CN115151634A - Application of sodium alginate-gelatin 3D scaffold in supporting differentiation of adipose precursor cells - Google Patents
Application of sodium alginate-gelatin 3D scaffold in supporting differentiation of adipose precursor cells Download PDFInfo
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- CN115151634A CN115151634A CN202280002824.5A CN202280002824A CN115151634A CN 115151634 A CN115151634 A CN 115151634A CN 202280002824 A CN202280002824 A CN 202280002824A CN 115151634 A CN115151634 A CN 115151634A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
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- A—HUMAN NECESSITIES
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Abstract
The application provides an application of a sodium alginate-gelatin 3D scaffold in supporting differentiation of adipose precursor cells. The method can differentiate the fat precursor cells into mature fat cells with a large number of fat droplets in a lower cost and higher efficiency method; in the sodium alginate-gelatin 3D support, the sodium alginate, the gelatin and the cross-linking agent are all safe and nontoxic components, are frequently used in a large amount in food, and can ensure the edible safety as part of a food product; the support has certain thickness and elasticity, so the support can provide certain taste, provides support for the taste of cell meat.
Description
Technical Field
The application belongs to the technical field of future food, and particularly relates to an application of a sodium alginate-gelatin 3D scaffold in supporting differentiation of adipose precursor cells.
Background
Animal meat, an indispensable food protein source, accounts for a considerable proportion of the world's dining table. The increasing demand for animal protein or animal meat by humans throughout the world is a great challenge for traditional animal husbandry and even for the ecology of the world. In this regard, the related art all over the world has been devoted to the development of cell meat, and has gradually formed a main line of development of cell culture meat.
The cell culture meat takes cells as a starting point and a dropping point, and is carried out in three stages of obtaining high-quality primary cells, editing and processing the cells to obtain seed cells capable of maintaining the division and differentiation capacity for a long time, and expanding the cells on a large scale and combining the expanded cells with other tissues or substances to form meat products with mouthfeel and flavor consistent with real animal meat. The technology comprises the steps of harvesting stem cells with the capacity of differentiating into muscle cells on different species, wherein the stem cells comprise Induced Pluripotent Stem Cells (iPSCs), embryonic Stem Cells (ESCs), related unipotent stem cells and the like; forming stable seed cells by the obtained cells in the modes of gene editing or small molecule induction and the like; using seed cells as a starting point, and amplifying the cell number in a large scale through a cell fermentation tank; under a certain cell number, cell meat with muscle texture and rich animal protein is obtained through differentiation induction.
For the third stage of cell culture meat, there have been a lot of studies in tissue engineering for life engineering. Among them, hydrogels containing gelatin or sodium alginate as a main component have been widely used, and such hydrogel components have been widely used in the research and development of organoids, scaffolds for cell culture, wound dressings, cultured meat, and the like. The hydrogel has the function principle of providing an environment for culturing cells in vitro on the premise of simulating the in vivo physicochemical environment and the spatial structure of the cells, and the cells still maintain the important physiological activity function under the environment, so that the cells can play roles in different directions subsequently.
Among them, fat has been attracting attention as an essential component in animal meat, both in terms of nutrition and flavor, and also in cell meat. The in vitro growth and differentiation of the fat precursor cells obtained from different species have quite stable results, so that a focus is put on how to more efficiently differentiate the fat precursor cells into mature fat cells with certain size and number of intracellular fat drops.
CN113768138A discloses a method for manufacturing an edible gellan gum/gelatin 3D scaffold for cell culture meat, the method can be directly used for cell culture without freeze drying, under the condition of not using toxic cross-linking agents, the 3D scaffold is formed through electrostatic interaction between gellan gum and gelatin, the obtained gellan gum/gelatin 3D scaffold has good cell adhesion and compatibility, skeletal muscle cell culture can be carried out, however, the scaffold cannot be used for supporting differentiation of lipo-precursor cells.
CN113208059A discloses a method for manufacturing an edible pectin/chitosan/collagen 3D scaffold for cell culture meat, which comprises the steps of uniformly mixing pectin (high methoxyl pectin and low methoxyl pectin), chitosan and collagen according to a certain proportion, fully reacting for more than 2 hours at room temperature, freezing overnight, and freeze-drying to obtain the pectin/chitosan/collagen 3D scaffold. According to the invention, the 3D scaffold is formed through electrostatic interaction among pectin, chitosan and collagen, the obtained pectin/chitosan/collagen 3D scaffold has good cell adhesion and compatibility, and the scaffold can not be used for supporting differentiation of fat precursor cells.
Therefore, there is a need to develop a lower cost, more efficient method to differentiate the adipocyte precursor cells into adipocytes with large lipid droplet maturation.
Disclosure of Invention
The application provides an application of a sodium alginate-gelatin 3D scaffold in supporting differentiation of an adipocyte precursor. The application provides a hydrogel scaffold for supporting differentiation of fat precursor cells, and further comprises a method for differentiating the fat precursor cells into intracellular lipid droplets with different sizes on the obtained scaffold.
In a first aspect, the present application provides the use of a sodium alginate-gelatin 3D scaffold to support differentiation of adipogenic precursor cells.
The application provides a sodium alginate-gelatin 3D scaffold, and finds that the scaffold can be used as a hydrogel scaffold for supporting differentiation of fat precursor cells, and further comprises a method for differentiating the fat precursor cells into intracellular lipid droplets with different sizes on the obtained scaffold. The present application differentiates adipogenic precursor cells into mature adipocytes with large lipid droplets in a less costly, more efficient process. And sodium alginate, gelatin and cross-linking agent are all safe and nontoxic components, are frequently used in large quantities in food, and can ensure edible safety as a part of food products. The sodium alginate-gelatin-contained bracket has certain thickness and elasticity, so that the bracket can provide a certain degree of mouthfeel and support the mouthfeel of cell meat; in addition, the sodium alginate-gelatin scaffold and the cells differentiated on the sodium alginate-gelatin scaffold are beneficial to entering the subsequent processes of mass production, storage, meat customization and the like in a fat cell-3D scaffold compound mode.
