Gelatin-based high internal phase emulsion as well as preparation method and application thereof

Abstract

The invention relates to a gelatin-based high internal phase emulsion as well as a preparation method and application thereof. The gelatin-based high internal phase emulsion comprises a water phase and an oil phase, the water phase comprises gelatin, an emulsifier and water, and the oil phase comprises an oily substance; the gelatin-based high internal phase emulsion is an oil-in-water type high internal phase emulsion, and the particle size dispersion index of oil phase liquid drops in the gelatin-based high internal phase emulsion is 0.05-0.40. The oil-phase liquid drops in the gelatin-based high-internal-phase emulsion are spherical and good in particle size uniformity, and the cell 3D culture carrier prepared from the gelatin-based high-internal-phase emulsion has the advantages of being good in aperture uniformity, good in biocompatibility and high in nutrition transfer efficiency.

Description

Technical Field

[0001] This invention relates to the field of high internal phase emulsion technology, and in particular to a gelatin-based high internal phase emulsion, its preparation method, and its application. Background Technology

[0002] 3D cell culture has become a core technology in fields such as drug screening and tissue engineering because it can more realistically simulate the physiological environment in vivo. However, existing 3D cell culture carriers have significant limitations: although block hydrogels have biocompatibility, their low internal porosity makes it difficult for nutrients to penetrate to the central area, leading to cell necrosis. In addition, their fixed size makes it impossible to achieve high-throughput culture. Fiber carriers have better size uniformity, but poor biocompatibility and are prone to cytotoxic residues. Moreover, the materials used are mostly difficult to degrade, and cell recovery requires high-intensity enzymatic hydrolysis or chemical treatment, which can easily damage cell viability.

[0003] High internal phase emulsions (HIPEs), also known as ultra-concentrated emulsions or gel emulsions, are a type of emulsion with a dispersed phase volume fraction of 74% or higher. The porous materials formed after solidification possess advantages such as high specific surface area, high porosity, and light weight, making them applicable to various fields including cell culture and daily chemical hygiene products. In the daily chemical hygiene product field, such as sanitary napkins and dressings, they offer advantages such as absorption, flow conduction, water retention, anti-backflow, and sustained release. In the cell culture field, they have the potential to simulate a three-dimensional microenvironment. However, traditional preparation methods (such as mechanical stirring) for HIPEs have two major problems: 1. Uncontrolled morphology and size: The dispersed phase easily forms lumps or irregular particles with uneven particle size, resulting in porous materials that cannot meet the uniformity requirements of high-throughput culture; 2. Insufficient biocompatibility: Reliance on high-concentration synthetic surfactants or chemical cross-linking agents leads to cytotoxicity, making them difficult to use directly in cell culture.

[0004] Therefore, there is a need to develop a 3D cell culture vector with good size uniformity, high nutrient delivery efficiency, and good biocompatibility. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a gelatin-based high internal phase emulsion, its preparation method, and its application. In the gelatin-based high internal phase emulsion, the oil phase droplets are spherical with good particle size uniformity. The cell 3D culture carrier made using the gelatin-based high internal phase emulsion has the advantages of good pore size uniformity, good biocompatibility, and high nutrient delivery efficiency.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a gelatin-based high internal phase emulsion, the gelatin-based high internal phase emulsion comprising an aqueous phase and an oil phase, the aqueous phase comprising gelatin, an emulsifier and water, and the oil phase comprising oily substances; the gelatin-based high internal phase emulsion is an oil-in-water type high internal phase emulsion, and the particle size distribution index (PDI) of the oil phase droplets in the gelatin-based high internal phase emulsion is 0.05~0.40 (e.g. 0.10, 0.15, 0.20, 0.25, 0.30 or 0.35, etc.), more preferably 0.05~0.20.

