pores after freezing, while the microspheres prepared by general commercial GelMA (synthesized with 300Bloomg jelly strength pigskin gelatin) have no pores after freezing.

[0022] According to a specific embodiment of the present invention, preferably, the aqueous phase solution contains methacrylated gelatin synthesized from low jelly strength pig skin gelatin and a blended phase for assisting cell culture.

[0023] According to a specific embodiment of the present invention, preferably, based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin is 2-20 g / 100 mL, more preferably 5-12.5 g / 100 mL.

[0024] According to a specific embodiment of the present invention, preferably, the aqueous phase solution contains methacrylated gelatin synthesized from pig skin gelatin with low jelly strength and a blended phase for assisting cell culture; based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin synthesized from pig skin gelatin with low jelly strength is 2.5-10 g / 100 mL, preferably 8-10 g / 100 mL; based on the volume of the solvent in the aqueous phase solution, the content of the blended phase is 2.5-10 g / 100 mL, preferably 8-10 g / 100 mL.

[0025] According to a specific embodiment of the present invention, the blended phase is selected from one or a combination of two or more of gelatin, hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan and fibrinogen.

[0026] According to a specific embodiment of the present invention, the aqueous phase solution further contains sugars, and the content of sugars is 1-10 g / 100 mL, preferably 4-8 g / 100 mL, based on the volume of the solvent in the aqueous phase solution.

[0027] According to a specific embodiment of the present invention, preferably, the sugar in the aqueous phase solution includes one or a combination of two or more of lactose, raffinose, and melezitose, preferably raffinose.

[0028] According to a specific embodiment of the present invention, preferably, the cell density in the aqueous solution is 1×10 3 -1×10 8 / mL, preferably 1×10 6 -2×10 7 .

[0029] According to a specific embodiment of the present invention, preferably, the cells include one or a combination of two or more of human mesenchymal stem cells, cancer cells, fibroblasts and endothelial cells.

[0030] In the preparation method of the present invention, the selected cells are cultured in a culture medium dedicated to each type of cells, the culture medium is replaced every 2 days, and after the cells are in the logarithmic growth phase, they are digested with 0.25% trypsin, collected by centrifugation, and then re-vortexed with a methacrylated gelatin solution prepared with a solvent and synthesized from pig skin gelatin with a content of 5-12.5 g / 100 mL of low jelly strength, as an aqueous phase solution for standby use. The prepared methacrylated gelatin solution is filtered and sterilized through a filter membrane (0.22 μm pores) and then added to the cells.

[0031] In the present invention, the culture medium used for each type of cell is a general commercial culture medium commonly used for the cell type in the art.

[0032] According to a specific embodiment of the present invention, preferably, the oil phase solution is obtained by mixing an oily material with a surfactant.

[0033] According to a specific embodiment of the present invention, preferably, the oily material includes one or a combination of two or more of mineral oil, fluorinated oil, and silicone oil.

[0034] According to a specific embodiment of the present invention, preferably, based on 100% volume of the oil phase solution, the content of the surfactant is 1-5%, more preferably 4%.

[0035] According to a specific embodiment of the present invention, preferably, the surfactant includes one or a combination of two or more of Span80, Tween 20, AEO-3, and FluoSurf.

[0036] According to a specific embodiment of the present invention, preferably, the method of preparing the aqueous phase solution and the oil phase solution into gel-state microdroplets includes droplet microfluidics, oil-water emulsion method, complex coacervation method or electrospray method.

[0037] According to a specific embodiment of the present invention, the present invention uses a droplet microfluidic method to prepare gel-state microdroplets with an aqueous solution and an oil-phase solution. Any microfluidic device in the art that can obtain gel-state microdroplets of the desired particle size can be used. Preferably, the specific steps of the droplet microfluidic method include: adding the aqueous solution and the oil-phase solution to the microfluidic device, adjusting the flow rate of the aqueous solution and the oil-phase solution respectively, and mixing. Among them, the flow rate of the aqueous solution and the oil-phase solution refers to the flow rate of the aqueous solution inlet and the oil-phase solution inlet respectively input into the chip, and the syringe is pushed and controlled by a precision pump. The pipeline specifications at the aqueous solution inlet and the oil-phase solution inlet during input can be set according to the particle size of the prepared gel-state microdroplets. Preferably, the outer diameter of the hose at the aqueous solution inlet and the oil-phase solution inlet during input is 0.76-2.29mm, and the inner diameter is 0.25-1.27mm; for example, the inner diameter of the hose at the aqueous solution inlet and the oil-phase solution inlet during input is 0.51mm, and the outer diameter is 1.52mm.

[0038] According to a specific embodiment of the present invention, the specifications of each pipeline in the droplet microfluidics device of the present invention can be set according to the particle size of the prepared gel-state microdroplets. Preferably, in the droplet microfluidics device, the diameter of the chip channel of the oil phase solution and the water phase solution is 100-400 microns; the diameter of the droplet pipeline after the oil phase solution and the water phase solution are mixed is 300-800 microns. For example, the diameter of the chip channel of the oil phase solution and the water phase solution is 300 microns; the diameter of the droplet pipeline after the oil phase solution and the water phase solution are mixed is 550 microns.

[0039] According to a specific embodiment of the present invention, preferably, the flow rate of the aqueous phase solution is 1-3 mL / h, and the flow rate of the oil phase solution is 4-10 mL / h.

[0040] In the preparation method of the present invention, by adjusting the flow rates of the aqueous solution and the oily solution of the microfluidic device, gel-state microdroplets containing or not containing cells with a particle size range of 150-400 microns can be stably obtained.

[0041] In the preparation method of the present invention, when the aqueous phase solution does not contain the cells to be preloaded, the prepared gel-state microdroplets remain stable at low temperatures and can be enriched by centrifugation, sedimentation, etc., and further added with a cleaning solution, such as a 0.9% sodium chloride solution, a 1× phosphate buffered saline solution, or ultrapure water, to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0042] In the preparation method of the present invention, when the aqueous solution contains cells to be preloaded, the prepared gel-state microdroplets remain stable at low temperatures, can be enriched by centrifugation, sedimentation, etc., and further added with a cleaning solution. In order to maintain good cell viability, a culture medium commonly used in the art that is compatible with the preloaded cells can be added as a cleaning solution to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0043] In the preparation method of the present invention, after the supernatant of the gel-state microdroplets is removed after centrifugation, a culture medium containing a photoinitiator is added; the gel-state microdroplets are placed in a refrigerator at 4°C to prevent the gel-state microdroplets from melting and ensure that the photoinitiator fully penetrates. Thereafter, the gel-state microdroplet solution is transferred to a culture dish and a freezing solution is added.

[0044] According to a specific embodiment of the present invention, preferably, the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphite (LAP) and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0045] According to a specific embodiment of the present invention, preferably, based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.1-0.5 g / 100 mL, preferably 0.1-0.3 g / 100 mL, and more preferably 0.15 g / 100 mL.

[0046] The preparation method of the liquid containing the photoinitiator in the present invention is as follows: 0.01-0.05 g of the photoinitiator is added to every 1 mL of solvent to prepare a photoinitiator mother solution with a concentration of 1-5 g / 100 mL. When used, the 1-5 g / 100 mL photoinitiator mother solution is diluted 10 times, and 1 mL of the photoinitiator mother solution is added to 9 mL of the solvent to obtain a liquid containing the photoinitiator.

[0047] According to a specific embodiment of the present invention, preferably, the volume ratio of the cryopreservation solution to the gel-state microdroplets is 1:(1-10), more preferably 1:(1-3), and further preferably 1:2.

[0048] According to a specific embodiment of the present invention, preferably, the step of gradient freezing comprises: standing at -10°C to -25°C for 20-60 minutes, transferring to -80°C to -196°C environment for storage for later use, and can be stored for a long time.

[0049] According to a specific embodiment of the present invention, preferably, the ultraviolet light irradiation time is 30-180s.

[0050] According to a specific embodiment of the present invention, preferably, based on the total volume of the cryopreservation solution, the components of the cryopreservation solution include: 0-15mL / 100mL of DMSO, 0-12g / 100mL of sugars, 0-40mL / 100mL of serum, 0.1-0.5g / 100mL of photoinitiator, and the rest is culture medium; more preferably, based on the total volume of the cryopreservation solution, the components of the cryopreservation solution include: 1-15mL / 100mL of DMSO, 1-12g / 100mL of sugar, 1-40mL / 100mL of serum, 0.1-0.5g / 100mL of photoinitiator, and the rest is culture medium; further preferably, based on the total volume of the freezing solution, the components of the freezing solution include: 5-15mL / 100mL of DMSO, 4-8g / 100mL of sugar, 20-40mL / 100mL of serum, 0.1-0.3g / 100mL of photoinitiator, and the rest is culture medium. As a typical preferred composition, the components of the freezing solution include: 10mL / 100mL of DMSO, 4g / 100mL of raffinose, 40mL / 100mL of serum, 0.15g / 100mL of photoinitiator, and the rest is culture medium.

