Freezable porous gel microspheres, and methods of making and using the same

Abstract

The application provides a kind of freezable porous gel microspheres and its preparation method and application.The method comprises: preparing water phase solution and oil phase solution into gel state microdroplets, the water phase solution contains low gel strength pigskin gelatin synthesized methacrylated gelatin; gel state microdroplets are soaked with liquid containing photoinitiator, after sufficient penetration, freezing is carried out, so that the water in the interior of gel state microdroplets forms ice crystal; using ultraviolet light irradiation to form ice crystal gel state microdroplets, crosslinking solidification is carried out, and freezable porous gel microspheres are obtained.The porous gel microspheres of the application support preloaded cells, get rid of the time urgency of present preparation and present use, and can be used at different times and spaces for cell printing and injection manufacturing; for patients, it can be extracted once, long-term frozen, and used as needed, to avoid repeated trauma.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine, particularly to the field of tissue engineering and biomanufacturing using gel microspheres as raw materials, specifically to a cryopreservable porous gel microsphere, its preparation method, and its application. Background Technology

[0002] The development of tissue engineering and regenerative medicine technologies has provided new solutions for the repair and regeneration of tissues and organs. Gel microspheres are a novel material form, composed of tightly packed sub-millimeter-sized gel spheres. The interstices between these microspheres have a higher porosity than traditional hydrogels, thus facilitating cellular physiological activities, including migration, proliferation, and differentiation. In clinical applications, biomaterials are often injected. The sub-micron size and weak physical interactions of gel microspheres give them significant shear-thinning behavior, allowing for easy injection through fine needles. After injection, the frictional forces between the microspheres rapidly induce self-repair of the structure. This physical form endows materials that are difficult to extrude with shear-thinning and self-repairing properties, enabling them to be extruded through needles and retain their shape after extrusion. Furthermore, gel microspheres can also be used as inks for bio-3D printing. Bio-3D printing, as an advanced technology in the field of biomanufacturing, can create biomimetic functional structures using cells, active molecules, and biomaterials as basic units, reconstructing the complexity of tissues and organs to a greater extent. Gel microsphere inks combined with bio-3D printing can construct customized tissue-engineered scaffolds. Therefore, gel microspheres have broad application prospects in clinical injection and tissue engineering.

[0003] The porosity of traditional gel microsphere inks is related to their packing density, approximately 15% for dense packing and approximately 25% for loose packing. Excessively loose packing leads to poor extrusion characteristics, making it impossible to print high-fidelity structures, and resulting in weaker bonding between microspheres. Some technologies modify the surface of gel microspheres to provide dynamic bonding for loosely packed microspheres, further reducing the required packing density threshold for printing and thus increasing porosity. However, despite the improved porosity compared to traditional bulk hydrogels, gel microsphere inks still have certain limitations in biological applications, including difficulty in cell extension within the microspheres and insufficient nutrient transport. More interconnected micropores and larger pore spaces not only facilitate nutrient transport, thereby increasing cell viability, but also promote cell extension without waiting for hydrogel degradation.

[0004] Several strategies have been used to create smaller-scale pore features within gel microspheres. Some techniques prepare lyophilized gel microspheres (also known as microcarriers) for stem cell expansion, but these lyophilized microspheres cannot be pre-loaded with cells and can only be used as microcarriers for cell seeding, resulting in limited seeding efficiency. Other techniques use aqueous two-phase precursor hydrogels to prepare gel microspheres, sacrificing one phase to obtain interconnected pores. This method can pre-load higher cell densities and achieve better pore connectivity, but is limited by the material system; currently, only a limited number of biocompatible material systems can achieve spontaneous liquid-liquid phase separation. Furthermore, no work has yet demonstrated that aqueous two-phase cell-loaded microspheres support storage; they can only be used immediately after manufacturing. In summary, achieving high-porosity, one-step-ready cell-loaded porous gel microspheres remains a challenge. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a cryopreservable porous gel microsphere, its preparation method, and its application. This porous gel microsphere has fewer material limitations, supports pre-loaded cells, eliminates the time constraints of current preparation and use, and can be accessed in different times and spaces.

[0006] To achieve the above objectives, the present invention provides a method for preparing cryopreservable porous gel microspheres, wherein the method includes:

[0007] Aqueous and oil phase solutions were used to prepare gel-like microdroplets; wherein the aqueous phase solution contained methacrylated gelatin (GelMA) synthesized from low gel strength porcine skin gelatin.

[0008] The gel-like microdroplets are soaked in a liquid containing a photoinitiator, and after full penetration, they are frozen, causing the water inside the gel-like microdroplets to form ice crystals.

[0009] Crosslinking and solidifying gel microdroplets that form ice crystals using ultraviolet light irradiation yields porous gel microspheres that can be frozen.

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

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

[0012] Aqueous and oil phase solutions were used to prepare gel-like microdroplets; wherein the aqueous phase solution contained methacrylated gelatin synthesized from low gel strength porcine skin gelatin, and also contained cells to be preloaded.

[0013] The gel microdroplets were soaked in a liquid containing a photoinitiator. After full penetration, the gel microdroplets were transferred to a cryopreservation solution and subjected to gradient freezing, which caused the water inside the gel microdroplets to form ice crystals.

[0014] Crosslinking and solidifying gel microdroplets that form ice crystals using ultraviolet light irradiation yields porous gel microspheres that can be frozen.

[0015] According to a specific embodiment of the present invention, the method for synthesizing methacrylated gelatin from low gel strength porcini gelatin includes: preparing a gelatin solution from porcini gelatin with a gel strength of 100-150 Bloomg; slowly adding methacrylic anhydride dropwise to the gelatin solution and stirring thoroughly to allow for complete reaction; after the reaction is complete, neutralizing the unreacted methacrylic anhydride with an alkaline solution (e.g., NaHCO3); and removing unreacted substances through a dialysis bag (molecular weight cutoff ~12000 Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000 Da); washing and drying the purified methacrylated gelatin solution to obtain the final methacrylated gelatin from low gel strength porcini gelatin.

[0016] According to a specific embodiment of the present invention, preferably, the temperature range of the low temperature is 0-10℃.

[0017] According to a specific embodiment of the present invention, preferably, when there are no cells to be preloaded in the aqueous solution, i.e., 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-carrying hydrogel precursor solution (i.e., aqueous solution) contains cells to be pre-loaded, i.e., when preparing cell-carrying porous gel microspheres, the cross-linking environment temperature is room temperature and the time is 2 minutes. However, before the porous gel microspheres are taken out after gradient freezing and cross-linked, they must always be kept below 4°C.

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

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

[0021] In the preparation method of this invention, material optimization was performed. GelMA microspheres, typically used for tissue culture, do not develop pores when frozen (without drying and dehydration). Therefore, this invention employs multiple schemes to optimize the material of the GelMA microspheres, enabling them to develop pores after freezing (without drying and dehydration), ultimately resulting in an optimized scheme: using GelMA synthesized from low-gel-strength porcine gelatin to prepare porous gel microspheres for in-situ pre-loaded cells. In this invention, the methacrylated gelatin is prepared by adding methacrylic anhydride (MA) to low-gel-strength porcine gelatin, resulting in a substitution reaction. Microspheres prepared by this method can form pores after freezing, while microspheres prepared from commercially available GelMA (synthesized from 300 Bloomg gel-strength porcine gelatin) do not have pores after freezing.

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

[0023] According to a specific embodiment of the present invention, preferably, 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 solution.

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

[0025] According to a specific embodiment of the present invention, the blend phase is selected from one or more combinations 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 solution further contains sugars, and the sugar content is 1-10 g / 100 mL, preferably 4-8 g / 100 mL, based on the volume of the solvent in the aqueous solution.

[0027] According to a specific embodiment of the present invention, preferably, the sugars in the aqueous solution include one or more of lactose, raffinose, and menotriose, with raffinose being the most preferred.

[0028] According to a specific embodiment of the present invention, preferably, the cell density in the aqueous solution is 1×10⁻⁶. 3 -1×10 8 Cells / mL, preferably 1×10⁻⁶ 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 human mesenchymal stem cells, cancer cells, fibroblasts, and endothelial cells.

[0030] In the preparation method of this invention, the selected cells are cultured in various types of cell-specific culture media, which are changed every 2 days. After the cells reach the logarithmic growth phase, they are digested with 0.25% trypsin, centrifuged, and then a low-gel-strength porcine gelatin-based methacrylated gelatin solution with a content of 5-12.5 g / 100 mL is prepared by re-vortexing and used as an aqueous solution. The prepared methacrylated gelatin solution is sterilized by filtration through a 0.22 μm pore membrane before being added to the cells.

