Controllable degradation porous microcarrier as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a water-soluble alginate microcarrier as well as a preparation method and application thereof. The alginate microcarrier comprises a carrier core structure and a shell surface modifier, the micro-carrier core layer is formed by carrying out plasma crosslinking on water-soluble alginate, ethylenediamine tetraacetic acid salt, nitrilotriacetic acid salt, diethylene triamine pentaacetic acid and water-soluble divalent metal salt, and light-sensitive alginate and a photoinitiator can also be added, so that the micro-carrier core layer has two crosslinking networks; the shell layer is composed of one of gelatin, collagen, fibrinogen, albumin and fibrin, or the gelatin, the collagen, the fibrinogen, the albumin and the fibrin and other biocompatible polymers; the obtained microcarrier has the characteristics of high transparency and rapid degradation, and adherent cells can be amplified in vitro; the microcarrier obtained in the invention can be used for mesenchymal stem cell culture, and can be rapidly degraded under a mild cell condition to harvest cells.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a controllably degradable porous microcarrier and a preparation method and application thereof. Background Art

[0002] With the rapid growth of tissue regeneration technology, cell therapy requires a large number of high-quality cells. It is estimated that each patient needs millions to billions of cells to treat the disease. This is due to the low cell retention rate in the defective area and the certain killing effect of the immune system on the cells. Therefore, it is necessary to develop optimized cell biomanufacturing schemes to produce enough cells for clinical use. A variety of methods for large-scale cell culture have been reported, and two-dimensional cell culture using culture flasks is the most common and simple method. The method of using horizontal expansion rather than vertical expansion requires a lot of space and consumes a lot of manpower and material resources. In order to solve these problems, microcarriers have gradually been used for large-scale cell culture, and have gradually replaced traditional two-dimensional cell culture methods, and have been found to exceed the performance of other expansion technologies.

[0003] Cell microcarriers are micro-nano particles with three-dimensional structures that can serve as scaffolds for cell attachment and provide support for the attachment and growth of adherent cells. Compared with traditional two-dimensional culture matrices, microcarriers are very small in size and can be suspended in culture medium with mild stirring, achieving large-scale cell proliferation in a short period of time while maintaining their specific morphology and phenotype. However, there are still many deficiencies in microcarriers in scientific research and clinical applications. For example, the particle size distribution of microcarriers is uneven and cannot be accurately regulated; in the later stage of cell collection, the introduction of various proteins and enzymes cannot be completely avoided, which brings difficulties to cell purification and increases the production cost of cell products.

[0004] During the preparation process of cell products used for clinical treatment, the introduction of various protein preparations and enzyme preparations should be avoided, which also brings challenges to traditional cell culture methods. Especially for the large-scale culture of cells that need to adhere to the wall to further grow and proliferate, trypsin digestion needs to be introduced during the collection process to redisperse the adherent cells in the culture medium before the next step of re-cultivation or concentration and enrichment. The introduction of trypsin will not only have a certain impact on the cells, but also increase the difficulty of the subsequent purification process of cell products. Therefore, a method is needed to avoid the additional introduction of trypsin to separate the cells from the microcarrier. Therefore, the development and design of microcarrier materials to prepare controllable degradable microcarriers can help reduce the introduction of trypsin. When the microcarrier is degraded, the adherent cells lose the supporting carrier, and the cells will be dispersed in the culture medium, and then they can be collected and concentrated by centrifugation.

[0005] Currently, microcarriers that can be used for cell culture include hyaluronic acid (HA) surface modified microcarriers, publication number CN116496970 A, which introduce H-glycyl-arginyl-glycyl-aspartyl-asparagine-proline-OH (RGD) sites and introduce sacrificial templates as pore-forming agents through surface modification. Hyaluronic acid is generally degraded under the action of hyaluronidase, which requires the introduction of enzymes and other protein substances for rapid degradation of microcarriers; there is also a scheme of using natural inorganic granular sea sand as a microcarrier culture matrix, publication number CN 115595288 B, which is obtained by soaking sea sand of about 60 mesh in a poly-lysine solution for surface modification. The main component of sea sand is silicon dioxide, which can hardly be degraded in a gentle way, so it is impossible to harvest cells without introducing trypsin; in addition, polyurethane matrix is ​​used as microcarrier culture matrix, publication number CN 115737924 A, polyurethane is a biologically inert material, lacking biologically active components, which is not conducive to cell attachment on its surface, and SIS powder is modified on its surface to improve the biological activity of microcarriers. However, the existing polyurethane degradation methods include hydrolysis, alcoholysis, aminolysis, acidolysis, alkaline hydrolysis and thermal degradation, which are not suitable for the degradation of cell microcarriers and will lead to problems such as cell death. Summary of the invention

[0006] The purpose of the present invention is to provide a controllably degradable porous microcarrier and a preparation method thereof. The microcarrier of the present invention has controllably degradable capability and a porous high specific surface area structure, which is helpful for in vitro culture and collection and purification of cells.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0008] The invention provides a porous microsphere carrier for cell suspension adherent culture. The porous microsphere carrier has a porous core-shell structure, wherein the core layer is formed by cross-linking water-soluble alginate to form a gel microsphere, and the shell structure material is a composite material composed of one or a combination of gelatin, collagen, fibrinogen, albumin and fibrin, or a composite material composed of one or a combination of gelatin, collagen, fibrinogen, albumin and fibrin and other biocompatible polymers; the shell layer has a cell adhesion site, which can make the cells adhere and then proliferate and grow; the porous structure microcarrier can be quickly dissolved in a degradation solution, so as to facilitate the rapid extraction of cells. The cross-linking method is ionic cross-linking, photocross-linking, glutaraldehyde cross-linking or carbodiimide cross-linking; preferably, the other biocompatible polymers are polyethylene glycol, polyvinyl alcohol, polylactic acid or polylysine; preferably, the water-soluble alginate is alginate, aldehyde-modified alginate, or a mixture of alginate and a water-soluble polymer, and the water-soluble polymer includes gelatin, methacrylic anhydride-modified gelatin or collagen.

[0009] In the above technical solution, an improvement is made in that the diameter of the alginate gel microspheres is 10 to 600 μm; the porous structure of the porous microsphere carrier is composed of small pores with a diameter of 10 to 40 μm and large pores with a diameter of 40 to 60 μm; the average diameter of the porous structure of the porous microsphere carrier is 30 μm to 50 μm.