[0051] According to a specific embodiment of the present invention, preferably, the sugar in the freezing solution includes one or a combination of two or more of lactose, raffinose and melezitose, preferably raffinose.

[0052] According to a specific embodiment of the present invention, preferably, the photoinitiator in the freezing solution includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0053] The present invention also provides porous gel microspheres that can be frozen obtained by the above preparation method, wherein the particle size of the porous gel microspheres is 150-400 microns, preferably, the particle size of the porous gel microspheres is 200-250 microns, and the average equivalent diameter of the internal pores of the porous gel microspheres is 20-80 microns.

[0054] The present invention also provides a biological ink, which is prepared by the cryogenic porous gel microspheres obtained by the above preparation method or the cryogenic porous gel microspheres.

[0055] The present invention also provides a biological 3D printed product, which is prepared by the cryogenic porous gel microspheres obtained by the above preparation method, the above cryogenic porous gel microspheres or the above biological ink.

[0056] The present invention utilizes the ice crystal pore-making principle to construct micron-scale interconnected pores inside the microparticles, and the material applicability of the preparation method provided by the present invention is wide, and the wet porous gel microspheres of proteins, polysaccharides and synthetic hydrogels can be manufactured. The technical solution of the present invention supports the in-situ loading of living cells and can be stored in a low-temperature environment for a long time. When used, through rapid cross-linking and thawing, stable pores are left inside the microspheres after the ice crystals melt, and interconnected porous gel microspheres with in-situ cell loading can be obtained. The low-temperature storage process can be extended to three months or longer, and the cell viability in the hydrogel microspheres can be maintained during the low-temperature storage process.

[0057] Compared with the traditional method, the technical solution of the present invention has the following beneficial effects:

[0058] 1. The present invention realizes the manufacture of porous gel microspheres that can be cryopreserved and loaded with cells in situ. This process can achieve porosity without freeze-drying (traditional method). Through material and parameter optimization, it can support high-density in situ loading of living cells.

[0059] 2. The present invention can be applied to clinical cell therapy, realizing a porous gel microsphere-based microtissue module that can be preloaded with high-density cells, support long-term freezing of cells, and be taken on demand, eliminating the tedious steps of temporary preparation of scaffolds and cell planting, supporting instant bedside access for injection, transplantation and other tissue repair treatment scenarios, and establishing a "on-demand access on the shelf" cell therapy paradigm, getting rid of the time urgency of current preparation and current use, and being able to be taken in different time and space. For patients, it can be extracted once, frozen for a long time, and taken on demand to avoid repeated trauma.

[0060] 3. The cryopreservable porous gel microspheres prepared by the present invention are also a novel biological 3D printing ink material, which can construct granular stress yield bio-ink and be applied to cell printing. Compared with traditional solid gel microsphere particle ink, it has better porosity.

[0061] 4. It is difficult to achieve the porosity of GelMA porous gel microspheres in the prior art. Since ice crystals are easy to melt, the efficiency of photocuring is very low under low-temperature freezing conditions. Therefore, it is difficult to stably solidify porous gel microspheres made by ice crystal pore making through photocrosslinking. Most prior arts use freeze drying or chemical crosslinking. The technical solution of the present invention achieves stable photocured porous gel microspheres with a high output rate by optimizing materials, cryopreservation solution formula and cryopreservation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 A diagram of a cross-shaped droplet microfluidic chip device used in the present invention.

[0063] Figure 2 It is a schematic diagram of the technical solution of the present invention.

[0064] Figure 3 These are the test results of various materials of porous cryogel microspheres and the statistical analysis and strategy comparison results of internal pores.

[0065] Figure 4 Results from gel microsphere material formulation optimization for porous cryogel microspheres.

[0066] Figure 5 Results of gel microsphere cryofluid optimization for porous cryogel microspheres.

[0067] Figure 6 Comparison results before and after porous cryogel microsphere process optimization.

[0068] Figure 7 A comparison of traditional microgels and wet porous cryogel microspheres.

[0069] Figure 8 Staining images of conventional microgels and wet frozen porous gel microspheres supporting the culture of a variety of cells.

[0070] Fig. 9 These are the results of Alizarin red staining of wet frozen porous gel microspheres after one week of culture and two weeks of osteogenic differentiation.

[0071] Fig.10 These are the results of the survival rate and proliferation rate test of long-term cryopreservation of wet porous cryogel microspheres loaded with cells.

[0072] Fig.11 This is the result of wet frozen porous gel microspheres being used for extrusion bio-3D printing.

[0073] Fig.12 These are the experimental results of subcutaneous injection of traditional microgels and wet porous cryogel microspheres in rats.

[0074] Fig.13 These are the experimental results of high interconnected pore permeability of wet frozen porous gel microspheres.

[0075] Description of reference numerals:

[0076] 301: oil phase solution inlet; 302: water phase solution inlet; 303: oil phase channel; 304: water phase main channel; 305: water phase branch channel; 306: dropper channel. DETAILED DESCRIPTION

[0077] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0078] In some specific embodiments of the present invention, the preparation of porous gel microspheres can be carried out according to the following steps:

[0079] (1) Preparation of methacrylated gelatin porous gel microspheres. In this embodiment, droplet microfluidics is used to prepare methacrylated gelatin porous gel microspheres. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 4-10 mL / hour, and the flow rate of the aqueous solution is 1-3 mL / hour, gel-state micro droplets with a particle size range of 150-400 microns can be stably obtained. The aqueous solution contains methacrylated gelatin synthesized from low jelly strength pig skin gelatin, and the solvent of the aqueous solution can be ultrapure water or 1× phosphate buffered saline (PBS). The chip structure of the droplet microfluidic device used in the present invention is as follows: Figure 1 As shown, the microfluidic device chip includes an oil phase solution inlet 301 and an aqueous solution inlet 302, the oil phase solution inlet 301 is connected to the oil phase channel 303, the aqueous solution inlet 302 is connected to the aqueous phase channel composed of the aqueous phase main channel 304 and two sections of aqueous phase branch channels 305, the terminal outlets of the two sections of aqueous phase branch channels 305 merge with the outlet of the oil phase channel 303, and are connected to the inlet of the dropper channel 306; the flow rate of the aqueous phase solution and the oil phase solution refers to the flow rate of the aqueous phase solution inlet 302 and the oil phase solution inlet 301 respectively input into the chip, and is controlled by pushing the syringe with a precision pump. The hose connected to the aqueous phase solution inlet has an outer diameter of 1.52 mm and an inner diameter of 0.51 mm, and the hose connected to the oil phase solution inlet has an outer diameter of 1.52 mm and an inner diameter of 0.51 mm. The diameter of the chip channel for the oil phase solution and the water phase solution is D1, which is equal to 300 microns; the diameter of the dropper channel is D2, which is equal to 550 microns.

[0080] The gel-state microdroplets remain stable at low temperatures of 0-10°C, and can be enriched by centrifugation, sedimentation, etc., and further added with a cleaning solution, such as 0.9% sodium chloride solution, 1× phosphate buffered saline (PBS) or ultrapure water, to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0081] (2) After centrifugation and removal of the supernatant, add a liquid containing a photoinitiator and place it in a refrigerator at 4°C to prevent the gel-state microdroplets from melting, thereby ensuring that the photoinitiator fully penetrates into the gel-state microdroplets. Then transfer the gel-state microdroplet solution to a culture dish and freeze it in a -80°C low-temperature refrigerator. The solvent in the above-mentioned liquid containing a photoinitiator can be 0.9% sodium chloride solution, 1× phosphate buffered saline (PBS) or ultrapure water. The content of the photoinitiator is 0.1-0.5 g / 100 mL based on the volume of the solvent in the liquid containing the photoinitiator.

[0082] (3) The porous gel microspheres are stored in a -80°C low-temperature refrigerator in a frozen form for a period of time. The freezing time affects the growth morphology of ice crystals in the hydrogel (preferably 90 minutes). Then, the culture dish is taken out of the -80°C low-temperature refrigerator, and the gel-state microdroplets are irradiated and solidified using a 405nm wavelength light source. The illumination time ranges from 30 to 180 seconds (preferably 120 seconds) to cross-link the methacrylated gelatin. Finally, wet porous gel microspheres are obtained.