[0008] In this invention, the oil phase droplets in the gelatin-based high internal phase emulsion exhibit good particle size uniformity. The 3D cell culture carrier made using the gelatin-based high internal phase emulsion has good pore size uniformity and three-dimensional microenvironment simulation capability. The gelatin in the gelatin-based high internal phase emulsion can achieve temperature-sensitive curing. After being made into a 3D cell culture carrier, the gelatin is also easily digestible, liquefying at temperatures above 37°C or rapidly degrading after trypsin treatment, facilitating cell recovery. The cell recovery efficiency can reach ≥90%, solving the problems of poor biocompatibility, insufficient nutrient delivery, and difficulty in cell recovery of traditional 3D cell culture carriers.

[0009] Preferably, the mass of the gelatin is 8% to 20% of the total mass of the aqueous phase, for example, 10%, 12%, 14%, 16% or 18%, etc.

[0010] Preferably, the emulsifier is 1% to 5% of the total mass of the aqueous phase, for example, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%, etc.

[0011] Preferably, the emulsifier comprises a nonionic surfactant, and more preferably, the nonionic surfactant comprises Tween 80.

[0012] In this invention, the aqueous phase is a compound of gelatin and a low-toxicity nonionic surfactant. By utilizing the amphiphilic properties of gelatin (hydrophilic groups such as amino and / or carboxyl groups, hydrophobic segments such as alanine and / or leucine) and the synergistic effect of Tween 80, the oil-water interfacial tension can be reduced, and the aggregation of oil droplets under high internal phase conditions can be inhibited.

[0013] Preferably, the aqueous phase further includes a crosslinking agent, and more preferably, the crosslinking agent includes genipin and / or glutaraldehyde.

[0014] Preferably, the crosslinking agent has a mass of 0.01% to 5% based on the total mass of the aqueous phase (100%), such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5%.

[0015] Preferably, the aqueous phase further includes a cell culture nutrient.

[0016] In this invention, cell culture nutrients are preferably added to the aqueous phase to provide cell nutrition and promote cell proliferation.

[0017] Preferably, the cell culture nutrient solution comprises 20% to 40% of the total mass of the aqueous phase, for example, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, or 38%, etc.

[0018] This invention does not impose specific limitations on the type of cell culture nutrient; any cell culture nutrient that is soluble in water and can supply cell nutrition and promote cell proliferation is applicable. This invention provides only illustrative examples:

[0019] Preferably, the cell culture nutrient comprises a peptide containing an arginine-glycine-aspartic acid sequence (RGD).

[0020] In this invention, the preferred addition of peptides containing the arginine-glycine-aspartic acid sequence can promote cell adhesion without cytotoxicity. The addition of peptides containing the arginine-glycine-aspartic acid sequence as nutrients can improve the initial cell survival rate.

[0021] Preferably, the cell culture nutrient further includes any one or a combination of at least two of glucose, L-glutamine, and basic fibroblast growth factor.

[0022] Preferably, the cell culture nutrient comprises the following components by mass percentage: 20%~30% (e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% or 29%) of peptides containing the arginine-glycine-aspartic acid sequence, 30%~50% (e.g., 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46% or 48%) of glucose, 10%~30% (e.g., 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26% or 28%) of L-glutamine, and 5%~25% (e.g., 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21% or 23%) of basic fibroblast growth factor, with the sum of the mass percentages of each component being 100%.

[0023] Preferably, the oily substance includes a biocompatible oil.

[0024] Preferably, the biocompatible oil includes any one or a combination of at least two of medium-chain triglyceride oil (MCT oil), mineral oil, or soybean oil.

[0025] For example, the medium-chain triglyceride oil is of type MCT-60, and the mineral oil is of type 15# medical mineral oil.

[0026] Preferably, the oil phase further includes growth factors.

[0027] Preferably, the growth factor is 0.1% to 0.5% of the total mass of the oil phase, for example, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% or 0.45%, etc.

[0028] Preferably, the growth factor includes a hydrophobic growth factor.

[0029] Preferably, the hydrophobic growth factor includes vitamin D and / or paclitaxel.

[0030] In this invention, the oil phase is selected from biocompatible oils that can load hydrophobic growth factors to achieve a sustained-release function and promote cell differentiation. The synergistic effect between the gelatin network formed by the solidification of the aqueous phase in the gelatin-based high internal phase emulsion and the microregions of the oil phase helps to improve nutrient diffusion efficiency and enhance cell proliferation activity.