[0010] In the above technical solution, the porous microsphere carrier has a narrow particle size distribution, and the coefficient of variation (CV) is less than 5%; (the coefficient of variation calculation formula = standard deviation / average value). The size of the gel microsphere depends on the chip size. The larger the channel size, the larger the gel microsphere produced. At the same time, the water-oil two-phase flow rate ratio affects the size of the gel microsphere. The larger the water phase flow rate, the larger the gel microsphere.

[0011] In the above technical solution, an improvement is made that in the porous microsphere carrier, the mass ratio of the core layer alginate gel microspheres to the shell layer material is distributed in a range of 1:1 to 20:1.

[0012] In the above technical solution, in one embodiment, the crosslinking method is ionic crosslinking or a combination of ionic crosslinking and photocrosslinking;

[0013] When the alginate microspheres are formed by ionic crosslinking, the water-soluble alginate is alginate, aldehyde-modified alginate or a mixture of alginate and a water-soluble polymer; the water-soluble polymer includes gelatin, methacrylic anhydride-modified gelatin or collagen;

[0014] The crosslinking agent used in the ionic crosslinking method includes one or more of ethylenediaminetetraacetate, nitrilotriacetate, diethylenetriaminepentaacetate or water-soluble divalent cations; preferably, the ethylenediaminetetraacetate is calcium-ethylenediaminetetraacetic acid, magnesium-ethylenediaminetetraacetic acid, ferrous-ethylenediaminetetraacetic acid, manganese-ethylenediaminetetraacetic acid or copper-ethylenediaminetetraacetic acid; the nitrilotriacetate is calcium-nitrilotriacetic acid, magnesium-nitrilotriacetic acid, ferrous-nitrilotriacetic acid, manganese-nitrilotriacetic acid or copper-nitrilotriacetic acid; the diethylenetriaminepentaacetate is calcium-diethylenetriaminepentaacetic acid, magnesium-diethylenetriaminepentaacetic acid, ferrous-diethylenetriaminepentaacetic acid, manganese-diethylenetriaminepentaacetic acid or copper-diethylenetriaminepentaacetic acid; the water-soluble divalent metal salt is one or a combination of ferrous chloride, calcium chloride, cupric chloride, zinc chloride, strontium chloride, barium chloride, nickel chloride or magnesium chloride;

[0015] Alginate microspheres formed by light crosslinking combined with ionic crosslinking, wherein the water-soluble alginate is a water-soluble photosensitive alginate, preferably, the alginate is one or a combination of methacrylic anhydride-modified alginate and methacryloyloxyethyl trimethylammonium chloride-modified alginate; the photoinitiator includes phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I-2959);

[0016] The concentration of the water-soluble alginate or water-soluble light-sensitive alginate used is 0.1-8w / v%, the concentration of the photoinitiator is 0.1-2w / v%, and the concentration of the ion cross-linking agent is 10mM-1200mM.

[0017] In the above technical solution, further, the method for preparing the porous microcarrier comprises the following steps:

[0018] (1) Preparation of the ion-crosslinked alginate gel microsphere solution

[0019] ①Preparation of aqueous solution:

[0020] During ion crosslinking, water-soluble alginate is dissolved in water as a raw material to prepare an alginate aqueous solution as an aqueous phase solution;

[0021] During photo-crosslinking, photo-crosslinked alginate is dissolved in water as a raw material to prepare an alginate aqueous solution, and a photo-crosslinking initiator is added as an aqueous phase solution;

[0022] When ionic crosslinking and photocrosslinking are combined, a water-soluble alginate and a photocrosslinking alginate are mixed as raw materials and dissolved in water, and a photocrosslinking initiator is added as an aqueous phase solution;

[0023] in:

[0024] The ion-crosslinked water-soluble alginate is alginate, aldehyde-modified alginate or a mixture of alginate and a water-soluble polymer; the water-soluble polymer includes gelatin, methacrylic anhydride-modified gelatin or collagen;

[0025] The ionic crosslinking reaction initiator is selected from one or more combinations of calcium-ethylenediaminetetraacetic acid, magnesium-ethylenediaminetetraacetic acid, ferrous-ethylenediaminetetraacetic acid, manganese-ethylenediaminetetraacetic acid, copper-ethylenediaminetetraacetic acid, calcium-nitrilotriacetic acid, magnesium-nitrilotriacetic acid, ferrous-nitrilotriacetic acid, manganese-nitrilotriacetic acid, copper-nitrilotriacetic acid, calcium-diethylenetriaminepentaacetic acid, magnesium-diethylenetriaminepentaacetic acid, ferrous-diethylenetriaminepentaacetic acid, manganese-diethylenetriaminepentaacetic acid, and copper-diethylenetriaminepentaacetic acid chelate aqueous solution;

[0026] The concentration of the ion-crosslinked water-soluble alginate is 0.1%-8w / v%, and the final concentration of the ion-crosslinking initiator is 30mM-400mM in terms of divalent metal ions;

[0027] The photo-crosslinked alginate is one or a combination of methacrylic anhydride-modified alginate and methacryloyloxyethyltrimethylammonium chloride-modified alginate;

[0028] The photocrosslinking initiator is one or a combination of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone;

[0029] The concentration of the photo-crosslinked alginic acid is 0.1% to 8w / v%, and the concentration of the photo-crosslinking initiator is 0.1% to 2w / v%;

[0030] The final concentration of the water-soluble alginate solution and the photo-crosslinked alginate solution crosslinked by the two crosslinking methods is 0.1% to 8w / v%, the final concentration of the ionic crosslinking initiator is 30mM to 400mM in terms of divalent metal ions, and the concentration of the photo-crosslinking initiator is 0.1% to 2w / v%;

[0031] ② Oil phase configuration: fluorinated oil, fluorinated surfactant, acidic substance are blended, or one or two of mineral oil and soybean oil are blended with non-ionic surfactant; the acidic substance is acetic acid, citric acid, oxalic acid or lactic acid; the non-ionic surfactant is Span-80 or Span-20;

[0032] ③ Collection phase configuration: one or more of fluorinated oil, perfluoroalcohol, and perfluoro acid are mixed with an acidic substance; or one or more of soluble divalent metal salts such as ferrous chloride, calcium chloride, cupric chloride, magnesium chloride, zinc chloride, strontium chloride, barium chloride, and nickel chloride solution; the acidic substance is acetic acid, citric acid, oxalic acid, or lactic acid;