In the present application, the adipogenic precursor cells are present in a form that adheres to the three-dimensional configuration of the scaffold. The 3D configuration in the hydrogel has a large number of loose porous structures, the interlaced microfilaments have pores which are communicated with each other, the fat precursor cells are supported by the microfilaments and are positioned in the pores formed by the fat precursor cells, and nutrient substances are obtained through the communicated pores, namely, the adhesion and nutrient environment required by the growth of the fat precursor cells are provided for the fat precursor cells.
In the present application, the differentiation of the adipogenic precursor cells on the 3D scaffold is more efficient. The three-dimensional culture system in the same basal area can simultaneously support more cells to exist compared with a two-dimensional plane. Meanwhile, the excellent interconnected pore structure enables components for inducing differentiation in the culture medium to act on the adipose-derived precursor cells more effectively and more fully, so that the adipose cells differentiated on the 3D scaffold have higher differentiation efficiency, namely, the number of the cells differentiated into the adipose-derived droplets is more.
In the present application, the adhesion of the adipogenic precursor cells in the 3D scaffold is not tight. Thereby providing more product forms for later production of cell meat. For example, by blowing the cell-3D scaffold complex with a pipette, pure mature adipocytes can be isolated; the myotube/myocyte-3D scaffold compound and the adipocyte-3D scaffold compound which are obtained by research in the laboratory are extruded and molded to obtain the fat and lean alternated cell meat.
In the present application, the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Mixing sodium alginate, gelatin and water, and solidifying to obtain hydrogel compound; then placing the hydrogel into a cross-linking agent solution for soaking and cross-linking to obtain a hydrogel support;
(b) Removing the residual cross-linking agent in the hydrogel support obtained in the step (a), soaking in ethanol, irradiating by using an ultraviolet lamp, and finally washing by using a buffer solution; and
(c) And (c) crushing the hydrogel stent treated in the step (b), and placing the crushed hydrogel stent in a buffer solution for oscillation to obtain the microfilament of the sodium alginate-gelatin 3D stent.
The sodium alginate-gelatin 3D scaffold at least comprises main components of sodium alginate and gelatin, cross-linking components of a cross-linking agent and ethanol, a three-dimensional structure formed by the interlacing of microfilaments and communicated pores formed by the interlacing of microfilaments.
In the present application, the mass ratio of sodium alginate, gelatin and water is (1-3): (1-4): (93-98), and can be 1.
Preferably, in step (a), the mixing temperature is 75-85 ℃, for example, can be 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃ and so on.
Preferably, in step (a), the solidification temperature is-85 to-75 ℃, and may be, for example, -85 ℃, -84 ℃, -83 ℃, -82 ℃, -81 ℃, -80 ℃, -79 ℃, -78 ℃, -77 ℃, -76 ℃, -75 ℃ and the like; the setting time is 8-16h, for example, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, etc.
Preferably, in step (a), the cross-linking agent solution is an aqueous solution of calcium chloride dihydrate, and the concentration of the aqueous solution of calcium chloride dihydrate is 400-600. Mu.M, and may be, for example, 400. Mu.M, 420. Mu.M, 440. Mu.M, 460. Mu.M, 480. Mu.M, 500. Mu.M, 520. Mu.M, 540. Mu.M, 560. Mu.M, 580. Mu.M, 600. Mu.M, or the like.
Preferably, in step (a), the temperature of said crosslinking is-25 to-15 ℃, and may be, for example, -25 ℃, -24 ℃, -23 ℃, -22 ℃, -21 ℃, -20 ℃, -19 ℃, -18 ℃, -17 ℃, -16 ℃, -15 ℃ and the like; the crosslinking time is 8-16h, for example, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h and the like.
Preferably, in step (b), the energy of the ultraviolet lamp is 20-40mJ/cm 2 For example, it may be 20mJ/cm 2 、22mJ/cm 2 、24mJ/cm 2 、26mJ/cm 2 、28mJ/cm 2 、30mJ/cm 2 、32mJ/cm 2 、34mJ/cm 2 、36mJ/cm 2 、38mJ/cm 2 、40mJ/cm 2 Etc.; the irradiation time is 1 to 3 hours, and may be, for example, 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours, 2 hours, 2.2 hours, 2.4 hours, 2.6 hours, 2.8 hours, 3 hours, or the like.
Preferably, in step (b), the buffer solution is PBS.
Preferably, in step (c), the crushing is performed by shearing, and the volume of the granular hydrogel fragments obtained by shearing is 1-9mm 2 For example, it may be 1mm 2 、2mm 2 、3mm 2 、4mm 2 、5mm 2 、6mm 2 、7mm 2 、8mm 2 、9mm 2 And the like.
Preferably, in step (c), the buffer solution is PBS and/or DMEM.
Preferably, in step (c), the oscillation is performed by using Lin Beier VORTEX-5 VORTEX mixer VORTEX-5.
Preferably, in step (c), the power of the oscillation is 40-60W, such as 40W, 42W, 44W, 46W, 48W, 50W, 52W, 54W, 56W, 58W, 60W, etc., and the oscillation time is 10-15min, such as 10min, 11min, 12min, 13min, 14min, 15min, etc.
Preferably, in step (c), the following operations are performed after the oscillation: and (3) oscillating and suspending the hydrogel fragments, wherein the hydrogel fragments comprise micro fragments obtained by oscillation and visible to the naked eye, and repeating the operations of manually suspending the fragments, standing for 1-3s (for example, 1s, 1.5s, 2s, 2.5s, 3s and the like) and taking out the upper micro fragments by utilizing the different settling speeds of the hydrogel fragments in the buffer solution.
In a second aspect, the present application provides a method of differentiation of adipogenic precursor cells, the method comprising the steps of: and inoculating the adipose precursor cells on the sodium alginate-gelatin 3D scaffold for differentiation to obtain mature adipocytes.