[0031] Preferably, the D50 particle size of the oil phase droplets in the oil-in-water emulsion is 10~1000 μm, such as 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm or 900 μm, and more preferably 50~500 μm.

[0032] In a second aspect, the present invention provides a method for preparing a gelatin-based high internal phase emulsion as described in the first aspect, the method comprising the following steps: mixing an aqueous phase and an oil phase by microfluidic control to obtain the gelatin-based high internal phase emulsion.

[0033] In this invention, the aqueous and oil phases of the gelatin-based high internal phase emulsion can remain liquid at room temperature (25°C), which facilitates microfluidic operation and subsequent preparation of solidified and shaped cell 3D culture carriers. By using microfluidics, gelatin-based high internal phase emulsions with high uniformity of oil phase droplet size can be prepared, solving the problem of uneven size of the dispersed phase in traditional stirring methods and meeting the uniformity requirements of high-throughput cell culture.

[0034] Preferably, the preparation method includes the following steps: (1) dissolving gelatin in water, adding emulsifier, optionally crosslinking agent and optionally cell culture nutrient, and mixing to obtain an aqueous phase; (2) mixing oily substances and optionally growth factors to obtain an oil phase; (3) injecting the aqueous phase obtained in step (1) and the oil phase obtained in step (2) into the continuous phase channel and dispersed phase channel of the microfluidic device, respectively, and the oil phase is sheared into uniform small spheres and dispersed in the aqueous phase in the focusing channel, and collected to obtain the gelatin-based high internal phase emulsion; wherein, steps (1) and (2) are performed in any order, or simultaneously.

[0035] Preferably, the dissolution temperature in step (1) is 60~70℃, such as 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃ or 69℃.

[0036] Preferably, the mixing in step (2) includes mixing using ultrasonic dispersion.

[0037] Preferably, the power of the ultrasonic dispersion is 100~200 W, such as 110 W, 120 W, 130 W, 140 W, 150 W, 160 W, 170 W, 180 W or 190 W.

[0038] Preferably, the ultrasonic dispersion time is 10-15 min, such as 10.5 min, 11 min, 11.5 min, 12 min, 12.5 min, 13 min, 13.5 min, 14 min or 14.5 min, etc.

[0039] Preferably, the temperature of the aqueous phase in step (3) is 35~45℃, such as 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃ or 44℃.

[0040] Preferably, the temperature of the oil phase in step (3) is 25~30℃, such as 25.5℃, 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 28.5℃, 29℃ or 29.5℃.

[0041] Preferably, the flow rate of the aqueous phase in step (3) is 50~500 μL / min, such as 100 μL / min, 150 μL / min, 200 μL / min, 250 μL / min, 300 μL / min, 350 μL / min, 400 μL / min or 450 μL / min.

[0042] Preferably, the flow rate of the oil phase in step (3) is 300~2000 μL / min, such as 500 μL / min, 700 μL / min, 900 μL / min, 1100 μL / min, 1300 μL / min, 1500 μL / min, 1700 μL / min or 1900 μL / min.

[0043] Preferably, the flow rate ratio of the water phase and the oil phase in step (3) is 1:(3~8), for example, 1:4, 1:5, 1:6 or 1:7, etc.

[0044] In this invention, microfluidics is used to precisely control the oil phase droplets to form small spheres, and to control the water phase flow rate, oil phase flow rate, and flow rate ratio, thereby precisely controlling the particle size of the oil phase droplets.

[0045] Thirdly, the present invention provides a 3D cell culture carrier, which is prepared from a gelatin-based high internal phase emulsion as described in the first aspect.

[0046] Fourthly, the present invention provides a method for preparing a 3D cell culture carrier as described in the third aspect, the method comprising the following steps: solidifying a gelatin-based high internal phase emulsion as described in the first aspect to obtain the 3D cell culture carrier.

[0047] Preferably, the curing includes static curing.