[0033] ④ Cleaning phase configuration: mixing fluorinated oil with one or more of perfluoroalcohol and perfluoroacid; or one or more of ethanol, acetone and isopropanol;

[0034] (2) Formation of alginate droplets

[0035] The aqueous solution obtained in step ① is injected into the aqueous channel of the microfluidic chip at a low flow rate, and the oil phase solution is injected into the oil phase channel of the microfluidic chip at a high flow rate. The aqueous solution and the oil phase solution are mixed through the emulsification channel to form water-in-oil emulsion droplets; preferably, the low flow rate is 0.1-5 mL / h, and the high flow rate is 1-30 mL / h;

[0036] The flow rate ratio of the water phase to the oil phase is 1:1 to 1:30, preferably 1:1 to 1:20; the microfluidic chip emulsification channel has a cross structure, a co-flow type, a T-shaped mixing structure or a fluid focusing structure, and has at least two-phase liquid input ports, an emulsification channel and an output channel;

[0037] (3) forming alginate microgels, including ion-crosslinked alginate microgels, photo-crosslinked alginate microgels, and ion-crosslinked combined with photo-crosslinked alginate microgels;

[0038] When forming ion-crosslinked alginate microgels, there are three ways to form them:

[0039] Method 1: The acidic substance reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous solution to release the soluble divalent metal ions, thereby completing the cross-linking in the collection pipeline of the microfluidic chip, and obtaining the alginate gel microspheres with the surfactant removed after washing with a washing phase; the collection phase solution is a mixture of one or more of fluorinated oil, perfluoroalcohol and perfluoro acid and the acidic substance;

[0040] Method 2: inputting the water-in-oil emulsion droplets prepared in step (2) into a collection phase solution to form alginate gel microspheres, and then washing with a washing phase to obtain alginate gel microspheres with the surfactant removed; the collection phase solution is a soluble divalent metal salt solution;

[0041] Method 3: The acidic substance reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous phase solution to release soluble divalent metal ions, thereby completing partial crosslinking in the collecting pipeline, and then the water-in-oil emulsion droplets prepared in step (2) are input into the collecting phase solution to form alginate gel microspheres, and the alginate gel microspheres with the surfactant removed are obtained after washing with the washing phase; the collecting phase solution is a soluble divalent metal salt solution;

[0042] The acidic substance includes citric acid, acetic acid, oxalic acid or lactic acid;

[0043] When forming a photo-crosslinked alginate microgel, the forming method is:

[0044] The photoinitiator generates free radicals under ultraviolet light irradiation, which triggers the free radical polymerization reaction of the photo-crosslinked alginate, so that the photo-crosslinked alginate is gelled in the collection pipeline of the microfluidic chip. After being cleaned with a cleaning phase, the photo-crosslinked alginate gel microspheres with the surfactant removed can be obtained.

[0045] When forming ion-crosslinked combined with photo-crosslinked alginate microgels, there are three formation methods:

[0046] Method 1: Acid reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in an aqueous solution to release soluble divalent metal ions, and ultraviolet light is used to irradiate the collecting pipeline, thereby completing ion crosslinking and photocrosslinking in the collecting pipeline, and alginate gel microspheres with the surfactant removed are obtained after cleaning with a cleaning phase; the collecting phase solution used is a mixture of one or more of fluorinated oil, perfluoroalcohol, and perfluoro acid and an acidic substance;

[0047] Method 2: using ultraviolet light to irradiate the collection pipeline to make the alginate droplets first complete photo-crosslinking, inputting the water-in-oil emulsion droplets prepared in step (2) into the collection phase solution to form alginate ion-crosslinked and photo-crosslinked gel microspheres, and obtaining the alginate gel microspheres with the surfactant removed after washing by the washing phase; the collection phase solution is a soluble divalent metal salt solution;

[0048] Method 3: Acid reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous phase solution to release soluble divalent metal ions, and ultraviolet light is used to irradiate the collecting pipeline to complete ion crosslinking and photocrosslinking in the collecting pipeline, and then the water-in-oil emulsion droplets prepared in step (2) are input into the collecting phase solution to further completely crosslink the alginate gel microspheres, and after washing with a washing phase, the alginate gel microspheres with the surfactant removed are obtained; the collecting phase solution is a soluble divalent metal salt solution;

[0049] (4) Surface modification of alginate microgels:

[0050] The washed alginate gel microspheres are coated with a protein shell layer. There are two preparation methods:

[0051] Method 1: Soak the prepared alginate gel microspheres doped with aldehyde-modified alginate in a shell protein aqueous solution, and then immerse them in a crosslinker aqueous solution for crosslinking after the shell protein is coated, and then centrifuge the above mixture at a speed of 1000-2000 rpm, remove the supernatant, and wash three times with deionized water; the mixing mass ratio of the aldehyde-modified alginate and the alginate gel microspheres is 0.1:10-10:1; the concentration of the crosslinker aqueous solution is 0.1-8w / v%, preferably 0.1-4w / v%; the crosslinker includes one or more combinations of glutaraldehyde, thrombin, and ethylenediamine; the shell protein aqueous solution includes one of gelatin, collagen, fibrinogen, albumin and fibrin, or the above proteins and other biocompatible polymers, and the concentration is 0.1-6%;

[0052] Method 2: The prepared alginate gel microspheres are immersed in an aqueous solution composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), the carboxyl groups on the alginate microspheres are activated, and then immersed in a shell protein aqueous solution for cross-linking, and then the mixture is centrifuged at a speed of 1000-2000 rpm, and after removing the supernatant, it is washed three times with deionized water; the coating aqueous solution includes one of gelatin, collagen, fibrinogen, albumin and fibrin, or the above proteins and other biocompatible polymers, and the concentration is 0.1-6%; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (NHS) is 0.1-5mM, preferably 0.1-2mM; other biocompatible polymers include polyethylene glycol, polyvinyl alcohol or polylactic acid and polylysine;

[0053] (5) Formation of porous ion-crosslinked alginate microgels

[0054] The alginate microspheres modified with shell proteins formed above are fully frozen at -20°C to -196°C, and then transferred to a freeze dryer for freeze drying to obtain porous microsphere carriers.