[0031] In this invention, the culture medium used for each type of cell is a commonly used commercial culture medium in the field for that type of cell.

[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, the content of the surfactant is 1-5% based on the volume of the oil phase solution as 100%, 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 for preparing the aqueous solution and oil solution into gel-like microdroplets includes droplet microfluidics, oil-water emulsion, composite coagulation, or electrospraying.

[0037] According to a specific embodiment of the present invention, the present invention utilizes a droplet microfluidic method to prepare gel-like microdroplets from an aqueous phase solution and an oil phase solution. Any microfluidic device in the art capable of obtaining gel-like microdroplets of the desired particle size can be used. Preferably, the specific steps of the droplet microfluidic method include: adding the aqueous phase solution and the oil phase solution into the microfluidic device, adjusting the flow rates of the aqueous phase solution and the oil phase solution respectively, and mixing them. The flow rates of the aqueous phase solution and the oil phase solution refer to the flow rates at the aqueous phase solution inlet and the oil phase solution inlet respectively input into the chip, controlled by a precision pump pushing the syringe. The pipe specifications at the aqueous phase solution inlet and the oil phase solution inlet can be set according to the particle size of the prepared gel-like microdroplets. Preferably, the outer diameter of the hose at the aqueous phase solution inlet and the oil phase solution inlet is 0.76-2.29 mm, and the inner diameter is 0.25-1.27 mm; for example, the inner diameter of the hose at the aqueous phase solution inlet and the oil phase solution inlet is 0.51 mm, and the outer diameter is 1.52 mm.

[0038] According to a specific embodiment of the present invention, the specifications of each channel in the droplet microfluidic device can be set according to the particle size of the prepared gel-like microdroplets. Preferably, in the droplet microfluidic device, the diameter of the chip channel for the oil phase solution and the aqueous phase solution is 100-400 micrometers; the diameter of the droplet channel after mixing the oil phase solution and the aqueous phase solution is 300-800 micrometers. For example, the diameter of the chip channel for the oil phase solution and the aqueous phase solution is 300 micrometers; the diameter of the droplet channel after mixing the oil phase solution and the aqueous phase solution is 550 micrometers.

[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 and oil phase solutions of the microfluidic device, gel-like microdroplets with or without cells and a particle size range of 150-400 micrometers can be stably obtained.

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

[0042] In the preparation method of this invention, when the aqueous solution contains cells to be preloaded, the prepared gel microdroplets remain stable at low temperatures and can be enriched by methods such as centrifugation and sedimentation, followed by the addition of a washing solution. To maintain good cell viability, a culture medium commonly used in the art, adapted to the preloaded cells, can be added as a washing solution to remove the oil phase components on the surface of the gel microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0043] In the preparation method of this invention, after centrifugation to remove the supernatant of the gel-like microdroplets, a culture medium containing a photoinitiator is added; the mixture is then placed in a refrigerator at 4°C to prevent the gel-like microdroplets from melting and to ensure sufficient penetration of the photoinitiator. The gel-like microdroplet solution is then transferred to a culture dish and cryopreservation solution is added.

[0044] According to a specific embodiment of the present invention, preferably, the photoinitiator comprises 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, the content of the photoinitiator is 0.1-0.5 g / 100 mL, more preferably 0.1-0.3 g / 100 mL, and even more preferably 0.15 g / 100 mL, based on the volume of the solvent in the liquid containing the photoinitiator.

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

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

[0048] According to a specific embodiment of the present invention, preferably, the gradient freezing step includes: standing at -10°C to -25°C for 20-60 minutes, then transferring to an environment of -80°C to -196°C for storage, which 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 composition of the cryopreservation solution includes: 0-15 mL / 100 mL of DMSO, 0-12 g / 100 mL of sugars, 0-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the remainder being culture medium; more preferably, based on the total volume of the cryopreservation solution, the composition of the cryopreservation solution includes: 1-15 mL / 100 mL of DMSO, The cryopreservation solution comprises 1-12 g / 100 mL of carbohydrates, 1-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the remainder being culture medium. More preferably, based on the total volume of the cryopreservation solution, the composition includes: 5-15 mL / 100 mL of DMSO, 4-8 g / 100 mL of carbohydrates, 20-40 mL / 100 mL of serum, 0.1-0.3 g / 100 mL of photoinitiator, and the remainder being culture medium. As a typical preferred composition, the cryopreservation solution comprises: 10 mL / 100 mL of DMSO, 4 g / 100 mL of raffinose, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the remainder being culture medium.

[0051] According to a specific embodiment of the present invention, preferably, the sugars in the cryopreservation solution include one or more of lactose, raffinose, and menotriose, preferably raffinose.

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

[0053] The present invention also provides a cryopreservable porous gel microsphere obtained by the above preparation method, wherein the particle size of the porous gel microsphere is 150-400 micrometers, preferably 200-250 micrometers, and the average equivalent diameter of the internal pores of the porous gel microsphere is 20-80 micrometers.

[0054] The present invention also provides a bio-ink, which is prepared from the frozen-storable porous gel microspheres obtained by the above preparation method or the frozen-storable porous gel microspheres described above.

[0055] The present invention also provides a bio-3D printed article, which is prepared from the cryopreservable porous gel microspheres obtained by the above preparation method, the cryopreservable porous gel microspheres mentioned above, or the bio-ink mentioned above.

[0056] This invention utilizes the principle of ice crystal pore formation to construct micron-level interconnected pores within microparticles. Furthermore, the preparation method provided by this invention has broad material applicability, enabling the fabrication of wet porous gel microspheres containing proteins, polysaccharides, and synthetic hydrogels. The technical solution of this invention supports in-situ loading of live cells and allows for long-term storage in low-temperature environments. During use, rapid cross-linking and thawing allow the ice crystals to melt, leaving stable pores within the microspheres, resulting in interconnected porous gel microspheres loaded with cells in situ. The low-temperature storage process can be extended to three months or longer, maintaining cell viability within the hydrogel microspheres during this period.

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

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

[0059] 2. This invention can be applied to clinical cell therapy by realizing a porous gel microsphere-based microtissue module that can be pre-loaded with high-density cells, supports long-term cell cryopreservation, and is available on demand. It eliminates the cumbersome steps of temporary scaffold preparation and cell seeding, and supports bedside immediate access for injection, transplantation, and other tissue repair treatments. It establishes a "shelf-mounted, on-demand" cell therapy paradigm, eliminating the time constraints of current preparation and use, and enabling access across time and space. For patients, it allows for one-time extraction, long-term cryopreservation, and on-demand access, avoiding repeated trauma.

[0060] 3. The cryopreservable porous gel microspheres prepared in this invention are also a novel bio-3D printing ink material, which can construct granular stress-yielding bio-inks for cell printing, and have better porosity than traditional solid gel microsphere granular inks.

[0061] 4. Achieving the desired porosity of GelMA porous gel microspheres in existing technologies is challenging. Because ice crystals melt easily, photocuring efficiency is very low under low-temperature freezing conditions. Therefore, porous gel microspheres created using ice crystal pore-forming methods are difficult to stabilize and cure through photocrosslinking. Most existing technologies employ freeze-drying or chemical crosslinking. The technical solution of this invention achieves a high yield of stable photocurable porous gel microspheres by optimizing materials, cryopreservation solution formulation, and cryopreservation process. Attached Figure Description

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

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

[0064] Figure 3 The figures show the test results of various materials for porous cryogel microspheres, as well as the statistical analysis of internal pores and the comparison of strategies.

[0065] Figure 4 The results of the optimized formulation of porous cryogel microspheres.

[0066] Figure 5 Results of optimizing the cryopreservation solution for porous cryogel microspheres.

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

[0068] Figure 7 This is a comparison image of traditional microgels and wet porous cryogel microspheres.

[0069] Figure 8 Staining images of traditional microgels and wet-frozen porous gel microspheres to support various cell cultures.

[0070] Figure 9 Alizarin Red staining results of wet-state frozen porous gel microspheres after one week of culture and two weeks of osteogenic differentiation.

[0071] Figure 10 The results show the survival rate and proliferation rate of cell-loaded wet porous cryogel microspheres after long-term cryopreservation.

[0072] Figure 11 Image showing the results of extruding wet-state frozen porous gel microspheres for bio-3D printing.

[0073] Figure 12 The results are from a rat subcutaneous injection experiment comparing traditional microgels and wet porous cryogel microspheres.