[0048] Preferably, the static curing temperature is 0~20℃, such as 2℃, 4℃, 6℃, 8℃, 10℃, 12℃, 14℃, 16℃ or 18℃.

[0049] Preferably, the static curing time is 10 to 30 minutes, such as 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, or 28 minutes.

[0050] Preferably, the step of heating and curing is further included before the static curing.

[0051] Preferably, the heating and curing temperature is 30~35℃, such as 30.5℃, 31℃, 31.5℃, 32℃, 32.5℃, 33℃, 33.5℃, 34℃ or 34.5℃, etc.

[0052] Preferably, the heating and curing time is 2 to 4 hours, such as 2.2 hours, 2.4 hours, 2.6 hours, 2.8 hours, 3.0 hours, 3.2 hours, 3.4 hours, 3.6 hours, or 3.8 hours.

[0053] Preferably, the curing process further includes washing and drying steps.

[0054] In this invention, when the prepared 3D cell culture carrier is used for short-term culture (≤7 days), no cross-linking agent needs to be added to the aqueous phase. The gelatin can be physically cured by standing at a low temperature of 0~20℃. If it is used for long-term culture (>7 days) or in scenarios that require frequent medium changes, a cross-linking agent can be added to the aqueous phase. The gelatin-based high internal phase emulsion can be cross-linked by heating and curing, which can improve the strength of the 3D cell culture carrier and ensure the integrity of the carrier.

[0055] Fifthly, the present invention provides the application of a gelatin-based high internal phase emulsion as described in the first aspect or a cell 3D culture carrier as described in the third aspect in cell culture, drug screening, tissue engineering scaffolds, or daily chemical hygiene products.

[0056] For example, the cell 3D culture carrier is used in the field of cell culture and can provide cells with a 3D environment that is closer to the natural tissue environment.

[0057] For example, the porous material obtained by curing and drying the gelatin-based high internal phase emulsion can be used in the field of sanitary napkins, dressings, and other daily chemical hygiene products. One or more layers of porous materials with the same or different pore sizes can be used as the core of sanitary napkins, dressings, and other daily chemical hygiene products. Through its high porosity, high connectivity, and controllable pore size, it can absorb, guide, lock in water, prevent backflow, or slowly release liquids such as water, menstrual blood, blood, body fluids, and nutrients, giving the product excellent properties such as thinness, dryness, long-lasting effect, and slow release.

[0058] Compared with the prior art, the present invention has at least the following beneficial effects:

[0059] The gelatin-based high internal phase emulsion of this invention exhibits good droplet size uniformity in the oil phase. The 3D cell culture carrier prepared using this gelatin-based high internal phase emulsion exhibits good pore size uniformity and three-dimensional microenvironment simulation capabilities. The synergistic effect of the aqueous gelatin network and the oil phase microregions enhances nutrient diffusion efficiency and improves cell proliferation activity. The gelatin in the gelatin-based high internal phase emulsion can achieve temperature-sensitive curing. After being prepared into a 3D cell culture carrier, the gelatin is also easily digestible, liquefying at temperatures above 37°C or rapidly degradable by trypsin treatment, facilitating cell recovery and achieving high cell recovery efficiency. This solves the problems of poor biocompatibility, insufficient nutrient delivery, and difficult cell recovery associated with traditional 3D cell culture carriers. The nutrient delivery efficiency of the 3D cell culture carrier is ≥68 μm / h, the cell adhesion rate of inoculated vascular endothelial cells is ≥88%, and the cell viability is ≥88%. Preferably, the nutrient delivery efficiency is ≥78 μm / h, the cell adhesion rate of inoculated vascular endothelial cells is ≥92%, and the cell viability is ≥90%. Attached Figure Description

[0060] Figure 1 SEM image of the cell 3D culture vector provided in Example 1. Detailed Implementation

[0061] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0062] Unless otherwise specified, the materials and equipment involved in the following detailed embodiments are all conventional materials and equipment in the art and will not affect the technical effects of the present invention.