[0083] In some specific embodiments of the present invention, the preparation of cell-loaded porous gel microspheres can be carried out according to the following steps:

[0084] (1) Porous gel microspheres can be loaded with different types of cells, including human mesenchymal stem cells, cancer cells, fibroblasts, endothelial cells, etc., with a cell density of 1×10 3 -1×10 8 / mL (preferably 10 7 The selected cells are cultured in a culture medium conventionally used for the cells in the art, and the culture medium is replaced every 2 days. After the cells are in the logarithmic growth phase, they are digested with 0.25% trypsin (Gibco 25200072), collected by centrifugation, and then re-spinned with a prepared aqueous solution containing methacrylated gelatin. The solvent of the aqueous solution can be ultrapure water or 1× phosphate buffered saline. The content of methacrylated gelatin is 5-12.5 g / 100 mL, preferably 10 g / 100 mL, based on the volume of the solvent in the aqueous solution. The aqueous solution also contains sugars. The content of sugars is 1-10 g / 100 mL, preferably 4-8 g / 100 mL, based on the volume of the solvent in the aqueous solution. The sugars are preferably raffinose, so as to improve the survival rate of the cells.

[0085] (2) Preparation of pre-loaded cell porous gel microspheres by droplet microfluidics. By adjusting the flow rate of the droplet microfluidics device, the flow rate of the oil phase solution is 4-10 mL per hour, and the flow rate of the aqueous solution is 1-3 mL per hour, gel-state micro-droplets with a particle size range of 150-400 microns can be stably obtained. In this embodiment, by fine-tuning the device parameters, gel-state micro-droplets with a particle size of about 300 microns can be stably obtained. The aqueous solution contains methacrylated gelatin synthesized from low jelly strength pigskin gelatin.

[0086] The gel-state microdroplets remain stable at low temperatures of 0-10°C and can be enriched by centrifugation, sedimentation, and further added with a cleaning solution. In order to maintain good cell viability, the culture medium is added as a cleaning solution to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0087] (3) After centrifugation and removal of the supernatant, add a culture medium containing a photoinitiator, and place it in a refrigerator at 4°C to prevent the gel microdroplets from melting, ensuring that LAP fully penetrates into the gel microdroplets. Then transfer the gel microdroplet solution to a culture dish, add the freezing solution, and place it in a low-temperature refrigerator for gradient freezing. The volume ratio of the freezing solution to the gel microdroplets is 1:(1-10); the gradient freezing step includes: standing at -10°C to -25°C for 20-60 minutes, and transferring to an environment of -80°C to -196°C for storage. Based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.1-0.5g / 100mL, preferably 0.15g / 100mL.

[0088] (4) The cell-loaded porous gel microspheres are stored in a -80°C freezer in a frozen form. When used, the microspheres are irradiated and cured with a 405 nm wavelength light source for 30-180 seconds (preferably 120 seconds) to cross-link the methacrylated gelatin. The liquid in the culture dish is then fully replaced with the preheated culture medium and transferred to a new culture dish / culture plate for culture. The culture medium is replaced every 48 hours and fresh culture medium is added.

[0089] In some specific embodiments of the present invention, in the preparation method of the above-mentioned porous gel microspheres, the aqueous phase solution contains methacrylated gelatin synthesized from low jelly strength pig skin gelatin, and the content of methacrylated gelatin is 2-20 g / 100 mL, more preferably 5-12.5 g / 100 mL, based on the volume of the solvent in the aqueous phase solution. Further preferably, the aqueous phase solution contains methacrylated gelatin synthesized from pig skin gelatin with low jelly strength and a blended phase for assisting cell culture. Based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin is 2.5-10 g / 100 mL; the content of the blended phase is 2.5-10 g / 100 mL; the blended phase is selected from one or a combination of two or more of gelatin, hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan and fibrinogen; preferably, the low jelly strength pig skin gelatin has a gel strength of 100-150 Bloomg.

[0090] In some specific embodiments of the present invention, in the preparation method of the above-mentioned porous gel microspheres, the oil phase solution is obtained by mixing an oily material with a surfactant; preferably, the oily material includes one or a combination of two or more of mineral oil, fluorinated oil, and silicone oil; based on the volume of the oil phase solution as 100%, the content of the surfactant is 1-5%, more preferably 4%; the surfactant includes one or a combination of two or more of Span80, Tween20, AEO-3, and FluoSurf.

[0091] In some specific embodiments of the present invention, in the preparation method of the porous gel microspheres, the components of the freezing solution include: 0-15mL / 100mL of DMSO, 0-12g / 100mL of sugar, 0-40mL / 100mL of serum, 0.1-0.5g / 100mL of photoinitiator, and the rest is culture medium; more preferably, based on the total volume of the freezing solution, the components of the freezing solution include: 1-15mL / 100mL of DMSO, 0-12g / 100mL of sugar, 0-40mL / 100mL of serum, 0.1-0.5g / 100mL of photoinitiator, and the rest is culture medium. mL of DMSO, 1-12g / 100mL of sugar, 1-40mL / 100mL of serum, 0.1-0.5g / 100mL of photoinitiator, and the rest is culture medium; further preferably, based on the total volume of the freezing solution, the components of the freezing solution include: 5-15mL / 100mL of DMSO, 4-8g / 100mL of sugar, 20-40mL / 100mL of serum, 0.1-0.3g / 100mL of photoinitiator, and the rest is culture medium. As a typical preferred composition, the components of the freezing solution include: 10mL / 100mL of DMSO, 4g / 100mL of raffinose, 40mL / 100mL of serum, 0.15g / 100mL of photoinitiator, and the rest is culture medium. The sugar includes one or a combination of two or more of lactose, raffinose and melezitose; the photoinitiator includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0092] The technical flow chart of the cryopreservable porous gel microspheres and their preparation method and application provided by the present invention is as follows: Figure 2 shown.

[0093] Material Optimization in the Preparation of Porous Gel Microspheres

[0094] The growth of ice crystals in hydrogel microspheres may be related to the length and three-dimensional conformation of the polymer chain. The length and three-dimensional conformation of the polymer chain of ice crystals affect the free energy of the microspheres. From a thermodynamic point of view, under stronger geometric constraints, the surface-to-volume ratio of the free energy of the microspheres increases, so the surface free energy increases significantly, which increases the free energy barrier for nucleation. Commercial methacrylated gelatin derived from pig skin in the common concentration range (5%-12.5%) used for cell culture cannot produce pores when frozen (not dried and dehydrated). This is because the molecular chain of this methacrylated gelatin is longer and has greater free energy. The elastic stress generated limits the growth of ice crystals, making it impossible to construct interconnected pores inside the microspheres.

[0095] In order to obtain a material that can stably form ice crystals in microspheres, the present invention prepares a variety of methacrylated gelatins with different molecular chain lengths and distributions: ① methacrylated gelatin derived from commercial pig skin; ② methacrylated gelatin derived from bacterially degraded pig skin (extensively degraded, with the shortest molecular chain and low molecular weight); ③ methacrylated gelatin derived from pig skin degraded by collagenase (specifically cleaving peptide bonds and decomposing into smaller peptide fragments and single α chains); ④ methacrylated gelatin derived from fish skin (wide molecular weight distribution, including a variety of chain structures with different molecular weights); ⑤ methacrylated gelatin synthesized from pig skin gelatin with low jelly strength (shorter molecular chains and lower molecular weight).

[0096] ①Methacrylated gelatin microspheres derived from commercial pig skin:

[0097] Synthesis of high jelly strength pig skin methacrylated gelatin. The purchased pig skin gelatin with a gel strength of 300 Bloomg (sigma, V900863-100G) was dissolved in 1× phosphate buffered saline (PBS) to prepare a gelatin solution. The gelatin content was 10 g / 100 mL based on the volume of PBS. The methacrylic anhydride was slowly added dropwise to the gelatin solution and stirred sufficiently to react. The mixture was reacted at 30 to 50°C for a period of time. After the reaction was completed, the unreacted methacrylic anhydride was neutralized with an alkaline solution (such as NaHCO3), and the unreacted substances were removed through a dialysis bag (molecular weight cutoff ~12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000Da). The purified methacrylated gelatin solution was washed and dried to obtain the final high jelly strength pig skin methacrylated gelatin product.

[0098] Preparation of methacrylated gelatin thermosensitive gel balls. In this embodiment, the Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin thermosensitive gel balls. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous solution is 2 mL / hour. Among them, based on the volume of the solvent in the aqueous solution, the content of methacrylated gelatin synthesized from high jelly strength pig skin gelatin is 10 g / 100 mL, and the solvent of the aqueous solution is 1× phosphate buffered saline (PBS). Based on the volume of the oil phase solution as 100%, the content of the surfactant Span is 4%, and the remaining components are mineral oil.

[0099] The gel-state microdroplets remain stable at low temperatures of 0-10°C, can be enriched by centrifugation, and further washed with 1× phosphate buffered saline (PBS). Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0100] After centrifugation, the supernatant was removed and a liquid containing a LAP photoinitiator was added; the solution was placed in a 4°C refrigerator to prevent the gel microdroplets from melting, and the solution was placed for 30 minutes to ensure that the photoinitiator fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish and frozen in a -80°C low-temperature refrigerator. The solvent in the liquid containing the photoinitiator was 1× phosphate buffered saline (PBS), and the content of the photoinitiator was 0.15 g / 100 mL based on its volume.