In the present application, the method for differentiation of the adipogenic precursor cells specifically comprises the steps of:
(1) Mixing the microfilament of the sodium alginate-gelatin 3D scaffold with a growth medium to obtain a mixture containing the microfilament and the growth medium;
(2) Dripping the mixture containing the microfilament and the growth medium obtained in the step (1) into the cell chamber, and removing the residual liquid on the upper layer when the microfilament is completely settled in the cell chamber;
(3) After resuspending the lipo-precursor cells, injecting the lipo-precursor cells into the cell chamber treated in the step (2), suspending the cell chamber in a pore plate, infiltrating the bottom of the cell chamber by adopting a growth culture medium, and performing contact inhibition; and
(4) And (4) replacing the growth culture medium in the step (3) with a differentiation culture medium, and inducing differentiation to obtain mature adipocytes.
Preferably, in the step (1) and the step (3), the growth medium consists of the following components in percentage by mass: DMEM 81-94%, FBS 5-15%, penicillin and streptomycin mixture 1-4%.
The DMEM may be 81 to 94%, for example, 81%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, etc., based on 100% of the total mass of the growth medium.
The content of FBS is 5 to 15%, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, etc., based on 100% by mass of the total growth medium.
The content of the mixture of penicillin and streptomycin is 1-4%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, etc., based on 100% by mass of the total growth medium.
Preferably, the mass ratio of the microfilament of the sodium alginate-gelatin 3D scaffold to the growth medium is 1 (1 × 10) 4 -6×10 4 ) For example, 1:1 × 10 4 、1:2×10 4 、1:3×10 4 、1:4×10 4 、1:5×10 4 、1:6×10 4 And the like.
Preferably, in the step (2), the dropping specifically comprises the following steps: the mixture containing the microfilaments and growth medium obtained in step (1) is extracted using a pasteur pipette, the pipette is left standing vertically for 3-5s, which may be, for example, 3s, 3.5s, 4s, 4.5s, 5s, etc., and the microfilaments are added dropwise to a 24-well cell chamber while they settle down at the pasteur pipette tip.
Preferably, in step (3), the density of the adipose precursor cells is 10 6 -10 8 Per mL, for example, may be 10 6 5X 10 units/mL 6 one/mL, 10 7 5X 10 units/mL 7 one/mL, 10 8 one/mL, etc.; the resuspension is carried out by using a growth medium solution, the rotation speed of the resuspension is 1000-1500rpm, such as 1000rpm, 1100rpm, 1200rpm, 1300rpm, 1400rpm, 1500rpm and the like, and the time of the resuspension is 5-10min, such as 5min, 6min, 7min, 8min, 9min, 10min and the like.
Preferably, in the step (3), the number of the adipose precursor cells injected into each chamber is 10 5 -10 7 May be, for example, 10 5 5X 10 cells/mL 5 cell/mL, 10 6 5X 10 units/mL 6 one/mL, 10 7 one/mL, etc.
Preferably, in step (3), the well plate is a 6-well plate.
Preferably, in step (3), the temperature of the contact inhibition is 36-38 ℃, for example, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃ and the like, and the time of the contact inhibition is 2-3 days, for example, 2 days, 2.5 days, 3 days and the like.
Preferably, in step (4), the specific operation of inducing differentiation is: replacing the growth medium in step (3) with a first differentiation medium, and inducing differentiation at 36-38 deg.C (such as 36 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C, 38 deg.C) for 2-3 days (such as 2 days, 2.5 days, 3 days); replacing the first differentiation medium with a second differentiation medium, inducing differentiation at 36-38 deg.C (such as 36 deg.C, 36.5 deg.C, 37 deg.C, 37.5 deg.C, 38 deg.C) for 8-10 days (such as 8 days, 9 days, 10 days), and replacing the second differentiation medium every 3-5 days (such as 3 days, 4 days, 5 days) within 8-10 days (such as 8 days, 9 days, 10 days);
wherein the first differentiation medium comprises: IBMX, dexamethasone, insulin, FBS and DMEM-f12 differentiation culture medium containing double antibodies; the second differentiation medium comprises: insulin, FBS, DMEM-f12 differential medium for double antibody.
Preferably, the first differentiation medium comprises the following components: 0.4-0.6mM IBMX, 0.2-0.3 μ M dexamethasone, 0.8-1.2 μ g/mL insulin, 8-12% FBS, 0.5-2% DMEM-f12 differentiation medium containing double antibody.
In the first differentiation medium of the present application, IBMX has a concentration of 0.4 to 0.6mM, and may be, for example, 0.4mM, 0.45mM, 0.5mM, 0.55mM, 0.6mM, or the like.
In the first differentiation medium of the present application, the concentration of dexamethasone is 0.2 to 0.3. Mu.M, and may be, for example, 0.2. Mu.M, 0.22. Mu.M, 0.24. Mu.M, 0.26. Mu.M, 0.28. Mu.M, 0.3. Mu.M, or the like.
In the first differentiation medium of the present application, the concentration of insulin may be 0.8 to 1.2. Mu.g/mL, and may be, for example, 0.8. Mu.g/mL, 0.9. Mu.g/mL, 1.0. Mu.g/mL, 1.1. Mu.g/mL, 1.2. Mu.g/mL, or the like.
In the first differentiation medium of the present application, the FBS content is 8 to 12% by volume, and may be, for example, 8%, 9%, 10%, 11%, 12%, or the like.
In the first differentiation medium of the present application, the content of diabody in DMEM-f12 differentiation medium containing diabody is 0.5-2% by weight, and may be, for example, 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2%, etc.
Preferably, the second differentiation medium comprises the following components: 0.8-1.2. Mu.g/mL insulin, 8-12% FBS, 0.5-2% DMEM-f12 differentiation medium containing the double antibody.
In the second differentiation medium of the present application, the concentration of insulin may be 0.8 to 1.2. Mu.g/mL, for example, 0.8. Mu.g/mL, 0.9. Mu.g/mL, 1.0. Mu.g/mL, 1.1. Mu.g/mL, 1.2. Mu.g/mL, or the like.