[0063] The sources of some of the raw materials used in the following examples and comparative examples are as follows:

[0064] Gelatin: Bloom strength 250, manufacturer is Shijiazhuang Jinda Gelatin;

[0065] Medium-chain triglyceride oil: Model number MCT-60, manufacturer: Heze Zhonghe Jianyuan;

[0066] Mineral oil: Type 15 medical mineral oil, manufactured by Maoming Petrochemical;

[0067] Soybean oil: Grade A soybean oil, manufactured by Golden Dragon Fish;

[0068] Peptide containing the arginine-glycine-aspartic acid sequence: model number IA4280, manufacturer Solarbio.

[0069] Genipin: Model G7790, manufacturer Solarbio.

[0070] Tween 80: Manufacturer is Croda, UK.

[0071] Example 1

[0072] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a 3D cell culture carrier. The gelatin-based high internal phase emulsion is prepared by the following method:

[0073] (1) Add gelatin to deionized water at 60°C and stir until completely dissolved. Add emulsifier (Tween 80) and cell culture nutrient solution and stir evenly to obtain an aqueous phase.

[0074] Based on the total mass of the above aqueous phase as 100%, the mass of gelatin is 15%, the mass of emulsifier is 3%, the mass of cell culture nutrient is 30%, and the remainder is deionized water.

[0075] The cell culture nutrient solution described above comprises the following components by weight percentage: 25% of a peptide (IA4280) containing the arginine-glycine-aspartic acid sequence, 40% glucose, 20% L-glutamine, and 15% basic fibroblast growth factor.

[0076] (2) Mix the oil (medium-chain triglyceride oil) and growth factor (vitamin D), and ultrasonically disperse for 15 min at a power of 100W to obtain the oil phase;

[0077] Based on the total mass of the oil phase being 100%, the mass of the growth factor is 0.3%.

[0078] (3) The aqueous phase obtained in step (1) at 40°C and the oil phase obtained in step (2) at 25°C are injected into the continuous phase channel and the dispersed phase channel of the microfluidic chip, respectively. The flow rate of the aqueous phase is 100 μL / min and the flow rate of the oil phase is 400 μL / min. The oil phase is sheared into uniform small spheres in the focusing channel and dispersed in the aqueous phase, and flows out from the outlet to obtain the gelatin-based high internal phase emulsion.

[0079] The cell 3D culture carrier was prepared by the following method: the gelatin-based high internal phase emulsion was allowed to stand at 20°C for 30 min to solidify, washed, and dried to obtain the cell 3D culture carrier.

[0080] Example 2

[0081] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a 3D cell culture carrier. The gelatin-based high internal phase emulsion is prepared by the following method:

[0082] (1) Add gelatin to deionized water at 65°C and stir until completely dissolved. Add emulsifier (Tween 80), crosslinking agent (Genipin) and cell culture nutrient solution and stir evenly to obtain an aqueous phase.

[0083] Based on the total mass of the above aqueous phase as 100%, the mass of gelatin is 20%, the mass of emulsifier is 1%, the mass of crosslinking agent is 5%, the mass of cell culture nutrient is 20%, and the remainder is deionized water.

[0084] The cell culture nutrient solution described above comprises the following components by weight percentage: 25% of a peptide (IA4280) containing the arginine-glycine-aspartic acid sequence, 40% glucose, 20% L-glutamine, and 15% basic fibroblast growth factor.

[0085] (2) Mix the oil (mineral oil) and growth factor (paclitaxel), and ultrasonically disperse for 12 min at a power of 150W to obtain the oil phase;

[0086] Based on the total mass of the oil phase being 100%, the mass of the growth factor is 0.1%.

[0087] (3) The aqueous phase obtained in step (1) at 35°C and the oil phase obtained in step (2) at 28°C are injected into the continuous phase channel and the dispersed phase channel of the microfluidic chip, respectively. The flow rate of the aqueous phase is 150 μL / min and the flow rate of the oil phase is 1200 μL / min. The oil phase is sheared into uniform small spheres in the focusing channel and dispersed in the aqueous phase, and flows out from the outlet to obtain the gelatin-based high internal phase emulsion.