[0055] The present invention also provides the use of the above-mentioned controllably degradable porous microcarrier in the field of large-scale suspension amplification culture of adherent cells.

[0056] In the above technical solution, further, the porous microcarrier is mixed with the cell suspension, left to stand for a period of time to allow the cells to adhere to the surface of the porous carrier, and then transferred to a shake flask or fermenter for suspension culture, and the cells can be proliferated on a large scale. After large-scale culture, the culture medium is removed, and the cells are detached from the porous microcarrier to complete cell harvesting.

[0057] In the above technical scheme, further, after the cells are cultured on a large scale, a degradation solution is added, and the cells quickly detach from the porous carrier, and can detach from the porous microcarrier within 15 minutes; the degradation solution includes a metal chelator and a protein degrading enzyme; the concentration of the metal ion chelator is 0.01-0.5w / v%, preferably 0.02%-0.3w / v%; the concentration of the protein degrading enzyme is 0.5-20mg / mL, preferably 1-10mg / mL; after the degradation solution is added to the microcarrier of the present invention, the cells can detach from the carrier in less than 15 minutes.

[0058] In the above technical scheme, further, the metal chelator includes one or more of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid (DTPA); the protein degrading enzyme is an enzyme that has the function of degrading extracellular matrix (ECM) proteins; preferably, it includes one or more of collagenase, trypsin, papain, gelatinase or matrix lysin.

[0059] The invention utilizes the shear force and surface tension between microfluids to pass alginate-ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate emulsions into a collection liquid, and the acid in the collection pipeline and the collection phase releases divalent metal ions (such as calcium ions, magnesium ions, ferrous ions, manganese ions, zinc ions, copper ions, barium ions, etc.) in ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate, so that the alginate is cross-linked to form alginate gel microspheres; then, a cleaning phase is used to remove the surfactant on the surface of the alginate gel microspheres, and then a cleaning phase solution remaining on the microspheres is washed with deionized water; the washed alginate microspheres are placed in a protein shell solution, and after the protein is coated on the surface of the gel microspheres, the solution is transferred to a cross-linking agent solution, and after the protein is coated on the surface of the gel microspheres, the solution is transferred to The method comprises the following steps: placing the cleaned alginate microspheres in a cross-linking agent solution, or placing the cleaned alginate microspheres in a 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solution, immersing the alginate gel microspheres in a shell protein aqueous solution for cross-linking after the alginate gel microspheres are activated, so as to achieve the coating of the protein shell layer; freezing the alginate gel microspheres with a protein shell layer at a temperature of -20°C to -196°C, placing the alginate gel microspheres in a vacuum freeze dryer for freeze drying after the microspheres are completely frozen, and removing ice crystals in the alginate gel microspheres by freeze drying to achieve the purpose of making pores on the alginate gel microspheres, thereby further increasing their specific surface area; the above method can prepare microcarriers with uniform size, high specific surface area and controllable degradation with a porous structure, which is significantly superior to the methods of the prior art.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The porous microspheres prepared by the present invention can be used to prepare porous microcarriers with different pore sizes, and the pore size distribution can be controlled by controlling the usage of ethylenediaminetetraacetate, nitrilotriacetate, diethylenetriaminepentaacetate, the concentration of acidic substances, the freezing rate, the concentration of photoinitiator and the concentration of light-sensitive alginate.

[0062] The controllably degradable porous microcarrier prepared by the present invention has two pore sizes, large and small, which further increases the specific surface area of ​​the microcarrier, provides more attachment area for cells, increases the number of proliferating cells on the microcarrier cells, and provides certain benefits for the migration of cells between microcarriers. Compared with existing cell culture microcarrier products, the microcarrier prepared by the present invention has the characteristics of rapid degradation and better cell proliferation effect.

[0063] The porous microcarrier prepared by the present invention can achieve the purpose of controlled degradation. The microcarrier can maintain long-term structural stability during cell proliferation. When cells are collected for purification and enrichment, the protein can be quickly degraded by adding degradation solution after removing the culture medium, and the cells have better cell activity. The number of cells in the microcarrier prepared by the present invention after five days of culture is 5.88×10 7 The amplification factor was 39.2 times, and the number of commercial gelatin microcarriers cultured for five days was 3.22×10 7 The amplification factor was 20.8 times.

[0064] The controllably degradable porous microcarrier prepared by the invention has high particle size monodispersity, can be pressed into tablets, occupies a small volume, and is convenient for storage and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Schematic diagram of preparing microcarriers by a microfluidic device according to an embodiment of the present invention;

[0066] Figure 2 Microspheres produced by microfluidic chips of different sizes in the embodiments of the present invention;

[0067] Figure 3 Producing microsphere size distribution for microfluidic chips of different sizes in embodiments of the present invention;

[0068] Figure 4 The macroscopic and scanning electron microscope images of the controllable degradable porous microcarrier prepared by the microfluidic technology in the embodiment of the present invention; a. macroscopic image, b. scanning electron microscope image;

[0069] Figure 5 The results of culturing fibrinogen cells coated with different ratios of oxidized alginate and alginic acid in the embodiments of the present invention; a. oxidized alginate:alginic acid is 1:10, b. oxidized alginate:alginic acid is 10:1;

[0070] Figure 6 Alginate microspheres prepared from different metal chelating salts in the embodiments of the present invention; a. calcium-ethylenediaminetetraacetic acid, b. calcium-nitrilotriacetic acid, c. calcium-diethylenetriaminepentaacetic acid;

[0071] Figure 7This is a scanning electron microscope image of alginate microcarrier after gelatin coating (glutaraldehyde) and freeze-drying in an embodiment of the present invention;

[0072] Figure 8 This is a light microscopic image of alginate microcarrier after gelatin coating (glutaraldehyde) freeze-dried and reconstituted in an embodiment of the present invention;

[0073] Fig. 9 This is a scanning electron micrograph of a gelatin-coated porous microcarrier (1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) method) in an embodiment of the present invention;

[0074] Fig.10 The pore size distribution of the gelatin-coated porous microcarrier (1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) method) in the embodiment of the present invention is as follows; the pore size distribution is 10 to 60 μm, and the average pore size is 30 to 50 μm;

[0075] Fig.11 This is an optical microscopic image of alginate microcarriers after freeze-drying and reconstitution of gelatin coated (1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) in an embodiment of the present invention; Fig.12 This is a confocal image of alginate microcarrier with fluorescein isothiocyanate modified gelatin A as the shell (1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) in an embodiment of the present invention;