[0074] Figure 13 The results are from experiments on the permeability of highly interconnected pores in wet-state frozen porous gel microspheres.

[0075] Explanation of reference numerals in the attached figures:

[0076] 301: Oil phase solution inlet; 302: Aqueous phase solution inlet; 303: Oil phase channel; 304: Aqueous phase main channel; 305: Aqueous phase branch channel; 306: Droplet tube channel. Detailed Implementation

[0077] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation 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 porous gel microspheres of methacrylated gelatin. In this embodiment, porous gel microspheres of methacrylated gelatin are prepared using droplet microfluidics. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution is 4-10 mL / h, and the flow rate of the aqueous phase solution is 1-3 mL / h, which can stably obtain gel-like microdroplets with a particle size range of 150-400 micrometers. The aqueous phase solution contains methacrylated gelatin synthesized from low-gel strength porcine skin gelatin, and the solvent of the aqueous phase solution can be ultrapure water or 1× phosphate-buffered saline (PBS). The chip structure of the droplet microfluidic device used in this invention is as follows: Figure 1 As shown, the microfluidic chip includes an oil phase solution inlet 301 and an aqueous phase solution inlet 302. The oil phase solution inlet 301 is connected to an oil phase channel 303, and the aqueous phase solution inlet 302 is connected to an aqueous phase channel composed of a main aqueous phase channel 304 and two aqueous phase branch channels 305. The outlets of the two aqueous phase branch channels 305 merge with the outlet of the oil phase channel 303 and connect to the inlet of the droplet channel 306. The flow rates of the aqueous and oil phase solutions refer to the flow rates input into the chip's aqueous phase solution inlet 302 and oil phase solution inlet 301, respectively, and are controlled by a precision pump pushing the injector. 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 also 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 aqueous phase solution is D1, which is equal to 300 micrometers; the diameter of the droplet channel is D2, which is equal to 550 micrometers.

[0080] Gel-state microdroplets remain stable at low temperatures of 0-10℃ and can be enriched by methods such as centrifugation and sedimentation. Further washing solutions, such as 0.9% sodium chloride solution, 1× phosphate-buffered saline (PBS), or ultrapure water, can be added 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 subsequent steps.

[0081] (2) After centrifugation and removal of the supernatant, add the liquid containing the photoinitiator and allow it to stand at 4°C to prevent the gel-like microdroplets from melting, ensuring that the photoinitiator fully penetrates into the gel-like microdroplets. Then, transfer the gel-like microdroplet solution to a petri dish and freeze it at -80°C. The solvent in the above-mentioned liquid containing the 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) After the porous gel microspheres are frozen and stored in a -80°C freezer 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 removed from the -80°C freezer, and the gel microdroplets are irradiated and solidified using a 405nm wavelength light source for 30-180 seconds (preferably 120 seconds) to crosslink 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 1×10⁻⁶. 3 -1×10 8 cells / mL (preferably 10) 7 The selected cells were cultured in a culture medium commonly used for this type of cell in the art, with the medium being changed every 2 days. After the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin (Gibco 25200072), centrifuged, and collected. They were then re-vortexed with a prepared aqueous solution containing methacrylated gelatin. The solvent for the aqueous solution could be ultrapure water or 1× phosphate buffer solution. The content of methacrylated gelatin, based on the volume of the solvent in the aqueous solution, was 5-12.5 g / 100 mL, preferably 10 g / 100 mL. The aqueous solution also contained sugars, based on the volume of the solvent in the aqueous solution, the content of sugars was 1-10 g / 100 mL, preferably 4-8 g / 100 mL. The sugars were preferably raffinose to improve cell viability.

[0085] (2) Preparation of pre-loaded cell-porous gel microspheres using droplet microfluidics. By adjusting the flow rate of the droplet microfluidic device, with the oil phase solution flow rate at 4-10 mL / h and the aqueous phase solution flow rate at 1-3 mL / h, gel-like microdroplets with a particle size range of 150-400 μm can be stably obtained. In this embodiment, by fine-tuning the device parameters, gel-like microdroplets with a particle size of approximately 300 μm can be stably obtained. The aqueous phase solution contains methacrylated gelatin synthesized from low-gel strength porcine skin gelatin.

[0086] Gel-like microdroplets remain stable at low temperatures (0-10℃) and can be enriched by methods such as centrifugation and sedimentation, followed by the addition of a washing solution. 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-like microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0087] (3) After centrifugation and removal of the supernatant, add culture medium containing the photoinitiator and incubate 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 cryopreservation solution, and place it in a low-temperature freezer for gradient freezing. The volume ratio of cryopreservation solution to gel microdroplets is 1:(1-10); the gradient freezing steps include: incubation at -10°C to -25°C for 20-60 minutes, followed by transfer to an environment of -80°C to -196°C for storage. Based on the volume of solvent in the liquid containing the photoinitiator, the content of the photoinitiator is 0.1-0.5 g / 100 mL, preferably 0.15 g / 100 mL.

[0088] (4) Cell-loaded porous gel microspheres were stored frozen at -80°C. Before use, the microspheres were irradiated with a 405nm wavelength light source for 30-180 seconds (preferably 120 seconds) to cure the microspheres and crosslink the methacrylated gelatin. The liquid in the culture dish was then completely replaced with preheated culture medium, and the microspheres were transferred to new culture dishes / well plates for incubation. The culture medium was changed every 48 hours with fresh medium.

[0089] In some specific embodiments of the present invention, in the above-mentioned method for preparing porous gel microspheres, the aqueous solution contains methacrylated gelatin synthesized from low gel strength pigskin 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 solution. More preferably, the aqueous solution contains methacrylated gelatin synthesized from low-gel strength porcine skin gelatin and a blend phase for assisting cell culture. The content of the methacrylated gelatin is 2.5-10 g / 100 mL based on the volume of the solvent in the aqueous solution; the content of the blend phase is 2.5-10 g / 100 mL; the blend phase is selected from one or more combinations of gelatin, hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan, and fibrinogen; preferably, the gel strength of the low-gel strength porcine skin gelatin is 100-150 Bloomg.

[0090] In some specific embodiments of the present invention, in the above-mentioned method for preparing porous gel microspheres, the oil phase solution is obtained by mixing an oily material with a surfactant; preferably, the oily material includes one 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 more of Span80, Tween20, AEO-3, and FluoSurf.

[0091] In some specific embodiments of the present invention, in the above-mentioned method for preparing porous gel microspheres, the cryopreservation solution comprises, based on the total volume of the cryopreservation solution, 0-15 mL / 100 mL of DMSO, 0-12 g / 100 mL of sugars, 0-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the remainder being culture medium; more preferably, based on the total volume of the cryopreservation solution, the cryopreservation solution comprises 1 .... The cryopreservation solution comprises: 5-15 mL / 100 mL DMSO, 4-8 g / 100 mL sugars, 20-40 mL / 100 mL serum, 0.1-0.3 g / 100 mL photoinitiator, and the remainder culture medium, based on the total volume of the cryopreservation solution. As a typical preferred composition, the cryopreservation solution comprises: 10 mL / 100 mL DMSO, 4 g / 100 mL raffinose, 40 mL / 100 mL serum, 0.15 g / 100 mL photoinitiator, and the remainder culture medium. The sugars include one or more of lactose, raffinose, and mesotriose; the photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) 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, their preparation method, and their applications provided by this invention is as follows: Figure 2 As shown.

[0093] Material optimization in the preparation of porous gel microspheres

[0094] The growth of ice crystals within hydrogel microspheres may be related to the length and three-dimensional conformation of the polymer chains. The length and three-dimensional conformation of the polymer chains in the ice crystals affect the free energy of the microspheres. From a thermodynamic perspective, under stronger geometric constraints, the surface-to-volume ratio of the free energy of the microspheres increases, thus significantly increasing the surface free energy and raising the free energy barrier for nucleation. Commercially available porcine skin-derived methacrylated gelatin, commonly used in cell culture at concentrations ranging from 5% to 12.5%, fails to generate pores during freezing (without drying and dehydration). This is because the relatively long molecular chains of this methacrylated gelatin result in a high free energy, and the resulting elastic stress restricts ice crystal growth, preventing the construction of interconnected pores within the microspheres.