In the second differentiation medium of the present application, FBS is contained in an amount of 8 to 12% by volume, and may be, for example, 8%, 9%, 10%, 11%, 12%, etc.
In the second differentiation medium of the present application, the content of diabody in the DMEM-f12 differentiation medium containing diabody is 0.5 to 2% by weight, and may be, for example, 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2%, etc.
Compared with the prior art, the method has the following beneficial effects:
(1) The application applies the sodium alginate-gelatin 3D scaffold to support the differentiation of the adipocyte precursor, and finds that the adipocyte precursor can be differentiated into mature adipocytes with a large number of intracellular lipid droplets by a lower-cost and higher-efficiency method.
(2) Sodium alginate, gelatin and cross-linking agent in the sodium alginate-gelatin 3D support provided by the application are all safe and nontoxic components, are components frequently used in a large amount in food, and can ensure edible safety as part of food products.
(3) The sodium alginate-gelatin-containing stent provided by the application has certain thickness and elasticity, so that the stent can provide a certain degree of mouthfeel and support the mouthfeel of cell meat.
(4) The sodium alginate-gelatin scaffold and the cells differentiated on the sodium alginate-gelatin scaffold are beneficial to entering the subsequent processes of batch production, storage, meat customization and the like in a fat cell-3D scaffold compound mode.
Drawings
FIG. 1 is a graph showing the differentiation efficiency and lipid droplet size effect of adipocytes differentiated on day 9 on the scaffold provided in example 1.
FIG. 2 is a graph showing the differentiation efficiency and lipid droplet size effect of adipocytes differentiated on the scaffold provided in example 2 at day 9.
FIG. 3 is a graph showing the differentiation efficiency and lipid droplet size effect of adipocytes differentiated on the 9 th day of the scaffold provided in example 3.
FIG. 4 is a graph showing the differentiation efficiency and lipid droplet size effect of adipocytes differentiated on day 9 of the scaffold provided in example 4.
FIG. 5 is a diagram of the scaffold components (gelatin-sodium alginate microfilaments) of simple example 1.
Detailed Description
The technical solution of the present application is further described below by means of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present application and should not be construed as a specific limitation of the present application.
The sources of the components in the following preparations and examples are as follows:
| name (R) | Manufacturer(s) of | Brand and/or specification |
| Sodium alginate | Aladdin | S100127-25g |
| Gelatin | sigma | C4243 |
| Calcium chloride dihydrate | Aladdin | C108379-25g |
| Ethanol | Aladdin | E111992-500mL |
| PBS buffer | Procell | PB180327 |
| DMEM | Hyclone | SH30243.01 |
| FBS | Gibco | 10270-106 |
| Penicillin and streptomycin mixture | 15140122 | C0222 |
| Adipose precursor cells | Primary isolation | 3 days old Bar Ma Zhu |
| IBMX | Sigma | I5879 |
| Dexamethasone | Sigma | D4902-25MG |
| Insulin | Sigma | I2643-25MG |
| DMEM-f12 differentiation medium | Gibco | DMEM/F12 |
Preparation example 1
The preparation example provides a sodium alginate-gelatin 3D scaffold, and the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Dissolving sodium alginate solid powder in pure water to prepare 2%w/v sodium alginate, dissolving gelatin solid particles in the 2%w/v sodium alginate pure water, and completely fusing the mixture in a water bath kettle at 80 ℃ to obtain a flowing colloid mixture of 2%w/v sodium alginate and 3%w/v gelatin; pouring the fluid colloid mixture into the bottom of a 6-hole dish, immediately placing the dish at the temperature of minus 80 ℃ for 12 hours, and solidifying and forming to obtain a hydrogel compound; then, 500. Mu.M CaCl was added to the coagulated hydrogel composite 2 Completely soaking the aqueous solution, and crosslinking the aqueous solution at the temperature of minus 20 ℃ for 12 hours to obtain a hydrogel bracket;
(b) Adding the remaining CaCl in the hydrogel scaffold 2 The aqueous solution was blotted dry and absolute ethanol was added to completely soak the scaffolds, the 6 well plate lid was opened and placed under the UV lamp of a biosafety cabinet at 20mJ/cm 2 Irradiating for 2h to obtain a scaffold with a loose porous structure, and finally washing the scaffold for 3 times by using PBS;
(c) Cutting the bracket processed in the step (b) into pieces with average area of 2mm as much as possible by using surgical scissors sterilized at high temperature and high pressure 2 Placing the hydrogel fragments into a 50mL centrifuge tube, adding 30mL PBS, and fully oscillating for 12min under the maximum power (30W) of an oscillator;
(d) And oscillating and suspending the hydrogel fragments which comprise micro fragments obtained by oscillation and visible to naked eyes, repeating the operations of manually suspending the fragments, standing for 1s and taking the upper micro fragments to obtain the raw material microfilament of the 3D hydrogel scaffold by utilizing different sedimentation speeds of the hydrogel fragments in a buffer solution due to different sizes, and storing the raw material microfilament at 4 ℃.
Preparation example 2
The preparation example provides a sodium alginate-gelatin 3D scaffold, and the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Dissolving sodium alginate solid powder in pure water to prepare 1%w/v sodium alginate, dissolving gelatin solid particles in 1%w/v sodium alginate pure water, and placing the mixture in a water bath kettle at 80 ℃ to completely fuse the mixture into a flowing colloid mixture of 1%w/v sodium alginate and 4%w/v gelatin; pouring the fluid colloid mixture into the bottom of a 6-hole dish, immediately placing the dish at the temperature of minus 80 ℃ for 12 hours, and solidifying and forming to obtain a hydrogel compound; then, 500. Mu.M CaCl was added to the coagulated hydrogel composite 2 Completely soaking the aqueous solution, and crosslinking for 12 hours at the temperature of minus 20 ℃ to obtain a hydrogel bracket;
(b) Adding the remaining CaCl in the hydrogel scaffold 2 The aqueous solution was blotted dry and absolute ethanol was added to completely soak the scaffolds, the 6 well plate lid was opened and placed under the UV lamp of a biosafety cabinet at 40J/cm 2 Irradiating for 2h to obtain a scaffold with a loose porous structure, and finally washing the scaffold for 3 times by using PBS;
(c) Cutting the bracket treated in the step (b) into pieces with average area of 1mm as much as possible by using surgical scissors sterilized at high temperature and high pressure 2 Putting the hydrogel fragments into a 50mL centrifuge tube, adding 30mL PBS, and fully oscillating for 12min under the maximum power (50W) of an oscillator;
(d) And oscillating and suspending the hydrogel fragments, wherein the hydrogel fragments comprise tiny fragments obtained by oscillation and visible to the naked eye, repeating the operations of manually suspending the fragments, standing for 2s and taking the tiny fragments on the upper layer to obtain the raw material microfilament of the 3D hydrogel scaffold by utilizing the fact that the hydrogel fragments have different sizes and cause different sedimentation speeds in a buffer solution, and storing the raw material microfilament at 4 ℃.