[0076] Fig.13 Light microscopic images of alginate microcarriers coated with gelatin (1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) at different concentrations in the embodiments of the present invention;

[0077] Fig.14 This is an optical microscopic image of alginate microcarriers after freeze-drying and reconstitution of chitosan coated (1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) in an embodiment of the present invention; Fig.15 This is a fluorescence image of human hair follicle stem cells dynamically cultured on a fibrinogen-coated controllably degradable microcarrier in an embodiment of the present invention;

[0078] Fig.16 The gelatin A coated porous microcarrier (EDC / NHS method) in the embodiment of the present invention can be used for cell culture on degradable microcarriers;

[0079] Fig.17 The chitosan shell porous microcarrier in the embodiment of the present invention can be used for degradable microcarrier cell culture;

[0080] Fig.18 The harvest of cells on the degradable microcarriers with a porous microcarrier with a fibrinogen shell in the embodiment of the present invention; a. Cell microcarrier culture diagram, b. Cell morphology after adding degradation solution;

[0081] Fig.19 The gelatin-shell porous microcarrier in the embodiment of the present invention can be harvested as a degradable microcarrier cell; a. Cell microcarrier culture diagram, b. Cell morphology after adding degradation solution;

[0082] Fig. 20 The microcarrier with gelatin coating is prepared by the mixed crosslinking method of ion crosslinking and photo crosslinking in the embodiment of the present invention;

[0083] Fig.21 This is a scanning electron microscope image of a freeze-dried microcarrier with a gelatin coating prepared by a mixed crosslinking method of ionic crosslinking and photocrosslinking in an embodiment of the present invention;

[0084] Fig. 22 This is a cell culture diagram of a methacrylic anhydride-modified gelatin (GelMA) porous microcarrier in the comparative example of the present invention;

[0085] Fig.23 This is a graph showing the degradation of the methacrylic anhydride-modified gelatin (GelMA) microcarrier in the ethylenediaminetetraacetic acid system in the comparative example of the present invention;

[0086] Fig.24 The alginate microspheres are produced by the emulsion method of the comparative example of the present invention;

[0087] Fig.25 Schematic diagram of cell culture on porous microcarriers of the present invention. DETAILED DESCRIPTION

[0088] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0089] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in combination with the specific content of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0090] Example 1 Preparation of Alginate Microspheres in Microfluidic Chip

[0091] This embodiment provides a method for preparing alginate microcarriers using fluorinated oil and alginate chips of different sizes. The specific preparation steps include:

[0092] 1. Prepolymerization liquid phase configuration

[0093] The calcium-ethylenediaminetetraacetic acid solution was mixed with a sodium alginate solution having a mass fraction of 4%, and the concentration of the calcium-ethylenediaminetetraacetic acid was controlled to be 100 mM, as the aqueous phase.

[0094] At 3M TM Novec TM Fluorinated surfactant is added to 7100 (patent CN202110546666.7

[0095] The temperature-sensitive fluorocarbon surfactant prepared in Example 1 was used as the oil phase, with the concentration of the surfactant controlled to be 0.2%.

[0096] At 3M TM Novec TM Add glacial acetic acid to 7100 to control the concentration of glacial acetic acid to 0.1%, and then add a fluorocarbon surfactant (the temperature-sensitive fluorocarbon surfactant prepared in Example 1 of patent CN202110546666.7) to control the concentration of the surfactant to 0.2% as the collecting phase.

[0097] Dissolve perfluorooctanol in 3M TM Novec TM In 7100, the concentration of perfluorooctanol is controlled at 20% as the cleaning phase.

[0098] 2. Preparation of alginate microspheres of different sizes:

[0099] After the water phase and oil phase are loaded into the syringe, they are installed on the syringe pump and injected into the microfluidic chip using a catheter. The chip uses a cross structure and the chip channel structure is as follows: Figure 1 The specific structure of the chip consists of an oil phase channel and a water phase channel. The oil phase channel and the water phase channel intersect to form a cross structure. After the oil phase and the water phase merge at the cross, oil-in-water droplets are generated under the action of the shear force of the oil phase channel. The specific preparation process is as follows Figure 1 The blended solution was injected into the oil phase channel of the microfluidic chip at an injection rate of 1-30 mL / h, and the injection rate in this embodiment was 10 mL / h; the aqueous phase was injected into the W channel of the microfluidic chip at an injection rate of 0.1-5 mL / h, and the injection rate in this embodiment was 1 mL / h. The diameters of the downstream channels were 50 μm and 300 μm. The aqueous phase and the oil phase were sheared in the microfluidic chip to obtain uniform-sized alginate pregel microspheres downstream of the channel, and the alginate chelated Ca was triggered by glacial acetic acid. 2+ The alginate microspheres are prepared by cross-linking. The alginate microspheres in the above-mentioned collection solution are collected by centrifugation and transferred to the washing phase to wash away the residual surfactant on the surface of the alginate microspheres, so that the microspheres can be completely dissolved in the water phase. By using chips of different sizes and controlling the flow rates of different water phases and oil phases, microspheres of different particle sizes can be prepared, such as Figure 2 shown.

[0100] Example 2 Preparation of microspheres of different sizes using microfluidic chips of different sizes

[0101] This embodiment provides a method for preparing microspheres of different sizes, and the specific preparation steps include:

[0102] The preparation method of alginate microspheres is the same as that of Example 1, except that different sizes of microfluidic chips are used. In this example, chips with a width of 70 μm and 350 μm are used, respectively, to prepare alginate microspheres with diameters of 77 μm and 386 μm, with coefficients of variation of 3.44% and 1.27%, respectively. Figure 3 The method can adjust the size and distribution of microspheres by changing the flow rate and flow rate ratio of the water phase and the oil phase, and can achieve uniform production of microspheres larger than 20 μm.

[0103] Example 3 Preparation of porous alginate microcarriers with fibrinogen as coating

[0104] This embodiment provides a method for preparing a controllably degradable porous microcarrier with alginate-calcium-ethylenediaminetetraacetic acid as a core and fibrinogen as an outer coating. The specific preparation steps include:

[0105] 1. Prepolymerization liquid phase configuration

[0106] The calcium-ethylenediaminetetraacetic acid solution was mixed with a sodium alginate solution having a mass fraction of 4%, and the concentration of the calcium-ethylenediaminetetraacetic acid salt was controlled to be 100 mM, as the aqueous phase.