[0101] The porous gel microspheres were stored in a -80°C freezer in a frozen form for 90 minutes, and then the culture dish was taken out of the -80°C freezer. The gel-state microdroplets were irradiated and cured for 120 seconds using a 405nm wavelength light source to finally obtain commercial pig skin-derived methacrylated gelatin gel microspheres.

[0102] ② Bacterial degradation of pig skin-derived methacrylated gelatin microspheres (extensively degraded, shortest molecular chain and low molecular weight):

[0103] Synthesize bacterially degraded methacrylated gelatin from pig skin. Prepare a gelatin solution by dissolving pig skin gelatin with a gel strength of 300 Bloomg in 1× phosphate buffered saline (PBS). Prepare a gelatin solution with a gelatin content of 10 g / 100 mL based on the volume of PBS. Slowly add methacrylic anhydride to the gelatin solution and stir it thoroughly to allow it to react fully. The mixture is reacted at 30 to 50°C for a period of time. After the reaction is completed, neutralize the unreacted methacrylic anhydride with an alkaline solution (such as NaHCO3) and remove the unreacted substances through a dialysis bag (molecular weight cutoff ~ 12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~ 10000Da). After dialysis, inoculate for 10 5 After the E. coli with a concentration of CFU / mL was degraded for 12 hours, the E. coli was filtered out with a filter bottle, and the purified methacrylated gelatin solution was washed and dried to obtain the final bacterially degraded pig skin methacrylated gelatin product.

[0104] Preparation of methacrylated gelatin thermosensitive gel balls. In this embodiment, the Figure 1The droplet microfluidic device shown is used to prepare methacrylated gelatin thermosensitive gel balls. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous solution is 2 mL / hour. Wherein, based on the volume of the solvent in the aqueous solution, the content of bacterially degraded methacrylated gelatin is 10 g / 100 mL, and the solvent of the aqueous solution is 1× phosphate buffered saline (PBS). Based on the volume of the oil phase solution as 100%, the content of the surfactant Span is 4%, and the remaining components are mineral oil. The gel-state microdroplets remain stable at low temperatures of 0-10°C, are enriched by centrifugation, and are further washed with 1× phosphate buffered saline (PBS) to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0105] After centrifugation and removal of the supernatant, a liquid containing a LAP photoinitiator was added, and the solution was placed in a 4°C refrigerator to prevent the gel microdroplets from melting, and the solution was placed for 30 minutes to ensure that the photoinitiator fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish and frozen in a -80°C low-temperature refrigerator. The solvent in the liquid containing the photoinitiator was 1× phosphate buffered saline (PBS), and the content of the photoinitiator was 0.15 g / 100 mL based on its volume.

[0106] The porous gel microspheres were stored in a -80°C freezer in a frozen form for 90 minutes, and then the culture dish was taken out of the -80°C freezer. The gel-state microdroplets were irradiated and solidified for 120 seconds using a 405 nm wavelength light source, thereby finally obtaining bacterially degraded porcine skin-derived methacrylated gelatin microspheres.

[0107] ③ Collagenase-degraded methacrylated gelatin microspheres derived from pig skin (specifically cleaved peptide bonds, broken down into smaller peptide fragments and single α chains);

[0108] Synthesis of methacrylated gelatin from pig skin with high jelly strength. Pig skin gelatin with a gel strength of 300 Bloomg was dissolved in 1× phosphate buffered saline (PBS) to prepare a gelatin solution. The gelatin content was 10 g / 100 mL based on the volume of PBS. Preparation: Slowly add methacrylic anhydride to the gelatin solution and stir it thoroughly to make it fully react. The mixture was reacted at 30-50°C for a period of time; after the reaction was completed, the unreacted methacrylic anhydride was neutralized with an alkaline solution (such as NaHCO3), and the unreacted substances were removed through a dialysis bag (molecular weight cutoff ~12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000Da); the purified methacrylated gelatin solution was washed and dried to obtain the final methacrylated gelatin product from pig skin with high jelly strength.

[0109] Preparation of methacrylated gelatin thermosensitive gel balls. In this embodiment, the Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin thermosensitive gel balls. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous solution is 2 mL / hour, and gel-state microdroplets with a particle size range of 150-400 microns can be stably obtained. Among them, based on the volume of the solvent in the aqueous solution, the content of methacrylated gelatin synthesized from high jelly strength pig skin gelatin is 10 g / 100 mL, and the solvent of the aqueous solution is 1× phosphate buffered saline (PBS). Based on the volume of the oil phase solution as 100%, the content of the surfactant Span is 4%, and the remaining components are mineral oil. The gel-state microdroplets remain stable at low temperatures of 0-10°C, are enriched by centrifugation, and further added with 1× phosphate buffered saline for washing to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0110] After centrifugation and removal of the supernatant, a liquid containing LAP photoinitiator and collagenase was added, and the mixture was placed in a 4°C refrigerator to prevent the gel-state microdroplets from melting. The mixture was placed for 30 minutes to ensure that the photoinitiator fully penetrated into the gel-state microdroplets, and the collagenase partially degraded the methacrylated gelatin from pig skin with high jelly strength to prepare the microgel. The gel-state microdroplet solution was then transferred to a culture dish and frozen in a -80°C low-temperature refrigerator. The solvent in the liquid containing the photoinitiator was 1× phosphate buffered saline (PBS), and the content of the photoinitiator was 0.15 g / 100 mL based on its volume; the content of the collagenase was 5 mg / mL based on the volume of the liquid containing the photoinitiator. The porous gel microspheres were stored in a -80°C low-temperature refrigerator in a frozen form for 90 minutes, and then the culture dish was taken out of the -80°C low-temperature refrigerator. The gel-state microdroplets were irradiated and solidified for 120 seconds using a 405nm wavelength light source, and finally methacrylated gelatin gel microspheres derived from collagenase-degraded pig skin were obtained.

[0111] ④ Methacrylated gelatin from fish skin (broad molecular weight distribution, including a variety of chain structures with different molecular weights):

[0112] Synthesis of fish skin methacrylated gelatin. Fish skin gelatin (Sigma, G7041) was dissolved in 1× phosphate buffered saline (PBS) to prepare a gelatin solution. The gelatin content was 10 g / 100 mL based on the volume of PBS. Methacrylic anhydride was slowly added dropwise to the gelatin solution and stirred thoroughly to allow it to react fully. The mixture was reacted at 30-50°C for a period of time. After the reaction was completed, the unreacted methacrylic anhydride was neutralized with an alkaline solution (such as NaHCO3), and the unreacted substances were removed through a dialysis bag (molecular weight cutoff ~ 12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~ 10000Da). The purified methacrylated gelatin solution was washed and dried to obtain the final fish skin methacrylated gelatin product.

[0113] In this embodiment, the use of Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin thermosensitive gel balls. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous solution is 2 mL / hour, and gel-state microdroplets with a particle size range of 150-400 microns can be stably obtained. Wherein, based on the volume of the solvent in the aqueous solution, the content of methacrylated gelatin synthesized from high jelly strength pigskin gelatin is 10 g / 100 mL, and the solvent of the aqueous solution is 1× phosphate buffered saline (PBS). Based on the volume of the oil phase solution as 100%, the content of the surfactant Span is 4%, and the remaining components are mineral oil. The gel-state microdroplets remain stable at low temperatures of 0-10°C, are enriched by centrifugation, and further added with 1× phosphate buffered saline (PBS) for washing to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0114] After centrifugation and removal of the supernatant, a liquid containing a photoinitiator is added, and the solution is placed in a 4°C refrigerator to prevent the gel microdroplets from melting, and the solution is placed for 30 minutes to ensure that the photoinitiator fully penetrates into the gel microdroplets. The gel microdroplet solution is then transferred to a culture dish and placed in a -80°C low-temperature refrigerator for freezing. The solvent in the liquid containing the photoinitiator is 1× phosphate buffered saline (PBS), and the content of the photoinitiator is 0.15 g / 100 mL based on its volume.

[0115] After the porous gel microspheres were stored in a -80°C low-temperature refrigerator in a frozen form for 90 minutes, the culture dish was taken out of the -80°C low-temperature refrigerator, and the gel-state microdroplets were irradiated and cured for 120 seconds using a 405nm wavelength light source to cross-link the methacrylated gelatin, thereby finally obtaining the methacrylated gelatin derived from fish skin.