Preparation example 3
The preparation example provides a sodium alginate-gelatin 3D scaffold, and the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Dissolving sodium alginate solid powder in pure water to prepare 3%w/v sodium alginate, dissolving gelatin solid particles in 3%w/v sodium alginate pure water, and placing the mixture in a water bath kettle at 80 ℃ to completely fuse the mixture into a flowing colloid mixture of 3%w/v sodium alginate and 3%w/v gelatin; pouring the fluid colloid mixture into the bottom of a 6-hole dish, immediately placing the dish at the temperature of minus 80 ℃ for 12 hours, and solidifying and forming to obtain a hydrogel compound; then, 500. Mu.M CaCl was added to the coagulated hydrogel composite 2 Completely soaking the aqueous solution, and crosslinking for 12 hours at the temperature of minus 20 ℃ to obtain a hydrogel bracket;
(b) Adding the remaining CaCl in the hydrogel scaffold 2 The aqueous solution was blotted dry and absolute ethanol was added to completely soak the scaffolds, the 6 well plate lid was opened and placed under the UV lamp of a biosafety cabinet at 35mJ/cm 2 Irradiating for 2h to obtain a scaffold with a loose porous structure, and finally washing the scaffold for 3 times by using PBS;
(c) Cutting the bracket processed in the step (b) into pieces with average area of 2mm as much as possible by using surgical scissors sterilized at high temperature and high pressure 2 Putting the hydrogel fragments into a 50mL centrifuge tube, adding 30mL DMEM, and fully oscillating for 12min under the maximum power (30W) of an oscillator;
(d) And oscillating and suspending hydrogel fragments which comprise micro fragments obtained by oscillation and visible to naked eyes, repeating the operations of shaking the suspended fragments by hand and taking the upper micro fragments after standing for 3s by utilizing the fact that the hydrogel fragments have different sizes to cause different sedimentation speeds in a buffer solution to obtain the raw material microfilament of the 3D hydrogel stent, and storing the raw material microfilament at 4 ℃.
Preparation example 4
The preparation example provides a sodium alginate-gelatin 3D scaffold, and the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Dissolving sodium alginate solid powder in pure water to prepare 2%w/v sodium alginate, dissolving gelatin solid particles in 2%w/v sodium alginate pure water, and placing the mixture in a water bath kettle at 80 deg.CFully fused into a flowable colloid mixture of 2%w/v sodium alginate and 3%w/v gelatin; pouring the flowing colloid mixture into the bottom of a 6-hole dish, immediately keeping the flowing colloid mixture at-80 ℃ for 12 hours, and solidifying and forming to obtain a hydrogel compound; then, 500. Mu.M CaCl was added to the coagulated hydrogel composite 2 Completely soaking the aqueous solution, and crosslinking for 12 hours at the temperature of minus 20 ℃ to obtain a hydrogel bracket;
(b) Adding the remaining CaCl in the hydrogel scaffold 2 The aqueous solution was blotted dry, the 6-well plate was uncovered and placed under the UV lamp of a biosafety cabinet at 30mJ/cm 2 Irradiating for 2h by using the energy of (1) to obtain a scaffold with a loose porous structure, and finally washing the scaffold for 3 times by using PBS;
(c) Cutting the bracket treated in the step (b) into pieces with average area of 1mm as much as possible by using surgical scissors sterilized at high temperature and high pressure 2 The hydrogel fragment is put into a 50mL centrifuge tube, 50mL of FBS/DMEM (the mass ratio of FBS/DMEM is 1;
(d) And oscillating and suspending the scaffold fragments, wherein the scaffold fragments comprise tiny fragments obtained by oscillation and visible to the naked eye, repeating the operations of oscillating and suspending the scaffold by hand, standing for 3S and taking the hydrogel fragments on the upper layer to obtain the raw material microfilament of the 3D hydrogel scaffold by utilizing different settling speeds of the scaffold fragments in the buffer solution due to different sizes of the scaffold fragments, and storing the raw material microfilament at 4 ℃.