[0107] At 3M TM Novec TM A fluorinated surfactant (the temperature-sensitive fluorocarbon surfactant prepared in Example 1 of fluorinated surfactant patent CN202110546666.7) was added to control the concentration of the surfactant to 0.2% as the oil phase.

[0108] At 3M TM Novec TM Add glacial acetic acid to control the concentration of glacial acetic acid to 0.1-1%, and then add a fluorocarbon surfactant (the temperature-sensitive fluorocarbon surfactant prepared in Example 1 of patent CN202110546666.7) to control the concentration of the surfactant to 0.2-1% as the collecting phase.

[0109] Dissolve perfluorooctanol in 3M TM Novec TM In the process, the concentration of perfluorooctanol is controlled to be 20-100% as the cleaning phase.

[0110] Fibrinogen was dissolved in deionized water to control the concentration of fibrinogen to 2 mg / mL, which was used as the external coating solution.

[0111] Thrombin was dissolved in deionized water to control the concentration of thrombin to 20 U / mL, which was used as the coating cross-linking solution.

[0112] 2. Preparation of Alginate Hydrogel Porous Microspheres:

[0113] After the water phase and oil phase are loaded into the syringe, they are installed in the syringe pump and injected into the microfluidic chip through the catheter. The chip channel structure is as follows Figure 1 The preparation process of microcarriers is as follows Figure 1 The dispersed phase was injected into the O+S oil phase channel of the microfluidic chip at an injection rate of 30 mL / h; the continuous phase was injected into the W channel of the microfluidic chip at an injection rate of 3 mL / h, and the downstream channel diameter was about 300 μm. The aqueous phase and the oil phase used the shearing effect of the fluid in the microfluidic chip to obtain alginate gel microspheres of uniform size downstream of the channel.

[0114] Downstream of the microfluidic chip, the prepared alginate gel microspheres were introduced into the collection phase through a catheter, and glacial acetic acid was used to further trigger the chelation of Ca by alginate. 2+ The completely cross-linked alginate microspheres were prepared by cross-linking.

[0115] The alginate microspheres in the above collection solution are collected by centrifugation and then transferred to the washing phase to wash away the residual surfactant on the surface of the alginate microspheres, so that the microspheres can be completely dissolved in the water phase.

[0116] 3. External coating cross-linking

[0117] The completely washed microspheres are collected, transferred to the outer shell aqueous solution, and soaked overnight at 37° C. so that the surface of the alginate microspheres is covered with fibrinogen.

[0118] The rehydrated alginate microspheres were washed with deionized water to remove uncoated fibrinogen, and then the alginate microspheres were transferred to the coating cross-linking solution by centrifugation at 1000-1500 rpm. After cross-linking for 15 minutes, thrombin was allowed to cross-link the fibrinogen to the surface of the alginate-calcium carbonate microspheres. The microspheres were washed three times with deionized water, and then centrifuged to enrich the microspheres to obtain controllably degradable porous microcarriers, which were stored in centrifuge tubes.

[0119] 4. Porous Formation

[0120] After the alginate microspheres were collected by centrifugation, the supernatant was removed and placed in a -80°C refrigerator for freezing overnight. After the alginate microspheres were completely frozen, they were transferred to a vacuum freeze dryer for freeze drying. After freeze drying for 24 hours, the microspheres were taken out and the scanning electron microscope showed that the surface of the microspheres had a porous structure, such as Figure 4 shown.

[0121] The diameter of the controllable degradable porous microcarrier depends on the control of the alginate-calcium-ethylenediaminetetraacetic acid microspheres during preparation through the microfluidic chip. As mentioned above, the effect of controlling the particle size of the microspheres is achieved by adjusting the width of the chip channel structure and the flow rate ratio of the dispersed phase and the continuous phase.

[0122] Through the above method, we can adjust the flow rate and flow rate ratio of the aqueous phase and the oil phase to achieve the preparation of microcarriers with a microsphere diameter greater than 20um, and the CV value can be less than 5% to adapt to the requirements of external conditions such as different types of cells and different culture times, and to achieve the preparation of porous microcarriers with uniform size.

[0123] Example 4 Preparation of porous aldehyde-containing alginate microcarriers with fibrinogen as shell

[0124] This embodiment provides a method for preparing a controllably degradable porous microcarrier containing aldehyde-formylated alginate-calcium-ethylenediaminetetraacetic acid as a core and fibrinogen as an outer coating, and the specific preparation steps include:

[0125] Disperse 1g of sodium alginate with high G fragment content in anhydrous ethanol, dissolve 40% sodium periodate in deionized water, then mix the two solution systems in the dark and stir, then add ethylene glycol to terminate the reaction, add deionized water for washing, and finally dialyze and freeze-dry the solution system to obtain aldehyde-modified sodium alginate. The high G fragment alginate has a G fragment content of 50-65%;

[0126] The prepared aldehyde-modified sodium alginate and sodium alginate are blended in a ratio of 10:1-1:10, and the solution is deionized water as a sodium alginate aqueous solution, and a aldehyde-modified alginate-alginate solution with a final concentration of 4%-8% is prepared, and the calcium-ethylenediaminetetraacetic acid solution is mixed. The specific operation in the next step is the same as that in Example 1. The prepared alginate-calcium-ethylenediaminetetraacetic acid calcium microspheres have the aldehyde group of aldehyde-modified alginate, and the adsorption capacity of proteins is significantly enhanced, so that fibrinogen can be better adsorbed, thereby enhancing the attachment and proliferation ability of cells. The results are shown in FIG. Figure 5 As shown, increasing the proportion of aldehyde-modified alginate can increase the amount of surface-coated fibrinogen, so cells can proliferate better on its surface.

[0127] Example 5 Preparation of Alginate Microspheres Using Different Types of Divalent Metal Chelating Agents

[0128] This embodiment provides a method for preparing alginate microcarriers of different types of metal chelators, and the specific preparation steps include:

[0129] The preparation method of alginate gel microspheres is the same as that of Example 1, except that the metal chelate compound salts selected are calcium-ethylenediaminetetraacetic acid, calcium-nitrilotriacetic acid and calcium-diethylenetriaminepentaacetic acid, and the concentration of the divalent metal chelate compound salt is controlled to 100 mM, so that alginate microspheres with uniform size can be prepared. Figure 6 The cross-linking method initiated by metal chelate salts is that the acid replaces the divalent metal ions in the divalent metal chelate salts to cross-link the alginate. Other types of divalent metal chelate salts can also achieve cross-linking of alginate through the above method.