[0116] ⑤Methacrylated gelatin microspheres synthesized from pig skin gelatin with low jelly strength (shorter molecular chains and lower molecular weight):

[0117] Synthesis of low jelly strength pig skin methacrylated gelatin. Pig skin gelatin (Aladdin, G108398) with a gel strength of 100 Bloom g was dissolved in 1× phosphate buffered saline (PBS) to prepare a gelatin solution. The gelatin content was 10 g / 100 mL based on the volume of PBS. Methacrylic anhydride was slowly added dropwise to the gelatin solution and stirred sufficiently to react. The mixture was reacted at 40°C for a period of time. After the reaction was completed, the unreacted methacrylic anhydride was neutralized with an alkaline solution (such as NaHCO3), and the unreacted substances were removed through a dialysis bag (molecular weight cutoff ~12000Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000Da). The purified methacrylated gelatin solution was washed and dried to obtain the final low jelly strength pig skin methacrylated gelatin product.

[0118] Preparation of methacrylated gelatin thermosensitive gel balls. In this embodiment, the Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin thermosensitive gel balls. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous solution is 2 mL / hour. Wherein, based on the volume of the solvent in the aqueous solution, the content of methacrylated gelatin synthesized from low jelly strength pigskin gelatin is 10 g / 100 mL, and the solvent of the aqueous solution is 1× phosphate buffered saline (PBS). Based on the volume of the oil phase solution as 100%, the content of the surfactant Span is 4%, and the remaining components are mineral oil. The gel-state microdroplets remain stable at low temperatures of 0-10°C, are enriched by centrifugation, and are further washed with 1× phosphate buffered saline (PBS) to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0119] After centrifugation and removal of the supernatant, a liquid containing a LAP photoinitiator was added, and the solution was placed in a 4°C refrigerator to prevent the gel microdroplets from melting, and the solution was placed for 30 minutes to ensure that the photoinitiator fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish and frozen in a -80°C low-temperature refrigerator. The solvent in the liquid containing the photoinitiator was 1× phosphate buffered saline (PBS), and the content of the photoinitiator was 0.15 g / 100 mL based on its volume.

[0120] The porous gel microspheres were stored in a -80°C low-temperature refrigerator in a frozen form for 90 minutes, then the culture dish was taken out of the -80°C low-temperature refrigerator, and the gel-state microdroplets were irradiated and solidified for 120 seconds using a 405nm wavelength light source, ultimately obtaining methacrylated gelatin gel microspheres synthesized from pig skin gelatin with low jelly strength, i.e., wet porous gel microspheres.

[0121] Qualitative and quantitative data of frozen porosity of microspheres prepared from different materials are shown in Figure 2. Figure 3 As shown. If the commonly used GelMA is used to prepare gel microspheres, pores cannot be obtained after freezing and photocrosslinking. Analysis shows that it may be because the ice crystals melt quickly during the photocrosslinking process and it is difficult to maintain the pore shape. Through the above test scheme, the present invention finally obtains a scheme for optimizing the microsphere preparation material, that is, using GelMA synthesized from low jelly strength pig skin gelatin to prepare porous gel microspheres, which can simply form relatively uniform and abundant interconnected micropores with good batch consistency.

[0122] Optimization of cryopreservation system in preparation of porous gel microspheres

[0123] When using gel microspheres to freeze cells, ice crystals formed during the freezing process can damage the cells. Therefore, in order to make the gel microspheres compatible with cell freezing, the present invention has explored a variety of freezing schemes, including the ratio of DMSO, the ratio of adding sugars, etc., and finally obtained the optimized results.

[0124] Optimization of gel microsphere material formulation for porous cryogel microspheres

[0125] (1) In this embodiment, human mesenchymal stem cells are selected as preload cells. Human mesenchymal stem cells are cultured in a special culture medium, and the culture medium is replaced every 2 days. After the cells are in the logarithmic growth phase, they are digested with 0.25% trypsin, collected by centrifugation, and then re-spinned with a prepared aqueous solution containing methacrylated gelatin synthesized from low jelly strength pig skin gelatin. The cell density is 10 7 / mL. Before adding cells, the methacrylated gelatin solution needs to be sterilized by filtering through a membrane (0.22 μm pores). The solvent of the aqueous solution is 1× phosphate buffered saline, and the content of methacrylated gelatin is 10 g / 100 mL based on the volume of the solvent in the aqueous solution. The aqueous solution also contains sugars, and the content of sugars is 4 g / 100 mL based on the volume of the solvent in the aqueous solution. The sugars are lactose, raffinose or melezitose.

[0126] (2) The flow rate was controlled by adjusting the parameters of the droplet microfluidic device. The flow rate of the oil phase solution was 6 mL per hour, and the flow rate of the water phase solution was 2 mL per hour, thereby obtaining gel-state microdroplets.

[0127] The gel-state microdroplets can be enriched by centrifugation, sedimentation and other methods, and further added with a washing solution. In order to maintain good cell viability, culture medium is added as a washing solution to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0128] (3) After centrifugation, remove the supernatant of the gel-state microdroplets, add the culture medium containing the photoinitiator, and place it in a refrigerator at 4°C to prevent the gel-state microdroplets from melting, so as to ensure that LAP fully penetrates into the gel-state microdroplets. Then transfer the gel-state microdroplet solution to a culture dish and add the optimized freezing solution. Then place it in a low-temperature refrigerator for gradient freezing, place it at -20°C for 20 minutes, and then transfer it to a -80°C refrigerator for long-term storage; wherein, based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.15g / 100mL. The diameter of the culture dish during freezing is 35mm, and the volume ratio of the gel-state microdroplets and the freezing solution in the culture dish during freezing is 1:2.

[0129] (4) The cell-loaded porous gel microdroplets are stored in a -80°C low-temperature refrigerator in a frozen form. When used, the gel microdroplets are irradiated and solidified with a 405nm wavelength light source for 120 seconds to cross-link methacrylated gelatin to obtain porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells; wherein, the ambient temperature and time for cross-linking are room temperature for 2 minutes, but it is necessary to ensure that it is always kept below 4°C from the time it is taken out of the low-temperature refrigerator to before cross-linking. At the same time, in order to increase the light reflection during cross-linking, tin foil needs to be added to the inner wall of the culture dish, and the process requires sterilization. Then, the liquid in the culture dish is fully replaced with the preheated culture medium, and the culture dish / culture plate is transferred to a new culture dish / culture well plate for culture. The culture medium is replaced every 48 hours, and fresh culture medium is added for cell culture.

[0130] In this embodiment, the hydrogel component in direct contact with cells, i.e., the aqueous phase solution, is optimized. Lactose, raffinose, or melezitose is added to the aqueous phase solution for preparing microspheres to optimize the gel microsphere material formula of porous cryogel microspheres, i.e., different sugars are added to the microsphere material GelMA, such as Figure 4 As shown, from left to right are raffinose (Raf), lactose (Lac), and melezitose (Mel). The results at this time have not been optimized for the freezing solution. Based on the total volume of the freezing solution, the components of the freezing solution include: 0-15mL / 100mL of DMSO and the rest is culture medium.

[0131] Figure 4 The results showed that adding raffinose to the aqueous solution for preparing microspheres can maintain the viability of the cells in this system to the greatest extent. Therefore, raffinose was used to optimize the subsequent freezing solution (i.e., the peripheral cryoprotective solution of microspheres).

[0132] Optimization of cryogel microsphere freezing solution for porous cryogel microspheres

[0133] (1) In this embodiment, human mesenchymal stem cells and human lung cancer cells are selected as preload cells. Human mesenchymal stem cells and human lung cancer cells are cultured in a special culture medium, and the culture medium is replaced every 2 days. After the cells are in the logarithmic growth phase, they are digested with 0.25% trypsin, collected by centrifugation, and re-spinned with an aqueous phase solution containing methacrylated gelatin synthesized from low jelly strength pig skin gelatin, and used as the aqueous phase for microfluidic experiments. The cell density is 10 7 Before adding cells, the methacrylated gelatin solution needs to be sterilized by filtering through a filter membrane (0.22 μm pores). The solvent of the aqueous phase solution is 1× phosphate buffered saline, and the content of methacrylated gelatin is 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution. The aqueous phase solution also contains raffinose, and the content of raffinose is 4 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0134] (2) The flow rate was controlled by adjusting the parameters of the droplet microfluidic device. The flow rate of the oil phase solution was 6 mL per hour, and the flow rate of the water phase solution was 2 mL per hour, thereby obtaining gel-state microdroplets.

[0135] The gel-state microdroplets can be enriched by centrifugation, sedimentation and other methods, and further added with a washing solution. In order to maintain good cell viability, culture medium is added as a washing solution to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0136] (3) After centrifugation, remove the supernatant of the gel-state microdroplets, add the culture medium containing the photoinitiator, and place it in a refrigerator at 4°C to prevent the gel-state microdroplets from melting, so as to ensure that LAP fully penetrates into the gel-state microdroplets. Then, transfer the gel-state microdroplet solution to a culture dish, add the optimized freezing solution, and then place it in a low-temperature refrigerator for gradient freezing, place it at -20°C for 20 minutes, and then transfer it to a -80°C refrigerator for long-term storage; wherein, based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.15g / 100mL. The diameter of the culture dish during freezing is 35mm, and the volume ratio of the gel-state microdroplets and the freezing solution in the culture dish during freezing is 1:2.