[0088] The cell 3D culture carrier was prepared by the following method: the gelatin-based high internal phase emulsion was heated and cured at 30°C for 4 h, allowed to stand at 20°C for 30 min, washed, and dried to obtain the cell 3D culture carrier.

[0089] Example 3

[0090] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a 3D cell culture carrier. The gelatin-based high internal phase emulsion is prepared by the following method:

[0091] (1) Add gelatin to deionized water at 70°C and stir until completely dissolved. Add emulsifier (Tween 80), crosslinking agent (glutaraldehyde) and cell culture nutrient (peptide containing arginine-glycine-aspartic acid sequence) and stir until homogeneous to obtain aqueous phase.

[0092] Based on the total mass of the above aqueous phase as 100%, the mass of gelatin is 10%, the mass of emulsifier is 5%, the mass of crosslinking agent is 1%, the mass of cell culture nutrient is 40%, and the remainder is deionized water.

[0093] The cell culture nutrient solution described above comprises the following components by weight percentage: 25% of a peptide (IA4280) containing the arginine-glycine-aspartic acid sequence, 40% glucose, 20% L-glutamine, and 15% basic fibroblast growth factor.

[0094] (2) Mix the oil (soybean oil) and growth factor (paclitaxel), and ultrasonically disperse for 10 min at a power of 200W to obtain the oil phase;

[0095] Based on the total mass of the oil phase being 100%, the mass of the growth factor is 0.5%.

[0096] (3) The aqueous phase obtained in step (1) at 45°C is added to the oil phase obtained in step (2) at 30°C and injected into the continuous phase channel and dispersed phase channel of the microfluidic chip respectively. The flow rate of the aqueous phase is 200 μL / min and the flow rate of the oil phase is 600 μL / min. The oil phase is sheared into uniform small spheres in the focusing channel and dispersed in the aqueous phase, and flows out from the outlet to obtain the gelatin-based high internal phase emulsion.

[0097] The cell 3D culture carrier was prepared by the following method: the gelatin-based high internal phase emulsion was heated and cured at 35°C for 2 h, allowed to stand at 20°C for 30 min, washed, and dried to obtain the cell 3D culture carrier.

[0098] Example 4

[0099] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a cell 3D culture carrier. The difference between this embodiment and Example 1 is that in step (1), the mass percentage of gelatin is adjusted to 5% based on the total mass of the aqueous phase as 100%, while other conditions are the same as in Example 1.

[0100] Example 5

[0101] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a cell 3D culture carrier. The difference between this embodiment and Example 1 is that in step (1), the mass percentage of gelatin is adjusted to 25% based on the total mass of the aqueous phase as 100%, while other conditions are the same as in Example 1.

[0102] Example 6

[0103] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a cell 3D culture carrier. The difference between this embodiment and Example 1 is that in step (3), the flow rate of the aqueous phase is adjusted to 100 μL / min, the flow rate of the oil phase is adjusted to 600 μL / min, and other conditions are the same as in Example 1.

[0104] Comparative Example 1

[0105] This embodiment provides a gelatin-based high internal phase emulsion, its preparation method, and a cell 3D culture carrier. The difference between this embodiment and Example 1 is that step (3) is adjusted to add the aqueous phase obtained in step (1) at 40°C at 5 mL / s to the oil phase obtained in step (2) at 25°C under stirring at 500 rpm. The volume ratio of the aqueous phase to the oil phase is 1:4. Stirring is continued at 500 rpm for 30 min to obtain the gelatin-based high internal phase emulsion. Other conditions are the same as in Example 1.

[0106] The gelatin-based high internal phase emulsions provided in Examples 1-6 and Comparative Example 1 were subjected to the following performance tests.

[0107] Particle size testing: Dynamic light scattering was performed using a Malvern Zetasizer Nano ZS at a temperature of 25°C. Each sample was tested three times and the average value was taken.

[0108] The cell 3D culture vectors provided in Examples 1-6 and Comparative Example 1 were subjected to the following performance tests.