[0095] To obtain materials capable of stabilizing ice crystal formation within microspheres, this invention prepares various methacrylated gelatins with different molecular chain lengths and distributions: ① commercially available methacrylated gelatin derived from pigskin; ② bacterially degraded methacrylated gelatin derived from pigskin (widely degraded, with the shortest molecular chains and low molecular weight); ③ collagenase-degraded methacrylated gelatin derived from pigskin (specifically cleaved peptide bonds, breaking down into smaller peptide fragments and individual α chains); ④ fish skin-derived methacrylated gelatin (broad molecular weight distribution, containing various chain structures with different molecular weights); and ⑤ methacrylated gelatin synthesized from low-gel strength pigskin gelatin (shorter molecular chains and lower molecular weight).

[0096] ① Commercially available methacrylated gelatin microspheres derived from pigskin:

[0097] Synthesizing high-gel-strength porcine methacrylic gelatin. Purchased porcine gelatin (Sigma, V900863-100G) 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 PBS volume). Methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring thoroughly to allow for complete reaction. The mixture was reacted at 30–50 °C for a period of time. After the reaction was complete, unreacted methacrylic anhydride was neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances were removed by dialysis (molecular weight cutoff ~12000 Da) or membrane ultrafiltration (molecular weight cutoff ~10000 Da). The purified methacrylic gelatin solution was washed and dried to obtain the final high-gel-strength porcine methacrylic gelatin product.

[0098] Preparation of methacrylated gelatin thermosensitive gel spheres. This embodiment utilizes, as follows: Figure 1 The droplet microfluidic device shown was used to prepare thermosensitive gel spheres of methacrylated gelatin. The flow rates of the droplet microfluidic device were adjusted: the oil phase solution flow rate was 6 mL / h, and the aqueous phase solution flow rate was 2 mL / h. The content of methacrylated gelatin synthesized from high-gel-strength pigskin gelatin was 10 g / 100 mL, based on the volume of the solvent in the aqueous phase solution. The solvent in the aqueous phase solution was 1× phosphate-buffered saline (PBS). Based on 100% of the oil phase solution volume, the content of the surfactant Span was 4%, and the remaining components were mineral oil.

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

[0100] After centrifugation and removal of the supernatant, a liquid containing LAP photoinitiator was added. The solution was then incubated at 4°C for 30 minutes to prevent the gel-like microdroplets from melting, ensuring the photoinitiator fully penetrated into the microdroplets. The gel-like microdroplet solution was then transferred to a petri dish and frozen at -80°C. The solvent in the photoinitiator-containing liquid was 1× phosphate-buffered saline (PBS), and the photoinitiator concentration was 0.15 g / 100 mL by volume.

[0101] After the porous gel microspheres were frozen and stored in a -80°C freezer for 90 minutes, the culture dish was removed from the -80°C freezer and the gel microdroplets were irradiated and solidified for 120 seconds using a 405nm wavelength light source, finally obtaining commercially available methacrylated gel microspheres derived from pigskin.

[0102] ② Bacterial-degraded porcine skin-derived methacrylated gelatin gel microspheres (widely degraded, with the shortest molecular chains and low molecular weight):

[0103] Methacrylated gelatin derived from pigskin and degraded by synthetic bacteria was prepared. A gelatin solution was prepared by dissolving pigskin gelatin with a gel strength of 300 Bloomg in 1× phosphate-buffered saline (PBS). The gelatin concentration was 10 g / 100 mL (based on PBS volume). Methacrylic anhydride was slowly added dropwise to the gelatin solution with thorough stirring to allow for complete reaction. The mixture was reacted at 30–50 °C for a period of time. After the reaction was complete, unreacted methacrylic anhydride was neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances were removed by dialysis using a dialysis bag (molecular weight cutoff ~12000 Da) or a membrane ultrafiltration system (molecular weight cutoff ~10000 Da). After dialysis, the mixture was inoculated with 10... 5 After 12 hours of degradation by CFU / mL E. coli, the E. coli was removed by filtration in a filter bottle. The purified methacrylated gelatin solution was then washed and dried to obtain the final bacterially degraded porcine skin methacrylated gelatin product.

[0104] Preparation of methacrylated gelatin thermosensitive gel spheres. This embodiment utilizes, as follows: Figure 1The droplet microfluidic device shown was used to prepare thermosensitive gel spheres of methacrylated gelatin. The flow rates of the droplet microfluidic device were adjusted: the oil phase solution flow rate was 6 mL / h, and the aqueous phase solution flow rate was 2 mL / h. The aqueous phase solution contained 10 g / 100 mL of bacterially degraded methacrylated gelatin, and the solvent was 1× phosphate-buffered saline (PBS). The oil phase solution contained 4% surfactant (Span) by volume, with the remainder being mineral oil. The gel-state microdroplets remained stable at a low temperature of 0-10°C. They were enriched by centrifugation and then washed with 1× phosphate-buffered saline (PBS) to remove the oil phase components from the surface of the gel-state microdroplets. Through this enrichment-washing process, the oil phase content was reduced to a level that would not affect subsequent steps.

[0105] After centrifugation and removal of the supernatant, a liquid containing LAP photoinitiator was added. The mixture was then incubated at 4°C for 30 minutes to prevent the gel-like microdroplets from melting, ensuring the photoinitiator fully penetrated into the microdroplets. The gel-like microdroplet solution was then transferred to a petri dish and frozen at -80°C. The solvent in the photoinitiator-containing liquid was 1× phosphate-buffered saline (PBS), and the photoinitiator concentration was 0.15 g / 100 mL by volume.

[0106] After the porous gel microspheres were frozen and stored in a -80°C freezer for 90 minutes, the culture dish was removed from the -80°C freezer and the gel microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, and finally bacterially degraded porcine skin-derived methacrylated gel microspheres were obtained.

[0107] ③ Methacrylated gel microspheres derived from pig skin and degraded by collagenase (the peptide bonds are specifically cleaved, breaking down into smaller peptide fragments and individual α chains);

[0108] Synthesized high-gel-strength methacrylated gelatin from pigskin. Pigskin 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. Methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring thoroughly to allow for complete reaction. The mixture was reacted at 30-50°C for a period of time. After the reaction was complete, unreacted methacrylic anhydride was neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances were removed by dialysis (molecular weight cutoff ~12000 Da) or membrane ultrafiltration (molecular weight cutoff ~10000 Da). The purified methacrylated gelatin solution was washed and dried to obtain the final high-gel-strength methacrylated gelatin product from pigskin.

[0109] Preparation of methacrylated gelatin thermosensitive gel spheres. This embodiment utilizes, as follows: Figure 1 The droplet microfluidic device shown was used to prepare thermosensitive gel spheres of methacrylated gelatin. By adjusting the flow rate of the droplet microfluidic device, with the oil phase solution at a flow rate of 6 mL / h and the aqueous phase solution at a flow rate of 2 mL / h, gel-like microdroplets with a particle size range of 150-400 μm could be stably obtained. Specifically, based on the volume of the solvent in the aqueous phase solution, the content of methacrylated gelatin synthesized from high-gel strength porcine skin gelatin was 10 g / 100 mL, and the solvent in the aqueous phase solution was 1× phosphate-buffered saline (PBS). Based on 100% of the volume of the oil phase solution, the content of the surfactant Span was 4%, and the remaining components were mineral oil. The gel-like microdroplets remained stable at a low temperature of 0-10°C. They were enriched by centrifugation and further washed with 1× phosphate-buffered saline to remove the oil phase components from the surface of the gel-like microdroplets. Through the enrichment-washing steps, the oil phase content could be reduced to a level that would not affect subsequent steps.

[0110] After centrifugation and removal of the supernatant, a liquid containing LAP photoinitiator and collagenase was added. The mixture was then incubated at 4°C to prevent the gel-like microdroplets from melting. After 30 minutes of incubation, the photoinitiator was allowed to fully penetrate the microdroplets, and the collagenase partially degraded to prepare a high-gel-strength porcine skin-derived methacrylated gelatin microgel. The gel-like microdroplet solution was then transferred to a petri dish and frozen at -80°C. The solvent in the photoinitiator-containing liquid was 1× phosphate-buffered saline (PBS), and the photoinitiator concentration was 0.15 g / 100 mL by volume; the collagenase concentration was 5 mg / mL by volume of the photoinitiator-containing liquid. After the porous gel microspheres were frozen and stored in a -80°C freezer for 90 minutes, the culture dish was removed from the -80°C freezer and the gel microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, and finally, methacrylated gel microspheres derived from pig skin and degraded by collagenase were obtained.