[0137] (4) The cell-loaded porous gel microdroplets are stored in a -80°C low-temperature refrigerator in a frozen form. When used, the gel microdroplets are irradiated and solidified with a 405nm wavelength light source for 120 seconds to cross-link methacrylated gelatin to obtain porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells; wherein, the ambient temperature and time for cross-linking are room temperature for 2 minutes, but it is necessary to ensure that it is always kept below 4°C from the time it is taken out of the low-temperature refrigerator to before cross-linking. At the same time, in order to increase the light reflection during cross-linking, tin foil needs to be added to the inner wall of the culture dish, and the process requires sterilization. Then, the liquid in the culture dish is fully replaced with the preheated culture medium, and the culture dish / culture plate is transferred to a new culture dish / culture well plate for culture. The culture medium is replaced every 48 hours, and fresh culture medium is added for cell culture.

[0138] like Figure 5 As shown in a, based on the total volume of the cryopreservation solution, the components of the cryopreservation solution include: 0, 5, 10, 15 mL / 100 mL of DMSO, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the rest is culture medium. The crtl group is not cryopreserved and directly cross-linked.

[0139] like Figure 5 As shown in b in the figure, based on the total volume of the freezing solution, the components of the freezing solution include: 0, 5, 10, 15 mL / 100 mL of DMSO, 0, 4, 8, 12 g / 100 mL of sugar, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the rest is culture medium. The freezing solution formula is as follows Figure 5 As shown in b.

[0140] like Figure 5 The results showed that the best combination of balanced sugar and DMSO concentrations in the cryopreservation solution was 10% DMSO + 4% raffinose, that is, based on the total volume of the cryopreservation solution, the composition of the cryopreservation solution included 10mL / 100mL DMSO, 4g / 100mL sugar, 40mL / 100mL serum, 0.15g / 100mL photoinitiator, and the rest was culture medium. The cell survival rate of the prepared pre-loaded cell porous gel microspheres (on the first day after recovery) was increased from 35% to 70%, and reached 90% after seven days of culture.

[0141] Process optimization in the preparation of porous gel microspheres

[0142] In the preparation of porous gel microspheres, even after material optimization, it is difficult to obtain frozen porous gel microspheres with high yield before process optimization, and no pores ( Figure 6 a) or the pores appear on the surface rather than inside ( Figure 6This is the case of b). Because tiny ice crystals melt quickly, and the gel-state microdroplets are in a solid state when frozen, the photocrosslinking reaction rate is greatly reduced in the solid state. At the same time, it is also difficult for light to pass through the gel-state microdroplets to make the area in the dish uniform.

[0143] In this embodiment, the use of Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin porous gel microspheres.

[0144] By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous phase solution is 1 mL / hour. In this embodiment, by fine-tuning the device parameters, a gel-state micro-droplet with a particle size of about 230 microns is stably obtained. The aqueous phase solution contains methacrylated gelatin synthesized from low jelly strength pigskin gelatin, and the solvent of the aqueous phase solution is 1× phosphate buffered saline. The content of methacrylated gelatin is 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0145] The gel-state microdroplets can be enriched by centrifugation, sedimentation and the like, and a cleaning solution, such as 0.9% sodium chloride solution, 1× phosphate buffered saline solution or ultrapure water, can be further added to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0146] After centrifugation, remove the supernatant of the gel-state microdroplets, and add a solution containing the photoinitiator LAP. The solution can use 0.9% sodium chloride solution, 1× phosphate buffered saline solution or ultrapure water as a solvent. Let it stand in a 4°C refrigerator to prevent the gel-state microdroplets from melting and ensure that LAP fully penetrates into the gel-state microdroplets. Then transfer the gel-state microdroplet solution to a culture dish and freeze it in a -80°C low-temperature refrigerator. Wherein, the content of the photoinitiator is 0.15g / 100mL based on the volume of the solvent in the liquid containing the photoinitiator. Before the process optimization, no tin foil was added to the inner wall of the culture dish, the diameter of the culture dish was 60mm, and the volume ratio of the gel-state microdroplets to the freezing solution in the culture dish was 1:4 when frozen. After the process optimization, tin foil was added to the inner wall of the culture dish, the diameter of the culture dish was 35mm, and the volume ratio of the gel-state microdroplets to the freezing solution in the culture dish was 1:2 when frozen.

[0147] The cell-free gel microdroplets were stored in a -80°C low-temperature refrigerator in a frozen form for 90 minutes, then taken out of the -80°C low-temperature refrigerator and placed in a 4°C refrigerator for irradiation and curing with a 405nm wavelength light source for 2 minutes, or directly irradiated and cured with a 405nm wavelength light source in a -80°C low-temperature refrigerator for 2 minutes. Finally, porous gel microspheres of different morphologies were obtained.

[0148] The present invention can obtain an optimized high yield by optimizing the size of the culture dish during freezing (preferably 35 mm), the ratio of gel-state microdroplets to freezing solution in the culture dish during freezing (preferably 1:2), the environmental temperature and time of cross-linking after freezing (preferably 4° C. and 2 minutes when there are no cells, and 2 minutes at room temperature when there are cells, but it is necessary to ensure that it is always kept below 4° C. from the time it is taken out of the low-temperature refrigerator to before cross-linking), and increasing the light reflection during cross-linking (preferably adding tin foil to the inner wall of the culture dish, and paying attention to the sterilization operation), such as Figure 6 As shown in (c), the optimized product has more uniform and moderately sized pores and better batch consistency.

[0149] In summary, the present invention obtains cryogel microspheres with stable pores by testing multiple materials and optimizing processes through complex scheme design.

[0150] Example 1 Preparation of porous gel microspheres

[0151] (1) Preparation of methacrylated gelatin porous microspheres. Figure 1 The droplet microfluidic device shown is used to prepare methacrylated gelatin porous gel microspheres. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 6 mL / hour, and the flow rate of the aqueous phase solution is 1 mL / hour. In this embodiment, by fine-tuning the device parameters, gel-state microdroplets with a particle size of about 230 microns are stably obtained. The solvent of the aqueous phase solution is 1× phosphate buffered saline solution, and the content of methacrylated gelatin synthesized from low jelly strength pig skin gelatin is 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0152] The gel-state microdroplets can be enriched by centrifugation, sedimentation and the like, and a cleaning solution, such as 0.9% sodium chloride solution, 1× phosphate buffered saline solution or ultrapure water, can be further added to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-cleaning steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0153] (2) After centrifugation, remove the supernatant of the gel-state microdroplets and add a solution containing the photoinitiator LAP. The solution can use 0.9% sodium chloride solution, 1× phosphate buffered saline solution or ultrapure water as the solvent. Place it in a refrigerator at 4°C to prevent the gel-state microdroplets from melting and ensure that LAP fully penetrates into the gel-state microdroplets. Then transfer the gel-state microdroplet solution to a culture dish and freeze it in a -80°C low-temperature refrigerator. Based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.15g / 100mL.

[0154] (3) The gel-state microdroplets were stored in a -80°C low-temperature refrigerator in a frozen form for 90 minutes, and then taken out of the -80°C low-temperature refrigerator. The gel-state microdroplets were irradiated and solidified using a 405 nm wavelength light source for 120 seconds to cross-link the methacrylated gelatin. Finally, porous gel microspheres were obtained. The microsphere morphology is as follows Figure 7 As shown in the wet porous frozen microgel.

[0155] Example 2 Preparation of porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells

[0156] This embodiment provides a method for preparing porous gel microspheres preloaded with human mesenchymal stem cells, and the specific steps are as follows:

[0157] (1) In this embodiment, human mesenchymal stem cells and human lung cancer cells were selected as preloaded cells. Human mesenchymal stem cells and human lung cancer cells were cultured in the culture medium of the cells conventionally used in the art. The culture medium was replaced every 2 days. After the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin, collected by centrifugation, and then re-spinned with the prepared methacrylated gelatin synthesized from low jelly strength pigskin gelatin as the aqueous phase solution. The cell density was 10 7 Before adding cells, the methacrylated gelatin solution needs to be sterilized by filtering through a filter membrane (0.22 μm pores). The solvent of the aqueous phase solution is 1× phosphate buffered saline, and the content of methacrylated gelatin is 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution. The aqueous phase solution also contains raffinose, and the content of raffinose is 4 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0158] (2) The flow rate is controlled by adjusting the parameters of the droplet microfluidic device. The flow rate of the oil phase solution is 4-10 mL per hour, and the flow rate of the water phase solution is 1-3 mL per hour. Stable gel-state microdroplets with a particle size of about 230 microns are obtained. The flow rate selection is related to the concentration of the water phase material. Calculated by the volume of the solvent in the aqueous phase solution, when the content of methacrylated gelatin is 5 g / 100 mL and the content of raffinose is 4 g / 100 mL, the flow rate of the oil phase solution is 8 mL per hour, and the flow rate of the aqueous phase solution is 2.5 mL per hour; calculated by the volume of the solvent in the aqueous phase solution, when the content of methacrylated gelatin is 10 g / 100 mL and the content of raffinose is 4 g / 100 mL, the flow rate of the oil phase solution is 6 mL per hour, and the flow rate of the aqueous phase solution is 2 mL per hour; calculated by the volume of the solvent in the aqueous phase solution, when the content of methacrylated gelatin is 12.5 g / 100 mL and the content of raffinose is 4 g / 100 mL, the flow rate of the oil phase solution is 9 mL per hour, and the flow rate of the aqueous phase solution is 2.5 mL per hour.