Preparation example 5
The preparation example provides a sodium alginate-gelatin 3D scaffold, and the sodium alginate-gelatin 3D scaffold is prepared by the following method:
(a) Dissolving sodium alginate solid powder in pure water to prepare 1%w/v sodium alginate, dissolving gelatin solid particles in 1%w/v sodium alginate pure water, and placing the mixture in a water bath kettle at 80 ℃ to completely fuse the mixture into a flowing colloid mixture of 1%w/v sodium alginate and 4%w/v gelatin; pouring the fluid colloid mixture into the bottom of a 6-hole dish, immediately placing the dish at the temperature of minus 80 ℃ for 12 hours, and solidifying and forming to obtain a hydrogel compound; then, 500. Mu.M CaCl was added to the coagulated hydrogel composite 2 Completely soaking the hydrogel in the aqueous solution, and crosslinking the hydrogel at the temperature of minus 20 ℃ for 12 hours to obtain the hydrogel scaffold；
(b) Adding the remaining CaCl in the hydrogel scaffold 2 The aqueous solution was blotted dry and absolute ethanol was added to completely soak the scaffolds, the 6 well plate lid was opened and placed under the UV lamp of a biosafety cabinet at 30mJ/cm 2 Irradiating for 2h to obtain a scaffold with a loose porous structure, and finally washing the scaffold for 3 times by using PBS;
(c) Cutting the bracket processed in the step (b) into pieces with average area of 0.5mm as much as possible by using surgical scissors sterilized at high temperature and high pressure 2 The hydrogel fragments are put into a 50mL centrifuge tube, 50mL PBS is added, and the mixture is fully shaken for 12min under the maximum power (50W) of a shaker to obtain the raw material of the 3D hydrogel stent, and the raw material is stored at 4 DEG C
Comparative preparation example 1
The preparation example provides a gellan gum/gelatin 3D scaffold, which is prepared by the following method:
(a) Dissolving gellan gum and gelatin in water to obtain gellan gum solution and gelatin solution with concentration of 2% and 3%, respectively; mixing gellan gum and gelatin solution according to the mass ratio of 1:2; keeping the temperature of the mixed solution above 40 ℃, adding the mixed solution into a mold, and standing at room temperature for reaction for more than 10min to obtain a 3D bracket;
(b) Cutting the 3D scaffold obtained in step (a) into pieces with average area of 1mm as much as possible by using surgical scissors sterilized under high temperature and high pressure 2 Putting the hydrogel fragments into a 50mL centrifuge tube, adding 30mL PBS, and fully oscillating for 12min under the maximum power (50W) of an oscillator;
(c) And oscillating and suspending the bracket, wherein the bracket comprises micro fragments which are obtained by oscillation and can be seen by naked eyes, repeating the operations of oscillating and taking the hydrogel fragments on the upper layer by utilizing different settling speeds of the fragments with different sizes to obtain the raw material microfilament of the 3D hydrogel bracket, and storing the raw material microfilament at 4 ℃.
Example 1
This example provides a method for differentiating an adipocyte precursor, which specifically includes the following steps:
(1) Aspirating the remaining liquid from the stent microwires provided in preparation example 1, adding fresh 89% dmem, 10% fbs, 12mL of a 1% penicillin and streptomycin mixture, and thoroughly pipetting the mixture using a pasteur pipette;
(2) Extracting the mixture of the microfilaments and the growth medium in the step (1) by using a pasteur pipette, vertically standing the pasteur pipette for 5 seconds, and adding the microfilaments into a cell chamber with 24 holes in a dropwise manner when the microfilaments are settled at the mouth of the pasteur pipette; removing the residual liquid on the upper layer when the microfilament is completely settled in the cell chamber;
(3) Mixing adipose precursor cells at 1 × 10 7 Density resuspension of cells/mL, including in particular cell count harvest 1X 10 7 After the cells were separated, the cells were placed in a 15mL centrifuge tube, centrifuged at 1200rpm for 5min to settle the cells at the bottom of the tube, the supernatant in the centrifuge tube was removed using a pipette gun, 1mL complete medium (10% FBS + DMEM) was added to the centrifuge tube, and the tube was carefully blasted with a pipette gun with a 1000. Mu.L range to a state of single cells without macroscopic particles and showing the cells separated from each other under a microscope. To each cell chamber 100. Mu.L of cell resuspension, i.e., 1X 10 6 A plurality of adipose precursor cells; the cell chambers were suspended in 6-well plates, to which 12mL of DMEM growth medium containing a mixture of 10% fbs, 1% penicillin and streptomycin was added, so that the bottom of the cell chambers were sufficiently infiltrated with the medium without air bubbles. The surface of the culture medium should not cross the pore above the cell chamber, so that the fat precursor cells are inhibited for 2 days at 37 ℃;
(4) Replacing the growth medium with DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25. Mu.M dexamethasone, 1. Mu.g/mL insulin, 10% FBS, 1% double antibody, and inducing differentiation of the cells in the differentiation medium at 37 ℃ for 2 days; the differentiation medium was replaced with DMEM-f12 differentiation medium containing 1. Mu.g/mL of insulin, 10% FBS, and 1% double antibody, and differentiation was continued at 37 ℃ for 9 days, with the solution being changed every 4 days.
Example 2
This example provides a method for differentiating an adipocyte precursor, which specifically includes the following steps:
(1) Aspirating the residual liquid from the stent microfilaments provided in preparative example 2, adding fresh 89% dmem, 10% fbs, 12mL of a 1% penicillin and streptomycin mixture, and thoroughly pipetting the solution using a pasteur pipette;
(2) Extracting the mixture of the microfilaments and the growth culture medium in the step (1) by using a pasteur pipette, vertically standing the pasteur pipette for 3s, and adding the microfilaments into a cell chamber with 24 holes in a dropwise manner when the microfilaments are settled at the mouth of the pasteur pipette; removing the residual liquid on the upper layer when the microfilament is completely settled in the cell chamber;
(3) Subjecting the adipose precursor cells to a treatment of differentiation at 1 × 10 7 Density resuspension of cells/mL, including cell count harvest 1X 10 7 After the cells were separated, the cells were centrifuged at 1200rpm in a 15mL centrifuge tube for 5min to settle the cells at the bottom of the tube, the supernatant in the centrifuge tube was removed using a pipette gun, 1mL of complete medium (10% FBS + DMEM) was added to the centrifuge tube, and the cells were gently blasted with a pipette gun with a 1000. Mu.L range until no particles were visible to the naked eye and were microscopically observed as single cells separated from each other. To each cell chamber was added 100. Mu.L, i.e., 1X 10 6 Individual adipose precursor cells; the cell chambers were suspended in 6-well plates, to which 12mL of DMEM growth medium containing a mixture of 10% fbs, 1% penicillin and streptomycin was added, so that the bottom of the cell chambers were sufficiently infiltrated with the medium without air bubbles. The surface of the culture medium needs to be submerged in the pore space above the cell chamber, so that the fat precursor cells are in contact with the culture medium for 2 days at 37 ℃;
(4) Replacing the growth medium with DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25. Mu.M dexamethasone, 1. Mu.g/mL insulin, 10% FBS, 1% double antibody, and inducing differentiation of the cells in the differentiation medium at 37 ℃ for 2 days; the differentiation medium was replaced with DMEM-f12 differentiation medium containing 1. Mu.g/mL of insulin, 10% FBS and 1% diabody, and differentiation was continued at 37 ℃ for 9 days, with the solution being changed every 4 days.