[0111] ④ Methacrylated gelatin derived from fish skin (broad molecular weight distribution, containing multiple chain structures of different molecular weights):

[0112] Synthesized 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 PBS volume). Methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring thoroughly to allow for complete reaction. The mixture was reacted at 30-50°C for a period of time. After the reaction was complete, unreacted methacrylic anhydride was neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances were removed by dialysis (molecular weight cutoff ~12000 Da) or membrane ultrafiltration (molecular weight cutoff ~10000 Da). The purified methacrylated gelatin solution was washed and dried to obtain the final fish skin methacrylated gelatin product.

[0113] This implementation plan utilizes, for example Figure 1 The droplet microfluidic device shown was used to prepare thermosensitive gel spheres of methacrylated gelatin. By adjusting the flow rate of the droplet microfluidic device, with the oil phase solution at a flow rate of 6 mL / h and the aqueous phase solution at a flow rate of 2 mL / h, gel-like microdroplets with a particle size range of 150-400 μm could be stably obtained. Specifically, based on the volume of solvent in the aqueous phase solution, the content of methacrylated gelatin synthesized from high-gel strength porcine skin gelatin was 10 g / 100 mL, and the solvent in the aqueous phase solution was 1× phosphate-buffered saline (PBS). Based on 100% of the volume of the oil phase solution, the content of surfactant Span was 4%, and the remaining components were mineral oil. The gel-like microdroplets remained stable at a low temperature of 0-10℃. They were enriched by centrifugation and further washed with 1× phosphate-buffered saline (PBS) to remove the oil phase components from the surface of the gel-like microdroplets. Through the enrichment-washing steps, the oil phase content could be reduced to a level that would not affect subsequent steps.

[0114] After centrifugation and removal of the supernatant, a liquid containing the photoinitiator was added. The mixture was then incubated at 4°C for 30 minutes to prevent the gel-like microdroplets from melting, ensuring the photoinitiator fully penetrated into the microdroplets. The microdroplet solution was then transferred to a petri dish and frozen at -80°C. The solvent in the photoinitiator-containing liquid was 1× phosphate-buffered saline (PBS), and the photoinitiator concentration was 0.15 g / 100 mL by volume.

[0115] Porous gel microspheres were frozen and stored at -80°C for 90 minutes. The culture dishes were then removed from the freezer, and the gel microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to cure them, which was then used to crosslink methacrylated gelatin. This yielded methacrylated gelatin derived from fish skin.

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

[0117] Synthesized low-gel-strength porcine methacrylic gelatin. A gel solution was prepared by dissolving porcine gelatin (Aladdin, G108398) with a gel strength of 100 Bloom g in 1× phosphate-buffered saline (PBS). The gelatin content was 10 g / 100 mL (based on PBS volume). Methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring thoroughly to allow for complete reaction. The mixture was reacted at 40°C for a period of time. After the reaction was complete, unreacted methacrylic anhydride was neutralized with an alkaline solution (e.g., NaHCO3), and unreacted substances were removed by dialysis (molecular weight cutoff ~12000 Da) or membrane ultrafiltration (molecular weight cutoff ~10000 Da). The purified methacrylic gelatin solution was washed and dried to obtain the final low-gel-strength porcine methacrylic gelatin product.

[0118] Preparation of methacrylated gelatin thermosensitive gel spheres. This embodiment utilizes, as follows: Figure 1 The droplet microfluidic device shown was used to prepare thermosensitive gel spheres of methacrylated gelatin. The flow rates of the droplet microfluidic device were adjusted: the oil phase solution flow rate was 6 mL / h, and the aqueous phase solution flow rate was 2 mL / h. The content of methacrylated gelatin synthesized from low-gel strength pigskin gelatin was 10 g / 100 mL, based on the volume of the solvent in the aqueous phase solution. The solvent in the aqueous phase solution was 1× phosphate-buffered saline (PBS). Based on 100% of the oil phase solution volume, the surfactant Span content was 4%, and the remaining components were mineral oil. The gel-state microdroplets remained stable at a low temperature of 0-10°C. They were enriched by centrifugation and further washed with 1× phosphate-buffered saline (PBS) to remove the oil phase components from the surface of the gel-state microdroplets. Through this enrichment-washing step, the oil phase content was reduced to a level that would not affect subsequent steps.

[0119] After centrifugation and removal of the supernatant, a liquid containing LAP photoinitiator was added. The mixture was then incubated at 4°C for 30 minutes to prevent the gel-like microdroplets from melting, ensuring the photoinitiator fully penetrated into the microdroplets. The gel-like microdroplet solution was then transferred to a petri dish and frozen at -80°C. The solvent in the photoinitiator-containing liquid was 1× phosphate-buffered saline (PBS), and the photoinitiator concentration was 0.15 g / 100 mL by volume.

[0120] After the porous gel microspheres were frozen and stored in a -80°C freezer for 90 minutes, the culture dish was removed from the -80°C freezer and the gel microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, and finally, methacrylated gel microspheres synthesized from pigskin gelatin with low gel strength were obtained, namely wet porous gel microspheres.

[0121] Qualitative and quantitative data on the frozen pore size of microspheres prepared from different materials, such as... Figure 3 As shown. If the commonly used GelMA is used to prepare gel microspheres, photocrosslinking after freezing fails to produce pores. This is likely because the rapid melting of ice crystals during photocrosslinking makes it difficult to maintain the pore shape. Through the above testing scheme, the present invention finally obtained an optimized scheme for microsphere preparation materials, namely: using GelMA synthesized from low gel strength pigskin gelatin to prepare porous gel microspheres, which can easily form relatively uniform and abundant interconnected micropores with good batch consistency.

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

[0123] When using gel microspheres to cryopreserve cells, the formation of ice crystals during the freezing process can damage the cells. Therefore, in order to make the gel microspheres compatible with cell cryopreservation, this invention has explored various cryopreservation schemes, including the ratio of DMSO and the ratio of added sugars, and finally obtained optimized results.

[0124] Optimization of Gel Microsphere Material Formulation for Porous Cryogel Microspheres

[0125] (1) This implementation plan selects human mesenchymal stem cells as pre-loaded cells. Human mesenchymal stem cells are cultured in a special culture medium, which is changed every two days. After the cells reach the logarithmic growth phase, they are digested with 0.25% trypsin, centrifuged, and then re-vortexed with a prepared aqueous solution containing methacrylated gelatin synthesized from low-gel strength porcine skin gelatin. The cell density is 102. 7 Cells / mL. Before adding cells, the methacrylated gelatin solution needs to be sterilized by passing it through a filter membrane (0.22 μm pore size). The solvent of the aqueous phase solution is 1× phosphate buffer solution, 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 sugars, and the content of sugars is 4 g / 100 mL based on the volume of the solvent in the aqueous phase solution. The sugars are lactose, raffinose, or menotriose.

[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 aqueous phase solution was 2 mL per hour to obtain gel-state microdroplets.

[0127] Gel-like microdroplets can be enriched by methods such as centrifugation and sedimentation, and then further cleaned with a washing solution. 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-like microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0128] (3) After centrifugation and removal of the supernatant from the gel microdroplets, culture medium containing the photoinitiator was added. The mixture was then kept in a 4°C freezer to prevent the gel microdroplets from melting, ensuring that LAP fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish, and the optimized cryopreservation solution was added. The dish was then subjected to gradient freezing at -20°C for 20 minutes, and then transferred to a -80°C freezer for long-term storage. The photoinitiator content was 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator. The diameter of the culture dish during freezing was 35 mm, and the volume ratio of the gel microdroplets to the cryopreservation solution in the culture dish during freezing was 1:2.

[0129] (4) Cell-loaded porous gel microdroplets were frozen and stored at -80°C. Before use, the microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, which was then used to crosslink methacrylated gelatin to obtain pre-loaded human mesenchymal stem cells and human lung cancer cells into porous gel microspheres. The crosslinking environment was room temperature for 2 minutes, but the temperature had to be kept below 4°C from removal from the freezer until crosslinking. To increase light reflection during crosslinking, aluminum foil was added to the inner wall of the culture dish. All processes required sterilization. The liquid in the culture dish was then completely replaced with preheated culture medium, and the cells were transferred to new culture dishes / well plates for culture. The culture medium was changed every 48 hours with fresh medium for cell culture.

[0130] This implementation scheme optimizes the hydrogel component that directly contacts the cells, namely the aqueous phase solution. Lactose, raffinose, or menotriose are added to the aqueous phase solution for preparing the microspheres to optimize the formulation of the porous cryogel microspheres. Specifically, different sugars are added to the GelMA microsphere material, such as... Figure 4 As shown, from left to right, they are raffinose (Raf), lactose (Lac), and melitriose (Mel). The results at this point have not yet been optimized by the cryopreservation solution. The total volume of the cryopreservation solution is used as the basis. The composition of the cryopreservation solution includes: 0-15 mL / 100 mL of DMSO, with the remainder being culture medium.