[0109] (1) Pore structure: The pore structure and pore size of the cell 3D culture carrier were observed by SEM (TESCAN MIRA3).

[0110] (2) Nutrient transfer efficiency: FITC fluorescent probe (1 mg / mL) was dropped onto the carrier surface, and the diffusion distance within 2 h was observed using a confocal microscope (Leica) to calculate the transfer rate (μm / h).

[0111] (3) Biocompatibility

[0112] ① Cell adhesion rate: The CCK-8 assay was used for testing. Endothelial cells of the same density were seeded onto a 3D cell culture carrier and a culture dish containing culture medium, serving as the test group and control group, respectively. After 24 h, unadhered endothelial cells were washed with phosphate-buffered saline (PBS) to remove them, and absorbance (OD value) was measured to obtain the OD values ​​of the test group and control group. The 3D cell culture carrier without endothelial cells was the first blank group, and its absorbance was measured and recorded as blank group OD1. The culture medium without endothelial cells was the second blank group, and its absorbance was measured and recorded as blank group OD2.

[0113] Cell adhesion rate = (OD of test group - OD1 of blank group) / (OD of control group - OD2 of blank group) × 100%.

[0114] ② Cell viability: The CCK-8 assay was used for testing. Endothelial cells of the same density were seeded onto a 3D cell culture medium and a culture dish containing culture medium, serving as the test group and control group, respectively. After 7 days of culture, unattached endothelial cells were removed by washing with phosphate-buffered saline (PBS), and absorbance (OD value) was measured to obtain the OD values ​​of the test group and control group. The 3D cell culture medium without endothelial cells served as the blank group, and its absorbance was measured and recorded as blank group OD1. The culture medium without endothelial cells served as the second blank group, and its absorbance was measured and recorded as blank group OD2.

[0115] Cell viability = (OD of test group - OD1 of blank group) / (OD of control group - OD2 of blank group) × 100%.

[0116] The test results are shown in Table 1 below.

[0117] Table 1

[0118]

[0119] According to the test results in Table 1, the particle size dispersion index of the oil phase droplets in the gelatin-based high internal phase emulsions provided in Examples 1 to 6 are all between 0.08 and 0.35, indicating good uniformity. The resulting 3D cell culture carriers have good pore size uniformity, good biocompatibility, and high nutrient delivery efficiency.

[0120] SEM image of the cell 3D culture vector provided in Example 1 is shown below. Figure 1 As shown, the aperture is relatively uniform.

[0121] Compared with Example 1, if the mass percentage of gelatin in the aqueous phase is too low (Example 4), the gelatin-based high internal phase emulsion has poor stability, increased particle size unevenness (PDI=0.35), insufficient mechanical strength of the prepared cell 3D culture carrier, and decreased biocompatibility and nutrient delivery efficiency.

[0122] Compared with Example 1, if the mass percentage of gelatin in the aqueous phase is too high (Example 5), the high viscosity of the aqueous phase will lead to an increase in the uneven particle size of the gelatin-based high internal phase emulsion (PDI=0.28), resulting in insufficient mechanical strength of the prepared 3D cell culture carrier and a decrease in biocompatibility and nutrient delivery efficiency.

[0123] Compared with Example 1, the gelatin-based high internal phase emulsion prepared by mixing aqueous and oil phases in Comparative Example 1 had poor particle size uniformity (PDI=0.85), resulting in decreased biocompatibility and nutrient delivery efficiency of the prepared cell 3D culture carrier.

[0124] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A gelatin-based high internal phase emulsion, characterized in that, The gelatin-based high internal phase emulsion comprises an aqueous phase and an oil phase. The aqueous phase includes gelatin, an emulsifier, and water, and the oil phase includes oily substances. The gelatin-based high internal phase emulsion is an oil-in-water type high internal phase emulsion, and the particle size dispersion index of the oil phase droplets in the gelatin-based high internal phase emulsion is 0.05~0.

40.