Example 3
This example provides a method for differentiating an adipocyte precursor, which specifically includes the following steps:
(1) Aspirating the residual liquid from the stent microwires provided in preparative example 3, adding fresh 89% dmem, 10% fbs, 1% penicillin and streptomycin mixture 12mL, and thoroughly pipetting the mixture using a pasteur pipette;
(2) Extracting the mixture of the microfilaments and the growth medium in the step (1) by using a pasteur pipette, vertically standing the pasteur pipette for 5 seconds, and adding the microfilaments into a cell chamber with 24 holes in a dropwise manner when the microfilaments are settled at the mouth of the pasteur pipette; removing the residual liquid on the upper layer when the microfilament is completely settled in the cell chamber;
(3) Mixing adipose precursor cells at 1 × 10 7 Density resuspension of cells/mL, including in particular cell count harvest 1X 10 7 After the cells were separated, the cells were centrifuged at 1200rpm in a 15mL centrifuge tube for 5min to settle the cells at the bottom of the tube, the supernatant in the centrifuge tube was removed using a pipette gun, 1mL of complete medium (10% FBS + DMEM) was added to the centrifuge tube, and the cells were gently blasted with a pipette gun with a 1000. Mu.L range until no particles were visible to the naked eye and were microscopically observed as single cells separated from each other. To each cell chamber was added 100. Mu.L, i.e., 1X 10 6 Individual adipose precursor cells; the cell chambers were suspended in 6-well plates, to which 12mL of DMEM growth medium containing 10% fbs, 1% penicillin and streptomycin mixture was added, so that the bottom of the cell chambers was well infiltrated by the medium, with no air bubbles present. The surface of the culture medium needs to be submerged in the pore space above the cell chamber, so that the fat precursor cells are in contact with the culture medium for 2 days at 37 ℃;
(4) Replacing the growth medium with DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25. Mu.M dexamethasone, 1. Mu.g/mL insulin, 10% FBS, 1% double antibody, and inducing differentiation of the cells in the differentiation medium at 37 ℃ for 2 days; the differentiation medium was replaced with DMEM-f12 differentiation medium containing 1. Mu.g/mL of insulin, 10% FBS and 1% diabody, and differentiation was continued at 37 ℃ for 9 days, with the solution being changed every 4 days.
Comparative example 1
This comparative example provides a method for differentiation of adipogenic precursor cells, which is different from example 1 only in that the scaffold microwire provided in preparation example 1 was replaced with the scaffold microwire provided in comparative preparation example 1.
Test example 1
Gel Strength test
Test samples: the sodium alginate-gelatin 3D scaffolds provided in preparation examples 1-5;
the test method comprises the following steps: measuring the elasticity of the bracket by taking the Young modulus as a model;
the specific test results are shown in table 1 below:
TABLE 1
As can be seen from the test results in Table 1, the sodium alginate-gelatin-containing scaffold provided by the application has a certain thickness, specifically 1-2mm, and a certain elasticity, specifically 1.0-2.0kPa, so that the scaffold can provide a certain degree of mouthfeel and support the mouthfeel of cell meat.
Test example 2
Characterization of differentiation results
Test samples: the method for differentiation of the adipogenic precursor cells provided in examples 1-3 and the method for differentiation of the adipogenic precursor cells provided in comparative example 1;
the test method comprises the following steps: a nile red staining method;
and (3) testing results: the adipogenic precursor cells undergoing differentiation on the scaffold appeared as numerous lipid droplets and were stained with nile red. FIGS. 1-4 are Hoechest/NileRed staining, where Hoechest is blue, indicating the size, shape and location of the nuclei; niled is red indicating the size, location and number of intracellular lipid droplets differentiated in the cytoplasm.
The specific test results are shown in table 2 below:
TABLE 2
| Sample(s) | Proportion of cells differentiating into lipid droplets (%) | Average particle size (. Mu.m) of fat droplets |
| Example 1 | 100％ | 10.2 |
| Example 2 | 100％ | 9.7 |
| Example 3 | 100％ | 10.3 |
| Comparative example 1 | 90％ | 5.6 |
As is clear from the test data in Table 2, the ratio of the cells differentiated into lipid droplets in the present application was 100%, and the average particle size of the large lipid droplets was 9.0 μm or more. It is fully demonstrated that the differentiation efficiency of the adipogenic precursor cells on the 3D scaffold is high. Compared with a two-dimensional plane, the three-dimensional culture system in the same area can simultaneously support more cells. Meanwhile, the excellent interconnected pore structure enables components for inducing differentiation in the culture medium to act on the adipose-derived precursor cells more effectively and more fully, so that the adipose cells differentiated on the 3D scaffold have higher differentiation efficiency, namely, the number of the adipose cells which are differentiated and differentiated at the same time is large. The applicant states that the application illustrates the application of the sodium alginate-gelatin 3D scaffold of the application in supporting differentiation of the adipogenic precursor cells through the above examples, but the application is not limited to the above examples, i.e. the application does not mean that the application has to rely on the above examples to be implemented. It should be understood by those skilled in the art that any modifications, equivalent substitutions of the raw materials of the product of the present application, and the addition of auxiliary components, selection of specific modes, etc., are all within the scope and disclosure of the present application.