[0159] The gel-state microdroplets can be enriched by centrifugation, sedimentation and other methods, and further added with a washing solution. In order to maintain good cell viability, culture medium is added as a washing solution to remove the oil phase components on the surface of the gel-state microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect the subsequent steps.

[0160] (3) After centrifugation, remove the supernatant of the gel microdroplets, add the culture medium containing the photoinitiator LAP, and place it in a refrigerator at 4°C to prevent the gel microdroplets from melting, so as to ensure that LAP fully penetrates into the gel microdroplets. Then transfer the gel microdroplet solution to a culture dish, add the optimized freezing solution, and based on the total volume of the freezing solution, the composition of the freezing solution includes 10mL / 100mL of DMSO, 4g / 100mL of sugars, 40mL / 100mL of serum, 0.15g / 100mL of photoinitiator, and the rest is culture medium. Then put it in a low-temperature refrigerator for gradient freezing, place it at -20°C for 20 minutes, and then transfer it to a -80°C refrigerator for long-term storage; wherein, based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.15g / 100mL. The diameter of the culture dish during freezing is 35mm, and the volume ratio of the gel microdroplets and the freezing solution in the culture dish during freezing is 1:2.

[0161] (4) The cell-loaded porous gel microdroplets are stored in a -80°C low-temperature refrigerator in a frozen form. When used, the gel microdroplets are irradiated and solidified with a 405nm wavelength light source for 120 seconds to cross-link methacrylated gelatin to obtain porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells; wherein, the ambient temperature and time for cross-linking are room temperature for 2 minutes, but it is necessary to ensure that it is always kept below 4°C from the time it is taken out of the low-temperature refrigerator to before cross-linking. At the same time, in order to increase the light reflection during cross-linking, tin foil needs to be added to the inner wall of the culture dish, and the process requires sterilization. Then, the liquid in the culture dish is fully replaced with the preheated culture medium, and the culture dish / culture plate is transferred to a new culture dish / culture well plate for culture. The culture medium is replaced every 48 hours, and fresh culture medium is added for cell culture.

[0162] Comparative Example 1: Preparation of traditional methacrylated gelatin microspheres.

[0163] This comparative example provides a traditional methacrylated gelatin microsphere preparation method, and the specific steps are as follows:

[0164] Steps (1)-(2): Same as Example 1.

[0165] (3) After centrifugation, the supernatant of the gel-state microdroplets is removed, and a culture medium containing a photoinitiator LAP is added, and the culture medium is placed in a refrigerator at 4° C. to prevent the gel-state microdroplets from melting, and to ensure that LAP fully penetrates into the gel-state microdroplets. The content of the photoinitiator is 0.15 g / 100 mL, based on the volume of the solvent in the liquid containing the photoinitiator.

[0166] (4) Then, the gel-state microdroplet solution was transferred to a culture dish and irradiated with a 405 nm wavelength light source to solidify the gel-state microdroplet for 120 seconds to cross-link the methacrylated gelatin. Figure 7 As shown in the traditional microgels.

[0167] Comparative Example 2 Preparation of traditional methacrylated gelatin preloaded cell gel microspheres.

[0168] This comparative example provides a preparation method of traditional methacrylated gelatin gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells, and the specific steps are as follows:

[0169] Steps (1)-(2): Same as Example 2.

[0170] (3) After centrifugation, the supernatant of the gel-state microdroplets is removed, and a culture medium containing a photoinitiator LAP is added, and the culture medium is placed in a refrigerator at 4° C. to prevent the gel-state microdroplets from melting, and to ensure that LAP fully penetrates into the gel-state microdroplets. The content of the photoinitiator is 0.15 g / 100 mL, based on the volume of the solvent in the liquid containing the photoinitiator.

[0171] (4) The gel-state micro-droplet solution is then transferred to a culture dish and irradiated with a 405 nm wavelength light source for 120 seconds to cross-link the methacrylated gelatin. The liquid in the culture dish is then fully replaced with the preheated culture medium and transferred to a culture dish / culture well plate for culture. The culture medium is replaced every 48 hours, and fresh culture medium is added for cell culture.

[0172] Culture of porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells and evaluation of cell growth status

[0173] The gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells prepared in Example 2 and Comparative Example 2 were added to fresh culture medium for culture, the culture medium was replaced every 48 hours, the cell status in the structure was checked regularly, and the cell growth status was detected and evaluated.

[0174] like Figure 8 As shown, Figure 8 a in the figure is a staining image of traditional microgels and wet frozen porous gel microspheres that support the culture of various cells. Figure 8Figure b is a cross-sectional view of wet frozen porous gel microspheres loaded with human lung cancer cells. Figure 8 As can be seen from a in the figure, it is difficult for cells to stretch inside the traditional gel microspheres, but in the porous gel microsphere system of the present invention, cells can grow well in three dimensions, human mesenchymal stem cells can stretch and spread, and human lung cancer cells can aggregate into clusters (such as Figure 8 The porous gel microspheres provided by the present invention have a more bionic morphology and are conducive to exerting cell functions. Figure 8 As shown in a, after the porous gel microspheres preloaded with human mesenchymal stem cells in Example 1 were cryopreserved for one month, the cells still maintained good vitality after recovery culture.

[0175] like Fig. 9 As shown in the figure, the results of Alizarin red staining of wet frozen porous gel microspheres after one week of culture and two weeks of osteogenic differentiation. The preloaded human mesenchymal stem cell porous gel microspheres prepared in Example 2 still maintained osteogenic differentiation ability after one month of cryopreservation, and the results of Alizarin red staining showed a large number of dense calcium nodules (solid particles in the dark area represent calcium nodules). This result indicates that the wet frozen porous gel microsphere system of the present invention is more conducive to osteogenic differentiation.

[0176] The method of the present invention has been optimized and has been proven to support the cryopreservation of a variety of cells, including stem cells and tumor cells, to maintain their vitality and function. These cells have good growth morphology in the porous gel microspheres of the present invention. Mesenchymal stem cells can stretch and spread, and tumor cells can aggregate into clusters with good growth potential.

[0177] Detection of cell survival and proliferation rate after long-term cryopreservation of porous gel microspheres preloaded with cells

[0178] It is difficult to achieve cryopreservation of in situ cell-loaded porous gel microspheres. The conventional methacrylated gelatin pre-loaded cell porous gel microspheres prepared in Comparative Example 2 and the porous gel microspheres pre-loaded with human mesenchymal stem cells and human lung cancer cells prepared in Example 2 by optimizing the materials, cryopreservation solution formula and cryopreservation process were tested.

[0179] The survival rate and proliferation rate of the wet porous cryogel microspheres carrying cells during long-term freezing (determined by CCK8 method), based on the volume of the solvent in the aqueous solution, Fig.10 (a), (b), and (c) are respectively MSC-loaded human mesenchymal stem cell porous microspheres prepared with methacrylated gelatin contents of 5 g / 100 mL, 10 g / 100 mL, and 12.5 g / 100 mL (i.e., 5, 10, and 12.5% ​​material concentrations). Fig.10 (d) is a porous microsphere loaded with A549 human lung cancer cells prepared with methacrylated gelatin having a content of 5 g / 100 mL and 10 g / 100 mL.

[0180] like Fig.10 As shown in (a) of Figure 2, the survival rate and proliferation rate of the sample with a methacrylated gelatin content of 5 g / 100 mL remained basically unchanged after one month of freezing. However, after three months of freezing, the survival rate dropped by about 10% and the proliferation rate decreased significantly. On the seventh day, the proliferation rate was only half of that of the group frozen for one month, which was equivalent to the non-frozen control group. Fig.10 As shown in (b), when the material concentration was 10%, the cell-loaded porous gel microsphere system showed the best long-term cryopreservation ability, maintaining a 74% survival rate after three months of freezing, and recovering to 90% after seven days of culture, and the proliferation rate was close to 500% of the first day, which was 1.5 times that of the non-frozen group. Fig.10 As shown in (c), when the material concentration is 12.5%, the decrease in water content may have an adverse effect on the cells, resulting in increased cell rigidity and stress. After three months of cryopreservation, the survival rate on the seventh day was 83%, and the proliferation rate was about 2.5 times.