2. The gelatin-based high internal phase emulsion according to claim 1, characterized in that, The gelatin content is 8% to 20% of the total mass of the aqueous phase, which is 100% of the total mass. Preferably, the emulsifier comprises 1% to 5% of the total mass of the aqueous phase (100%). Preferably, the emulsifier comprises a nonionic surfactant, and more preferably, the nonionic surfactant comprises Tween 80; Preferably, the aqueous phase further includes a crosslinking agent, and more preferably, the crosslinking agent includes genipin and / or glutaraldehyde; Preferably, the crosslinking agent comprises 0.01% to 5% of the total mass of the aqueous phase (100%). Preferably, the aqueous phase further includes a cell culture nutrient solution; Preferably, the cell culture nutrient solution comprises 20% to 40% of the total mass of the aqueous phase (100%).

3. The gelatin-based high internal phase emulsion according to claim 1 or 2, characterized in that, The oily substances include biocompatible oils; Preferably, the biocompatible oil includes any one or a combination of at least two of medium-chain triglyceride oil, mineral oil, or soybean oil; Preferably, the oil phase further includes growth factors; Preferably, the growth factor comprises 0.1% to 0.5% of the total mass of the oil phase (100%). Preferably, the growth factor includes a hydrophobic growth factor; Preferably, the hydrophobic growth factor includes vitamin D and / or paclitaxel.

4. The gelatin-based high internal phase emulsion according to any one of claims 1 to 3, characterized in that, The D50 particle size of the oil phase droplets in the water-in-oil emulsion is 10~1000 μm, more preferably 50~500 μm.

5. A method for preparing a gelatin-based high internal phase emulsion as described in any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: mixing the aqueous phase and the oil phase by microfluidics to obtain the gelatin-based high internal phase emulsion.

6. The preparation method according to claim 5, characterized in that, The preparation method includes the following steps: (1) Dissolve gelatin in water, add emulsifier, optionally crosslinking agent and optionally cell culture nutrient solution to obtain an aqueous phase; (2) Mix the oily substances with optional growth factors to obtain the oil phase; (3) The aqueous phase obtained in step (1) and the oil phase obtained in step (2) are injected into the continuous phase channel and the dispersed phase channel of the microfluidic device, respectively. The oil phase is sheared into uniform small balls in the focusing channel and dispersed in the aqueous phase. The oil phase is collected to obtain the gelatin-based high internal phase emulsion. In this case, steps (1) and (2) are performed in any order, or simultaneously. Preferably, the dissolution temperature in step (1) is 60~70℃; Preferably, the mixing in step (2) includes mixing using ultrasonic dispersion; Preferably, the power of the ultrasonic dispersion is 100~200 W; Preferably, the ultrasonic dispersion time is 10-15 min; Preferably, the temperature of the aqueous phase in step (3) is 35~45℃; Preferably, the temperature of the oil phase in step (3) is 25~30℃; Preferably, the flow rate of the aqueous phase in step (3) is 50~500 μL / min; Preferably, the flow rate of the oil phase in step (3) is 300~2000 μL / min; Preferably, the flow rate ratio of the aqueous phase and the oil phase in step (3) is 1:(3~8).

7. A 3D cell culture vector, characterized in that, The cell 3D culture carrier is prepared from a gelatin-based high internal phase emulsion as described in any one of claims 1 to 4.

8. A method for preparing a 3D cell culture carrier as described in claim 7, characterized in that, The preparation method includes the following steps: solidifying the gelatin-based high internal phase emulsion as described in any one of claims 1 to 4 to obtain the cell 3D culture carrier.

9. The preparation method according to claim 8, characterized in that, The curing includes static curing; Preferably, the temperature for static curing is 0~20℃; Preferably, the static curing time is 10~30 min; Preferably, the step of heat curing is further included before the static curing; Preferably, the temperature for heat curing is 30~35℃; Preferably, the heating and curing time is 2-4 hours; Preferably, the curing process further includes washing and drying steps.

10. The application of a gelatin-based high internal phase emulsion as described in any one of claims 1 to 4 or a cell 3D culture carrier as described in claim 7 in cell culture, drug screening, tissue engineering scaffolds, or daily chemical hygiene products.

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