[0130] Example 6 Preparation of porous alginate microcarriers with gelatin as shell (glutaraldehyde method)

[0131] This embodiment provides a method for preparing a controllably degradable porous microcarrier with alginate-calcium-ethylenediaminetetraacetic acid as a core and gelatin as an outer shell. The specific preparation steps include:

[0132] The preparation method of alginate microspheres is the same as that in Example 1, except that after the preparation and cleaning of the alginate microspheres are completed, they are added to a gelatin A solution with a concentration of 0.3% and soaked overnight at 37°C. Since alginate itself is negatively charged, it can undergo electrostatic adsorption with the positively charged gelatin A, so gelatin A can be well coated on the surface of the alginate microspheres; after the above-mentioned gelatin-coated alginate microspheres are washed with deionized water, the excess gelatin A solution is washed off, and then the gelatin-coated alginate microspheres are transferred to a 5% glutaraldehyde coating cross-linking solution by centrifugation. After cross-linking for 15 minutes, after glutaraldehyde cross-links gelatin A to the surface of the alginate microspheres, the microspheres are washed with deionized water, and the microspheres are enriched to obtain alginate microcarriers with a gelatin shell layer, which are stored in a centrifuge tube and placed in a refrigerator for freezing and then freeze-drying. The freeze-dried alginate microcarriers are as follows Figure 7 As shown in Figure 2, the microcarriers have a porous structure after freeze-drying. After reconstitution, the microcarriers have uniform size, as shown in Figure 2. Figure 8 As shown in FIG. 1 , the optical microscopy image of the freeze-dried alginate microcarrier after reconstitution shows that freeze-drying does not destroy the structure of the alginate microcarrier, and the integrity of the carrier can still be maintained after reconstitution.

[0133] Example 7 Preparation of Gelatin Shell Porous Alginate Microcarriers (1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) Method)

[0134] This embodiment provides a method for preparing a controllably degradable porous microcarrier with alginate as a core and gelatin as an outer shell. The specific preparation steps include:

[0135] The preparation method of alginate gel microspheres is the same as that in Example 1. Add them to a 1 mM solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and soak them at 37°C overnight to activate the carboxyl groups on the alginate. Then add 3 w / v% gelatin solution. Since alginate itself is negatively charged, it can undergo electrostatic adsorption with the positively charged gelatin A. Therefore, gelatin A can be well coated on the surface of the alginate-calcium carbonate microspheres, and the carboxyl groups can react with the amino groups after activation to form amide bonds, thereby further fixing the gelatin on the surface of the alginate microspheres. The above-mentioned microspheres are repeatedly washed with deionized water, and after washing away the excess gelatin A solution, the microspheres are enriched to obtain controllable degradable porous microcarriers, which are stored in centrifuge tubes. And put them in a refrigerator for freezing and then freeze-drying. The freeze-dried alginate microcarriers are as follows. Fig. 9 As shown in Figure 2, alginate microcarriers have a good porous structure with a pore size range of 10-60 μm and an average pore size of 30-50 μm. Fig.10 After reconstitution, the microcarriers have uniform size, as shown in Fig.11 As shown in FIG. 1 , the optical microscopy image of the freeze-dried alginate microcarrier after reconstitution shows that freeze-drying does not destroy the structure of the alginate microcarrier, and the integrity of the carrier can still be maintained after reconstitution.

[0136] In order to confirm that gelatin is indeed used as the shell of the alginate core, fluorescein isothiocyanate (FITC)-modified gelatin A was used to replace the above gelatin A. The confocal images are shown in Figure 2. Fig.12 As shown, the results show that gelatin is indeed used as the shell of alginate microspheres. The internal fluorescence intensity is low and the external fluorescence intensity is high, proving that the shell gelatin A will not be detached with the washing of deionized water.

[0137] Example 8 Preparation of porous alginate microcarriers with different concentrations of gelatin coating (1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) method)

[0138] This embodiment provides a method for preparing a controllably degradable porous microcarrier with alginate as a core and gelatin of different concentrations as an outer shell. The specific preparation steps include:

[0139] The preparation method of alginate microspheres with gelatin as the outer shell is the same as that of Example 7, except that the gelatin concentration is 0-6 w / v%, and different concentrations of gelatin are added to obtain alginate with different concentrations of gelatin shells, such as Fig.13 As shown, as the gelatin concentration increases, the gelatin-alginate microspheres become more opaque. The above results indicate that gelatin shells with different degrees of modification can be obtained by controlling the concentration of gelatin A.

[0140] Example 9 Preparation of porous microcarriers with chitosan as coating

[0141] This embodiment provides a method for preparing a controllably degradable porous microcarrier with alginate as a core and chitosan as an outer coating. The specific preparation steps include:

[0142] The preparation method of alginate gel microspheres is the same as that of Example 1, and the preparation of the surface shell layer is the same as that of Example 6, except that chitosan is dissolved in a dilute acetic acid solution to obtain a chitosan solution with a concentration of 2%, and the chitosan solution prepared above is blended with 2 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and alginate microspheres activated by N-hydroxysuccinimide (NHS), and the specific operation of activation and subsequent treatment is the same as that of Example 7. The prepared alginate microspheres have a chitosan coating, and the microcarriers have uniform size after redissolution, such as Fig.14 As shown in FIG. 1 , the optical microscopy image of the freeze-dried alginate microcarrier after reconstitution shows that freeze-drying does not destroy the structure of the alginate microcarrier, and the integrity of the carrier can still be maintained after reconstitution.

[0143] Example 10 Preparation of porous alginate microcarriers with different proteins as shell layers

[0144] This embodiment provides a method for preparing different types of proteins as the shell layer of alginate microcarriers, and the specific preparation steps include:

[0145] The preparation method of alginate gel microspheres is the same as that in Example 1, and the preparation of the surface shell is the same as that in Example 6, except for the selection of the solution and concentration of the protein shell. The protein shell solution includes gelatin, collagen, fibrinogen, albumin, fibrin and other biocompatible polymers, and the shell protein concentration is selected to be 0.1-5 w / v%. The above proteins can be bound to the alginate surface to form a shell by chemical (forming amide bonds through amino groups and carboxyl groups) or physical crosslinking (electrostatic interaction).