[0131] Figure 4 The results showed that adding raffinose to the aqueous solution used to prepare the microspheres maximized the preservation of cell viability. Therefore, raffinose was subsequently used to optimize the cryopreservation solution (i.e., the cryoprotective solution around the microspheres).

[0132] Optimization of cryopreservation solution for porous cryogel microspheres

[0133] (1) In this implementation plan, human mesenchymal stem cells and human lung cancer cells were selected as pre-loaded cells. Human mesenchymal stem cells and human lung cancer cells were cultured in a special culture medium, which was changed every two days. After the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin, centrifuged, and then re-vortexed with an aqueous solution containing methacrylated gelatin synthesized from low-gel strength porcine skin gelatin. This solution was used as the aqueous phase for microfluidic experiments. The cell density was 10-1. 7 (cells / mL). Before adding cells, the methacrylated gelatin solution needs to be sterilized by passing it through a filter membrane (0.22 μm pore size). The solvent of the aqueous phase solution is 1× phosphate buffer solution, 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 aqueous phase solution was 2 mL per hour to obtain gel-state microdroplets.

[0135] Gel-like microdroplets can be enriched by methods such as centrifugation and sedimentation, and then further cleaned with a washing solution. 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-like microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0136] (3) After centrifugation and removal of the supernatant from the gel microdroplets, culture medium containing the photoinitiator was added. The mixture was then kept in a 4°C freezer to prevent the gel microdroplets from melting, ensuring that LAP fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish, and the optimized cryopreservation solution was added. The dish was then placed in a low-temperature freezer for gradient freezing at -20°C for 20 minutes, and then transferred to a -80°C freezer for long-term storage. The photoinitiator content was 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator. The diameter of the culture dish during freezing was 35 mm, and the volume ratio of the gel microdroplets to the cryopreservation solution in the culture dish during freezing was 1:2.

[0137] (4) Cell-loaded porous gel microdroplets were frozen and stored at -80°C. Before use, the microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, which was then used to crosslink methacrylated gelatin to obtain pre-loaded human mesenchymal stem cells and human lung cancer cells into porous gel microspheres. The crosslinking environment was room temperature for 2 minutes, but the temperature had to be kept below 4°C from removal from the freezer until crosslinking. To increase light reflection during crosslinking, aluminum foil was added to the inner wall of the culture dish. All processes required sterilization. The liquid in the culture dish was then completely replaced with preheated culture medium, and the cells were transferred to new culture dishes / well plates for culture. The culture medium was changed every 48 hours with fresh medium for cell culture.

[0138] like Figure 5 As shown in a, the cryopreservation solution, based on its total volume, comprises: 0, 5, 10, or 15 mL / 100 mL of DMSO, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the remainder being culture medium. The CRT1 group was not cryopreserved but directly cross-linked.

[0139] like Figure 5 As shown in b, based on the total volume of the cryopreservation solution, the composition of the cryopreservation solution includes: 0, 5, 10, or 15 mL / 100 mL of DMSO, 0, 4, 8, or 12 g / 100 mL of sugars, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the remainder being culture medium. The cryopreservation solution formulation is as follows: Figure 5 As shown in b in the figure.

[0140] like Figure 5 The results showed that the optimal combination of sugar and DMSO concentrations in the cryopreservation solution was 10% DMSO + 4% raffinose. Specifically, based on the total volume of the cryopreservation solution, the composition of the cryopreservation solution included 10 mL / 100 mL of DMSO, 4 g / 100 mL of sugar, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the remainder being culture medium. This combination increased the cell viability (on the first day after thawing) of the prepared pre-loaded cell porous gel microspheres 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 high-yield frozen porous gel microspheres before process optimization, resulting in the formation of non-porous microspheres. Figure 6 a) or the pores appear on the surface rather than inside ( Figure 6The situation described in b) is problematic because tiny ice crystals melt rapidly, while gel-like microdroplets are solid when frozen. In the solid state, the rate of photocrosslinking reaction is significantly reduced, and ensuring uniform light transmission within the dish is also a challenge.

[0143] This implementation plan utilizes, for example Figure 1 The droplet microfluidic device shown is used to prepare porous gel microspheres of methacrylated gelatin.

[0144] By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution was 6 mL / h, and the flow rate of the aqueous phase solution was 1 mL / h. In this embodiment, by fine-tuning the device parameters, gel-like microdroplets with a particle size of approximately 230 micrometers were stably obtained. The aqueous phase solution contained methacrylated gelatin synthesized from low-gel strength pigskin gelatin. The solvent of the aqueous phase solution was a 1× phosphate buffer solution, and the content of methacrylated gelatin was 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0145] Gel-state microdroplets can be enriched by methods such as centrifugation and sedimentation, and then a cleaning solution, such as 0.9% sodium chloride solution, 1× phosphate buffer solution, or ultrapure water, can be 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 subsequent steps.

[0146] After centrifugation and removal of the supernatant from the gel-like microdroplets, a solution containing the photoinitiator LAP is added. The solution can be 0.9% sodium chloride solution, 1× phosphate buffer solution, or ultrapure water. The solution is incubated at 4°C to prevent melting of the gel-like microdroplets and ensure sufficient penetration of LAP into them. The gel-like microdroplet solution is then transferred to a petri dish and frozen at -80°C. The photoinitiator content is 0.15 g / 100 mL, based on the volume of solvent in the photoinitiator-containing liquid. Before process optimization, the petri dish was 60 mm in diameter and without aluminum foil on the inner wall. The volume ratio of gel-like microdroplets to cryopreservation solution was 1:4 during freezing. After process optimization, aluminum foil is added to the inner wall of the petri dish, the diameter is 35 mm, and the volume ratio of gel-like microdroplets to cryopreservation solution is 1:2 during freezing.

[0147] Cell-free gel microdroplets were frozen and stored at -80°C for 90 minutes. They were then removed from the -80°C freezer and placed in a 4°C freezer where they were irradiated with a 405nm wavelength light source for 2 minutes to cure. Alternatively, the gel microdroplets could be directly irradiated with a 405nm wavelength light source at -80°C for 2 minutes to cure. This resulted in porous gel microspheres with different morphologies.

[0148] This invention achieves optimized high yields by optimizing the size of the culture dish during freezing (preferably 35 mm), the ratio of gel microdroplets to cryopreservation solution within the culture dish during freezing (preferably 1:2), and the cross-linking environmental temperature and time after freezing (preferably 4°C for 2 minutes when cell-free, and room temperature for 2 minutes when carrying cells, but ensuring the temperature remains below 4°C from removal from the freezer until cross-linking). It also increases light reflection during cross-linking (preferably by adding tin foil to the inner wall of the culture dish, paying attention to sterilization procedures). Figure 6 As shown in c, the optimized product exhibits more uniform and appropriately sized pores, as well as better batch consistency.

[0149] In summary, through testing various materials and optimizing processes, and through complex scheme design, this invention has obtained cryogel microspheres with stable pore formation.

[0150] Example 1: Preparation of porous gel microspheres

[0151] (1) Preparation of methacrylated gelatin porous gel microspheres. This embodiment utilizes, as shown in the example... Figure 1 The droplet microfluidic device shown was used to prepare porous gel microspheres of methacrylated gelatin. By adjusting the flow rate of the droplet microfluidic device, the flow rate of the oil phase solution was 6 mL / h, and the flow rate of the aqueous phase solution was 1 mL / h. In this embodiment, by fine-tuning the device parameters, gel-state microdroplets with a particle size of approximately 230 micrometers were stably obtained. The solvent of the aqueous phase solution was a 1× phosphate buffer solution, and the content of methacrylated gelatin synthesized from low-gel strength pigskin gelatin was 10 g / 100 mL based on the volume of the solvent in the aqueous phase solution.

[0152] Gel-state microdroplets can be enriched by methods such as centrifugation and sedimentation, and then a cleaning solution, such as 0.9% sodium chloride solution, 1× phosphate buffer solution, or ultrapure water, can be 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 subsequent steps.

[0153] (2) After centrifugation and removal of the supernatant from the gel-like microdroplets, add a solution containing the photoinitiator LAP. The solution can be 0.9% sodium chloride solution, 1× phosphate buffer solution, or ultrapure water. Incubate at 4°C to prevent the gel-like microdroplets from melting, ensuring that LAP fully penetrates into the microdroplets. Then transfer the gel-like microdroplet solution to a petri dish and freeze at -80°C. The photoinitiator content is 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator.