Claims (10)
1. An application of a sodium alginate-gelatin 3D scaffold in supporting differentiation of fat precursor cells.
2. The use of claim 1, wherein the sodium alginate-gelatin 3D scaffold is prepared by the following preparation method:
(a) Mixing sodium alginate, gelatin and water, and solidifying to obtain hydrogel compound; then placing the hydrogel into a cross-linking agent solution for soaking and cross-linking to obtain a hydrogel support;
(b) Removing the residual cross-linking agent in the hydrogel support obtained in the step (a), soaking in ethanol, irradiating by using an ultraviolet lamp, and finally washing by using a buffer solution; and
(c) And (c) crushing the hydrogel stent treated in the step (b), and placing the crushed hydrogel stent in a buffer solution for oscillation to obtain the microfilament of the sodium alginate-gelatin 3D stent.
3. The use of claim 2, wherein in the step (a), the mass ratio of the sodium alginate to the gelatin to the water is (1-3) to (1-4) to (93-98);
preferably, in step (a), the temperature of the mixing is 75-85 ℃;
preferably, in the step (a), the solidification temperature is-85 to-75 ℃, and the solidification time is 8 to 16 hours;
preferably, in step (a), the cross-linking agent solution is an aqueous solution of calcium chloride dihydrate, and the concentration of the aqueous solution of calcium chloride dihydrate is 400-600 μ M;
preferably, in the step (a), the temperature of the crosslinking is-25 to-15 ℃, and the time of the crosslinking is 8 to 16 hours.
Preferably, in step (b), the energy of the ultraviolet lamp is 20-40mJ/cm 2 The irradiation time is 1-3h;
preferably, in step (b), the buffer solution is PBS;
preferably, in step (c), the crushing is performed by shearing, and the volume of the granular hydrogel fragments obtained by shearing is 1-9mm 3 ；
Preferably, in step (c), the buffer solution is PBS and/or DMEM;
preferably, in the step (c), the oscillation is performed by using a Lin Beier VORTEX-5 VORTEX mixer VORTEX-5;
preferably, in the step (c), the power of the oscillation is 40-60W, and the time of the oscillation is 10-15min;
preferably, in step (c), the following operation is performed after the oscillation: and oscillating and suspending the hydrogel fragments, wherein the hydrogel fragments comprise micro fragments obtained by oscillation and visible to the naked eye, and repeating the operations of manually suspending the fragments, standing for 1-3s and taking the upper micro fragments by utilizing different settling speeds of the hydrogel fragments in a buffer solution due to different sizes.
4. A method of differentiation of an adipocyte precursor comprising the steps of: seeding the lipoblasts on the sodium alginate-gelatin 3D scaffold of any one of claims 1 to 3 for differentiation to obtain mature adipocytes.
5. The method for differentiation of adipogenic precursor cells according to claim 4, wherein said method comprises in particular the steps of:
(1) Mixing the microfilament of the sodium alginate-gelatin 3D scaffold with a growth medium to obtain a mixture containing the microfilament and the growth medium;
(2) Dripping the mixture containing the microfilament and the growth medium obtained in the step (1) into the cell chamber, and removing the residual liquid on the upper layer when the microfilament is completely settled in the cell chamber;
(3) After resuspending the lipo-precursor cells, injecting the lipo-precursor cells into the cell chamber treated in the step (2), suspending the cell chamber in a pore plate, infiltrating the bottom of the cell chamber by adopting a growth culture medium, and performing contact inhibition; and
(4) And (4) replacing the growth culture medium in the step (3) with a differentiation culture medium, and inducing differentiation to obtain mature adipocytes.
6. The method for differentiating the adipogenic precursor cell as claimed in claim 5, wherein the growth medium in the step (1) and the step (3) is composed of the following components by mass percent: DMEM 81-94%, FBS 5-15%, penicillin and streptomycin mixture 1-4%;
preferably, the mass ratio of the microfilament of the sodium alginate-gelatin 3D scaffold to the growth medium is 1 (1 × 10) 4 -6×10 4 )。
7. The method for differentiation of adipogenic precursor cells as claimed in claim 5 or 6, wherein the step (2) of dropping comprises the following specific steps: and (2) extracting the mixture containing the microfilaments and the growth medium obtained in the step (1) by using a Pasteur pipette, vertically standing the pipette for 3-5s, and adding the microfilaments into a cell chamber with 24 holes in a dropwise manner when the microfilaments are settled at the mouth of the Pasteur pipette.
8. The method for differentiating the adipogenic precursor cell according to any of claims 5 to 7, wherein the density of the adipogenic precursor cell in step (3) is 10 6 -10 8 The cell/mL, the heavy suspension adopts growth medium solution, the rotation speed of the heavy suspension is 1000-1500rpm, and the time of the heavy suspension is 5-10min;
preferably, in the step (3), the number of the adipose precursor cells injected into each chamber is 10 5 -10 7 A plurality of;
preferably, in step (3), the well plate is a 6-well plate;
preferably, in the step (3), the temperature of the contact inhibition is 36-38 ℃, and the time of the contact inhibition is 2-3 days.
9. The method for differentiation of adipogenic precursor cells as claimed in any of claims 5-8, wherein in step (4), the specific operation of inducing differentiation is: replacing the growth culture medium in the step (3) with a first differentiation culture medium, and inducing differentiation for 2-3 days at 36-38 ℃; replacing the first differentiation culture medium with a second differentiation culture medium, inducing differentiation at 36-38 deg.C for 8-10 days, and replacing the second differentiation culture medium every 3-5 days within 8-10 days;
wherein the first differentiation medium comprises: IBMX, dexamethasone, insulin, FBS and DMEM-f12 differentiation culture medium containing double antibodies; the second differentiation medium comprises: insulin, FBS and double-antibody DMEM-f12 differentiation medium.
10. The method for differentiation of adipogenic precursor cells according to claim 9, wherein the first differentiation medium comprises the following components: 0.4-0.6mM IBMX, 0.2-0.3 μ M dexamethasone, 0.8-1.2 μ g/mL insulin, 8-12% FBS, 0.5-2% DMEM-f12 differentiation medium containing double antibody;
the second differentiation culture medium comprises the following components: 0.8-1.2 μ g/mL insulin, 8-12% FBS, 0.5-2% DMEM-f12 differentiation medium containing double antibody.
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