[0181] The 10% MSC-loaded human mesenchymal stem cell porous gel is most suitable for long-term cryopreservation, which greatly reduces the damage to cells caused by cryopreservation with medium, especially for stem cells. The value of cryopreservation is to get rid of the time urgency of current preparation and current use, and can be used in different time and space. For patients, it can be extracted once, cryopreserved for a long time, and used on demand to avoid repeated trauma.

[0182] like Fig.10 As shown in (d), the material concentration in the figure is the GelMA material concentration of the microspheres synthesized from low jelly strength pig skin gelatin, and the A549 cells used are human lung cancer cells (tumor cells). After freezing for two months, the survival rate of A549 human lung cancer cells (tumor cells) was 70%, which is in line with the usual level in the field. After seven days of culture, the survival rate was 85%, which is similar to the traditional microgels loaded with A549, exceeding 90%. It shows that the cell-carrying porous gel microsphere system of the present invention is also applicable to other cells.

[0183] Application of preloaded cell porous gel microspheres in in vivo injection

[0184] (1) Preparation of bio-ink. Cell-loaded porous gel microspheres can be selected for stem cell therapy and the like in combination with the purpose of treatment. The prepared traditional cell-loaded microgel (Comparative Example 2) and porous gel microsphere solution (Example 2) were used for in vivo injection for testing. The microspheres used were microspheres before being cured by ultraviolet irradiation, and they remained in a gel state at a relatively low temperature (0-10°C). A 70μm cell sieve was placed on an uncovered 50mL centrifuge tube, and the cell-loaded gel microsphere solution was then transferred to the cell sieve. The liquid outside the microspheres was removed by low-speed centrifugation at 50×g for 2 minutes to enrich the microspheres to obtain bio-ink.

[0185] (2) Extrusion injection. The bio-ink was loaded into a syringe, an equal volume of physiological saline was added, and the microspheres were delivered subcutaneously to the back of four-week-old male SD rats through a needle. The wet frozen porous gel microsphere ink provided by the present invention has good extrusion properties, such as Fig.11 shown.

[0186] In rats, conventional microgels were injected subcutaneously and wet frozen porous gel microspheres were cultured for two weeks ( Fig.12 The white dotted line in the figure indicates the undegraded material). Fig.12 As shown, the wet frozen porous gel microspheres provided by the present invention formed collagen fibers and uniform tissue ingrowth after two weeks. The subcutaneous injection experiment of rats proved good biocompatibility and accelerated blood vessel and tissue ingrowth.

[0187] Application of preloaded cell porous gel microspheres combined with biological 3D printing in the construction of porous scaffolds

[0188] (1) Preparation of bio-ink. The prepared cell-loaded porous gel microsphere solution was centrifuged through a cell sieve to enrich the microspheres as bio-ink. A 70 μm cell sieve was placed on an uncovered 50 mL centrifuge tube, and then the cell-loaded porous gel microsphere solution was transferred to the cell sieve. The solution was centrifuged at a low speed of 50 × g for 2 minutes to remove the liquid outside the microspheres, and the microspheres were enriched to obtain bio-ink.

[0189] (2) Bio-3D printing. The enriched microspheres are loaded as bio-ink onto the nozzle of an extrusion printer for printing. After printing, the hydrogel structure is selectively irradiated with a 405nm wavelength light source, immersed in culture medium and cultured in a 37°C incubator. Fig.13 As shown, the pores of the wet frozen porous gel microspheres provided by the present invention are highly interconnected, and the hydrogel structure has a porous structure, which supports the rapid transport of nutrients.

Claims

1. A method for preparing porous gel microspheres that can be frozen, wherein: The method comprises: The aqueous phase solution and the oil phase solution are prepared into gel-state microdroplets; wherein the aqueous phase solution contains methacrylated gelatin synthesized from pig skin gelatin with low jelly strength; The gel-state micro-droplets are soaked in a liquid containing a photoinitiator, and after being fully infiltrated, they are frozen so that the water inside the gel-state micro-droplets forms ice crystals; Ultraviolet light is used to cross-link and solidify the gel-state microdroplets that form ice crystals, thereby obtaining porous gel microspheres that can be frozen.

2. The method according to claim 1, wherein: The method comprises: The aqueous solution contains cells to be preloaded; The freezing is to transfer the gel-state microdroplets into a freezing solution to perform gradient freezing.

3. The method according to claim 1 or 2, wherein: The low jelly strength pigskin gelatin has a jelly strength of 100-150 Bloomg.

4. The method according to claim 1 or 2, wherein: Calculated based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin is 2-20 g / 100 mL.

5. The method according to claim 1 or 2, wherein: The aqueous phase solution contains methacrylated gelatin synthesized from pigskin gelatin with low jelly strength and a blended phase for assisting cell culture; And / or, based on the volume of the solvent in the aqueous phase solution, the content of the methacrylated gelatin is 2.5-10 g / 100 mL.

6. The method according to claim 5, wherein: The content of the blended phase is 2.5-10 g / 100 mL based on the volume of the solvent in the aqueous phase solution; And / or, the blended phase is selected from one or a combination of two or more of gelatin, hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan and fibrinogen.

7. The method according to claim 2, wherein: The aqueous phase solution further contains sugars, and the content of the sugars is 1-10 g / 100 mL based on the volume of the solvent in the aqueous phase solution; And / or, the sugars include one or a combination of two or more of lactose, raffinose and melezitose.

8. The method according to claim 2, wherein: The cell density in the aqueous solution is 1×10 3 -1×10 8 Pieces / mL; And / or, the cells include one or a combination of two or more of human mesenchymal stem cells, cancer cells, fibroblasts and endothelial cells.

9. The method according to claim 1 or 2, wherein: The oil phase solution is obtained by mixing an oily material with a surfactant; And / or, the oily material includes one or a combination of two or more of mineral oil, fluorinated oil, and silicone oil.

10. The method according to claim 9, wherein: Based on the volume of the oil phase solution being 100%, the content of the surfactant is 1-5%; And / or, the surfactant includes one or a combination of two or more of Span80, Tween20, AEO-3, and FluoSurf.

11. The method according to claim 1 or 2, wherein: The method for preparing the aqueous phase solution and the oil phase solution into gel-state micro-droplets includes a droplet microfluidics method, an oil-water emulsion method, a complex coacervation method or an electrospray method.

12. The method according to claim 11, wherein: The specific steps of the droplet microfluidics method include: adding the aqueous solution and the oil phase solution into a microfluidics device, adjusting the flow rates of the aqueous solution and the oil phase solution respectively, and mixing them; And / or, the flow rate of the aqueous solution is 1-3 mL / h, and the flow rate of the oil solution is 4-10 mL / h.

13. The method according to claim 1 or 2, wherein: The photoinitiator includes phenyl (2,4,6-trimethylbenzoyl) lithium phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone; And / or, based on the volume of the solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.1-0.5 g / 100 mL.

14. The method according to claim 2, wherein: The volume ratio of the freezing solution to the gel-state microdroplets is 1:(1-10); And / or, the volume ratio of the freezing solution to the gel-state microdroplets is 1:(1-3).

15. The method according to claim 2, wherein: The step of gradient freezing comprises: standing at -10°C to -25°C for 20-60 minutes, and transferring to -80°C to -196°C for storage.

16. The method according to claim 1 or 2, wherein: The ultraviolet light irradiation time is 30-180s.

17. The method according to claim 2, wherein: Based on the total volume of the freezing solution, the components of the freezing solution include: 0-15 mL / 100 mL of DMSO, 0-12 g / 100 mL of sugar, 0-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the rest is culture medium; And / or, based on the total volume of the freezing solution, the components of the freezing solution include: 1-15 mL / 100 mL of DMSO, 1-12 g / 100 mL of sugar, 1-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the rest is culture medium; And / or, the sugars include one or a combination of two or more of lactose, raffinose, and melezitose; And / or, the photoinitiator includes phenyl(2,4,6-trimethylbenzoyl) lithium phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

18. The cryopreservable porous gel microspheres prepared by the method according to any one of claims 1 to 17, wherein: The particle size of the porous gel microspheres is 150-400 microns; and / or, the particle size of the porous gel microspheres is 200-250 microns; And / or, the average equivalent diameter of the internal pores of the porous gel microspheres is 20-80 microns.

19. A biological ink, which is prepared by the cryogenic porous gel microspheres prepared by the method according to any one of claims 1 to 17 or the cryogenic porous gel microspheres according to claim 18.

20. A biological 3D printed product, which is prepared by the cryogenic porous gel microspheres obtained by the method described in any one of claims 1 to 17, the cryogenic porous gel microspheres described in claim 18, or the biological ink described in claim 19.

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