[0154] (3) The gel-state microdroplets were frozen in a -80°C freezer for 90 minutes, then removed and irradiated with a 405nm wavelength light source for 120 seconds to cure the microdroplets and crosslink the methacrylated gelatin. Porous gel microspheres were finally obtained. The morphology of the microspheres is as follows: Figure 7 The wet porous frozen microgel is shown in the figure.

[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, the specific steps of which are as follows:

[0157] (1) In this embodiment, human mesenchymal stem cells and human lung cancer cells were selected as pre-loaded cells. The human mesenchymal stem cells and human lung cancer cells were cultured in a conventional cell culture medium used in the art, with the medium changed every two days. After the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin, centrifuged, and then re-vortexed with a prepared aqueous solution containing methacrylated gelatin synthesized from low-gel strength porcine skin gelatin. The cell density was 10-1. 7 (cells / mL). Before adding cells, the methacrylated gelatin solution must be sterilized by passing it through a filter membrane (0.22 μm pore size). The solvent for the aqueous phase solution is 1× phosphate buffer solution, 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 was controlled by adjusting the parameters of the droplet microfluidic device. The flow rate of the oil phase solution was 4-10 mL per hour, and the flow rate of the aqueous phase solution was 1-3 mL per hour, so that gel-like microdroplets with a particle size of about 230 micrometers could be stably obtained. The flow rate selection is related to the concentration of the aqueous phase material. Based on the volume of solvent in the aqueous 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 / h and the flow rate of the aqueous phase solution is 2.5 mL / h; based on the volume of solvent in the aqueous 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 / h and the flow rate of the aqueous phase solution is 2 mL / h; based on the volume of solvent in the aqueous 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 / h and the flow rate of the aqueous phase solution is 2.5 mL / h.

[0159] Gel-like microdroplets can be enriched by methods such as centrifugation and sedimentation, and then further cleaned with a washing solution. 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-like microdroplets. Through the enrichment-washing steps, the oil phase content can be reduced to a level that does not affect subsequent steps.

[0160] (3) After centrifugation and removal of the supernatant from the gel microdroplets, culture medium containing the photoinitiator LAP was added. The mixture was then kept in a 4°C freezer to prevent the gel microdroplets from melting, ensuring that LAP fully penetrated into the gel microdroplets. The gel microdroplet solution was then transferred to a culture dish, and an optimized cryopreservation solution was added. Based on the total volume of the cryopreservation solution, the composition of the cryopreservation solution included 10 mL / 100 mL of DMSO, 4 g / 100 mL of sugars, 40 mL / 100 mL of serum, 0.15 g / 100 mL of photoinitiator, and the remainder being culture medium. The dish was then placed in a low-temperature gradient freezer, placed at -20°C for 20 minutes, and then transferred to a -80°C freezer for long-term storage. The photoinitiator content was 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator. The diameter of the culture dish during freezing was 35 mm, and the volume ratio of the gel microdroplets to the cryopreservation solution in the culture dish during freezing was 1:2.

[0161] (4) Cell-loaded porous gel microdroplets were frozen and stored at -80°C. Before use, the microdroplets were irradiated with a 405nm wavelength light source for 120 seconds to solidify them, which was then used to crosslink methacrylated gelatin to obtain pre-loaded human mesenchymal stem cells and human lung cancer cells into porous gel microspheres. The crosslinking environment was room temperature for 2 minutes, but the temperature had to be kept below 4°C from removal from the freezer until crosslinking. To increase light reflection during crosslinking, aluminum foil was added to the inner wall of the culture dish. All processes required sterilization. The liquid in the culture dish was then completely replaced with preheated culture medium, and the cells were transferred to new culture dishes / well plates for culture. The culture medium was changed every 48 hours with fresh medium for cell culture.

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

[0163] This comparative example provides a conventional method for preparing methacrylated gelatin gel microspheres, with the following specific steps:

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

[0165] (3) After centrifugation, remove the supernatant from the gel-like microdroplets, add the culture medium containing the photoinitiator LAP, and let it stand at 4°C to prevent the gel-like microdroplets from melting, ensuring that LAP fully penetrates into the gel-like microdroplets. The photoinitiator content is 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator.

[0166] (4) The gel-state microdroplet solution was then transferred to a petri dish and cured by irradiation with a 405 nm wavelength light source for 120 seconds, for crosslinking methacrylated gelatin. The microsphere morphology is as follows: Figure 7 As shown in the traditional microgels.

[0167] Comparative Example 2: Preparation of preloaded cell gel microspheres using conventional methacrylated gelatin.

[0168] This comparative example provides a traditional methacrylated gel microsphere preparation method for preloading human mesenchymal stem cells and human lung cancer cells. The specific steps are as follows:

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

[0170] (3) After centrifugation, remove the supernatant from the gel-like microdroplets, add the culture medium containing the photoinitiator LAP, and let it stand at 4°C to prevent the gel-like microdroplets from melting, ensuring that LAP fully penetrates into the gel-like microdroplets. The photoinitiator content is 0.15 g / 100 mL, based on the volume of solvent in the liquid containing the photoinitiator.

[0171] (4) The gel microdroplet solution was then transferred to a culture dish and irradiated with a 405 nm wavelength light source for 120 seconds to solidify the gel microdroplets, which was used to crosslink the methacrylated gelatin. The liquid in the culture dish was then completely replaced with preheated culture medium, and the solution was transferred to a culture dish / well plate for culture. The culture medium was changed every 48 hours with fresh medium added for cell culture.

[0172] Culture and evaluation of pre-loaded human mesenchymal stem cells and human lung cancer cells in porous gel microspheres

[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 and cultured. The culture medium was changed 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 In the image, 'a' represents the staining images of traditional microgels and wet-state frozen porous gel microspheres that support various cell cultures. Figure 8Image b in the image is a stained cross-sectional view of wet-state frozen porous gel microspheres carrying human lung cancer cells. From... Figure 8 As can be seen from 'a', cells struggle to extend within traditional gel microspheres, while in the porous gel microsphere system of this invention, cells can grow well in three dimensions. Human mesenchymal stem cells can extend and spread out, and human lung cancer cells can aggregate into clusters (e.g., Figure 8 (As shown in b) The porous gel microspheres provided by this invention have a more biomimetic morphology, which is beneficial for cell function. Figure 8 As shown in a, in Example 1, the pre-loaded human mesenchymal stem cell porous gel microspheres remained viable after being cryopreserved for one month and then revived and cultured.

[0175] like Figure 9 The image shows the Alizarin Red staining results of the wet-frozen porous gel microspheres after one week of culture and two weeks of osteogenic differentiation. The pre-loaded human mesenchymal stem cell porous gel microspheres prepared in Example 2 retained their osteogenic differentiation capacity after one month of cryopreservation, and the Alizarin Red staining results showed a large number of dense calcium nodules (solid particles in dark areas 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, through optimization, has been proven to support the cryopreservation of a variety of cells while maintaining their viability and function, including stem cells and tumor cells. These cells grow well in the porous gel microspheres of the present invention, with mesenchymal stem cells able to spread out and tumor cells able to aggregate and exhibit good growth.

[0177] Detection of cell viability and proliferation rate after long-term cryopreservation of pre-loaded porous gel microspheres

[0178] The cryopreservation of in situ cell-loaded porous gel microspheres is challenging. The traditional methacrylated gelatin preloaded cell porous gel microspheres prepared in Comparative Example 2 were compared with the porous gel microspheres preloaded with human mesenchymal stem cells and human lung cancer cells prepared in Example 2 by optimizing the materials, cryopreservation solution formulation and cryopreservation process.

[0179] The survival rate and proliferation rate of cell-loaded wet porous cryogel microspheres after long-term cryopreservation (determined by CCK8 assay), based on the volume of solvent in the aqueous solution, Figure 10 (a), (b), and (c) in the figures represent porous microspheres loaded with human mesenchymal stem cells (MSCs) prepared with methacrylated gelatin at concentrations of 5 g / 100 mL, 10 g / 100 mL, and 12.5 g / 100 mL (i.e., material concentrations of 5%, 10%, and 12.5%), respectively. Figure 10 In the example, (d) represents porous microspheres loaded with A549 human lung cancer cells prepared with methacrylated gelatin contents of 5 g / 100 mL and 10 g / 100 mL.

[0180] like Figure 10 As shown in (a), the survival rate and proliferation rate of samples with a methacrylated gelatin content of 5 g / 100 mL remained essentially unchanged after one month of freezing. However, after three months of freezing, the survival rate decreased by approximately 10%, and the proliferation rate significantly decreased. On day seven, the proliferation rate was only half that of the group frozen for one month, comparable to the non-frozen control group. Figure 10 As shown in (b), at a material concentration of 10%, the cell-loaded porous gel microsphere system exhibited the best long-term cryopreservation capability, maintaining a 74% survival rate after three months of freezing, recovering to 90% after seven days of culture, and with a proliferation rate approaching 500% of that on the first day, which is 1.5 times that of the non-frozen group. Figure 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, leading to 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] Porous gels containing MSCs (human mesenchymal stem cells) at a material concentration of 10% are ideal for long-term cryopreservation, significantly reducing cell damage caused by cryopreservation with a medium, especially for stem cells. The value of cryopreservation lies in overcoming the time constraints of immediate preparation and use, enabling access at different times and locations. For patients, it allows for single extraction, long-term cryopreservation, and on-demand use, avoiding repeated trauma.

[0182] like Figure 10 As shown in (d), the material concentration in the figure represents the concentration of GelMA material synthesized from pigskin gelatin with low gel strength for the microspheres. The A549 cells used are human lung cancer cells (tumor cells). After two months of freezing, the survival rate of the A549 human lung cancer cells (tumor cells) was 70%, which is in line with the usual level in the art. After seven days of culture, the survival rate was 85%, similar to that of conventional microgels loaded with A549, exceeding 90%. This indicates that the cell-loaded porous gel microsphere system of the present invention is also applicable to other cell types.

[0183] Application of pre-loaded cell porous gel microspheres in in vivo injection

[0184] (1) Preparation of bio-ink. Cell-loaded porous gel microspheres can be used in conjunction with therapeutic purposes, such as stem cell therapy. The prepared cell-loaded conventional 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, which remained in a gel state at a low temperature (0-10℃). A 70μm cell sieve was placed on an open 50mL centrifuge tube, and then the cell-loaded gel microsphere solution was transferred to the cell sieve. The tube was centrifuged at 50×g for 2 minutes to remove the liquid outside the microspheres, and the microspheres were enriched 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 extruded subcutaneously into the backs of male SD rats via a needle. The wet-state frozen porous gel microsphere ink provided by this invention has excellent extrusion properties, such as… Figure 11 As shown.

[0186] Rats were subcutaneously injected with conventional microgels and wet-state frozen porous gel microspheres and cultured for two weeks. Figure 12 The white dashed circle indicates undegraded material, such as... Figure 12 As shown, the wet-state frozen porous gel microspheres provided by this invention formed collagen fibers and uniform tissue ingrowth after two weeks. Subcutaneous injection experiments in rats demonstrated good biocompatibility and accelerated vascular and tissue ingrowth.

[0187] Application of pre-loaded cell-porous gel microspheres combined with bioprinting 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 open 50 mL centrifuge tube, and the cell-loaded porous gel microsphere solution was transferred to the cell sieve. The tube was centrifuged at 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. Enriched microspheres were loaded as bio-ink onto the nozzle of an extrusion printer for printing. After printing, the hydrogel structure was selectively irradiated with a 405nm wavelength light source, immersed in a culture medium, and incubated at 37°C. Figure 13 As shown, the wet-state frozen porous gel microspheres provided by the present invention have highly interconnected pores, and the hydrogel structure has a porous structure, which supports the rapid transport of nutrients.

Claims

1. A method for preparing cryopreservable porous gel microspheres, wherein, The method includes: Aqueous and oil phase solutions were used to prepare gel-like microdroplets; wherein the aqueous phase solution contained methacrylated gelatin synthesized from low gel strength pigskin gelatin. The gel-like microdroplets are soaked in a liquid containing a photoinitiator, and after full penetration, they are frozen, causing the water inside the gel-like microdroplets to form ice crystals. Cross-linking and solidification of gel microdroplets that form ice crystals were carried out by ultraviolet light irradiation to obtain porous gel microspheres that can be frozen. The gel strength of the low gel strength pigskin gelatin is 100-150 Bloom g; The content of methacrylated gelatin is 2-20 g / 100 mL, based on the volume of solvent in the aqueous solution. The specific steps of the droplet microfluidic method include: adding the aqueous solution and the oil solution into the microfluidic device, adjusting the flow rates of the aqueous solution and the oil solution respectively, and mixing them; 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. The porous gel microspheres have a particle size of 150-400 micrometers.

2. The method according to claim 1, wherein, The method includes: The aqueous solution contains cells to be preloaded; The freezing process involves transferring gel-like microdroplets into a cryopreservation solution and then subjecting them to gradient freezing.

3. The method according to claim 1 or 2, wherein, The aqueous solution contains methacrylated gelatin synthesized from low gel strength porcine skin gelatin and a blend phase for assisting cell culture.

4. The method according to claim 1 or 2, wherein the content of the methacrylated gelatin is 2.5-10 g / 100mL based on the volume of the solvent in the aqueous solution.

5. The method according to claim 3, wherein, The content of the blended phase is 2.5-10 g / 100mL, based on the volume of the solvent in the aqueous solution.

6. The method according to claim 3, wherein, The blend phase is selected from one 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 solution also contains sugars, and the sugar content is 1-10 g / 100 mL based on the volume of the solvent in the aqueous solution.

8. The method according to claim 7, wherein, The sugars include one or more of lactose, raffinose, and mesotriose.

9. The method according to claim 2, wherein, The cell density in the aqueous solution is 1×10⁻⁶. 3 -1×10 8 per mL.

10. The method according to claim 2, wherein, The cells include one or more of human mesenchymal stem cells, cancer cells, fibroblasts, and endothelial cells.

11. The method according to claim 1 or 2, wherein, The oil phase solution is obtained by mixing an oily material with a surfactant.

12. The method according to claim 11, wherein, The oily material includes one or more of mineral oil, fluorinated oil, and silicone oil.

13. The method according to claim 11, wherein, The content of the surfactant is 1-5% based on the volume of the oil phase solution as 100%.

14. The method of claim 11, wherein, The surfactant includes one or more of Span80, Tween 20, AEO-3, and FluoSurf.

15. The method according to claim 1 or 2, wherein, The photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

16. The method according to claim 1 or 2, wherein, The content of the photoinitiator is 0.1-0.5 g / 100mL, based on the volume of the solvent in the liquid containing the photoinitiator.

17. The method according to claim 2, wherein, The volume ratio of the cryopreservation solution to the gel microdroplets is 1:(1-10).

18. The method according to claim 2, wherein, The volume ratio of the cryopreservation solution to the gel microdroplets is 1:(1-3).

19. The method according to claim 2, wherein, The gradient freezing process includes: standing at -10°C to -25°C for 20-60 minutes, and then transferring to an environment of -80°C to -196°C for storage.

20. The method according to claim 1 or 2, wherein, The duration of ultraviolet light irradiation is 30-180 seconds.

21. The method according to claim 2, wherein, Based on the total volume of the cryopreservation solution, the cryopreservation solution comprises: 0-15 mL / 100 mL of DMSO, 0-12 g / 100 mL of sugars, 0-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the remainder being culture medium.

22. The method of claim 21, wherein, Based on the total volume of the cryopreservation solution, the cryopreservation solution comprises: 1-15 mL / 100 mL of DMSO, 1-12 g / 100 mL of sugars, 1-40 mL / 100 mL of serum, 0.1-0.5 g / 100 mL of photoinitiator, and the remainder being culture medium.

23. The method according to claim 21, wherein the sugar comprises one or more of lactose, raffinose, and menotriose.

24. The method according to claim 21, wherein, The photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphite and / or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

25. The cryopreservable porous gel microspheres prepared by the method of any one of claims 1-24, wherein, The porous gel microspheres have a particle size of 200-250 micrometers.

26. The porous gel microspheres according to claim 25, wherein, The average equivalent diameter of the internal pores of the porous gel microspheres is 20-80 micrometers.

27. A bio-ink prepared from cryopreservable porous gel microspheres prepared by the method of any one of claims 1-24 or cryopreservable porous gel microspheres as described in claim 25 or 26.

28. A bio-3D printed article, which is prepared from the cryopreservable porous gel microspheres obtained by the method of any one of claims 1-24, the cryopreservable porous gel microspheres of claim 25 or 26, or the bio-ink of claim 27.

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