[0146] Example 11 Cell culture on porous alginate microcarriers

[0147] Taking RA22B0003 cells as an example, the controllably degradable porous microcarriers prepared in Examples 1, 4, 7 and 9 of the present application were used for culture, and the process was as follows:

[0148] 1. Cell Culture on Porous Alginate Microcarriers with Fibrinogen as Shell

[0149] The fibrinogen alginate microcarriers were transferred to a low-viscosity centrifuge tube. After centrifugation to make the microcarriers densely packed, a high-concentration cell suspension was added dropwise. 50,000 cells were added to every 1 ml of microcarrier. After standing for 3 hours, the microcarrier and cell mixture was transferred to a shaking culture bottle. The shaking speed was 300 rpm. On the first day of culture, the cells gradually adhered to the surface of the microcarriers. On the second day of culture, the cells spread and began to proliferate. On the third day of culture, the cells had covered the surface of the microcarriers and began to gradually transfer to the adjacent microcarriers. On the fourth day of culture, the cells on the adjacent microcarriers began to spread and proliferate. On the fifth day of culture, more microcarrier surfaces were covered with cells. Fig.15 .

[0150] 2. Gelatin-coated porous microcarriers (1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC)

[0151] and N-hydroxysuccinimide (NHS) method) for cell culture on porous microcarriers

[0152] Transfer the above-mentioned freeze-dried gelatin-alginate porous microcarriers to a low-viscosity centrifuge tube (taking 1.5% gelatin-coated microcarriers as an example), add culture medium to dissolve and centrifuge to make the microcarriers densely packed, then add high-concentration cell suspension dropwise, add 50,000 cells per 1 ml of microcarriers, let it rest for 3 hours, then transfer the microcarrier and cell mixture to a shaking culture bottle, and shake at 300RPM for cell culture. The culture results on the fifth day are as follows: Fig.16 As shown, cells can proliferate well on microcarriers.

[0153] 3. Preparation of Porous Microcarriers with Chitosan as Coating

[0154] The freeze-dried chitosan alginate porous microcarriers were transferred to a low-viscosity centrifuge tube, and the culture medium was added for re-dissolution and centrifuged to make the microcarriers densely packed. Then, a high-concentration cell suspension was added dropwise, and 50,000 cells were added per 1 ml of microcarriers. After 3 hours of rest, the microcarrier and cell mixture was transferred to a shaking culture bottle with a shaking speed of 300 RPM for cell culture. The culture results on the fifth day are shown in Figure 1. Fig.17 As shown, cells can proliferate well on microcarriers.

[0155] In summary, the microcarrier used in the controllably degradable porous microcarrier and the preparation method thereof of the present application has a porous structure, which further increases the specific surface area of ​​the microcarrier and provides a spreading and adhesion space for cells; the external coating can provide adhesion sites for cells to promote cell proliferation.

[0156] Example 12 Cell harvesting from porous alginate microcarriers

[0157] This embodiment provides a method for rapidly harvesting cells from porous alginate microcarriers, and the specific preparation steps include:

[0158] 1. Preparation of degradation solution:

[0159] Mix 0.05 w / v% EDTA solution with 2 mg / mL collagenase and alginate, and adjust the pH to 7.2-7.4.

[0160] 2. Cell Harvesting on Porous Alginate Microcarriers with Fibrinogen as Shell

[0161] The cell culture microcarriers cultured for 5 days were centrifuged at 1000 RPM to make the microcarriers densely packed, and then 5 mL of the degradation solution prepared in step 1 was added and treated for 15 minutes. Fig.18 As shown, the results indicate that microcarriers can achieve rapid degradation of cells in the degradation solution.

[0162] 3. Cell Harvesting of Porous Alginate Microcarriers with Gelatin as Shell

[0163] The cell culture microcarriers cultured for 5 days were centrifuged at 1000 RPM to make the microcarriers densely packed, and then 5 mL of the degradation solution prepared in step 1 was added and treated for 15 minutes. Fig.19 As shown, the results indicate that microcarriers can achieve cell degradation under trypsin-free conditions.

[0164] 4. Cell Harvesting on Porous Alginate Microcarriers with Other Proteins or Biocompatible Polymers as Shells

[0165] The above-mentioned porous alginate microcarrier cell harvesting method of fibrinogen and gelatin can use the following degradation solution formula, use protein degrading enzymes or other enzymes that can degrade the surface shell, such as collagenase, trypsin, papain, gelatinase, matrix lysin, etc., after the enzyme degrades the surface protein or surface biocompatible polymer, the divalent metal ions in the ion-crosslinked alginate are chelated by metal chelators, such as ethylenediaminetetraacetic acid, nitrilotriacetic acid or diethylenetriaminepentaacetic acid, so that the alginate gel network is destroyed, thereby achieving rapid degradation of the alginate core.

[0166] Example 13: Shell modification of alginate microspheres formed by different cross-linking methods

[0167] This example provides a method for harvesting cells on alginate microcarriers with different cross-linking methods. The specific preparation steps include:

[0168] The preparation method of alginate gel microspheres is the same as that of Example 1, and the preparation of the surface shell is the same as that of Example 8, except that the crosslinking initiator and the aqueous solution are different. The aqueous solution is sodium alginate and sodium alginate modified with methacrylic anhydride, and the ionic crosslinking initiator is a mixture of calcium-ethylenediaminetetraacetate and a photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite. The ionic crosslinking initiator can simultaneously initiate crosslinking of sodium alginate modified with methacrylic anhydride and sodium alginate solution, and the photoinitiator can initiate polymerization of sodium alginate monomer modified with methacrylic anhydride. The concentration of the water-soluble sodium alginate solution is 2w / v%, the concentration of the methacrylic anhydride modified sodium alginate solution is 2w / v%, and the mixed concentration is 4w / v%. The microspheres produced are shown in FIG. Fig. 20 ,Different cross-linking methods do not affect the subsequent shell modification process. ,Alginate has a large number of carboxyl groups, which cannot be completely cross-linked. ,Thus, the subsequent shell modification related reactions can occur with the uncross-linked carboxyl groups. ,The results after surface shell modification are as follows. Fig.21 The above results show that the mixed cross-linking of the two cross-linking methods does not affect the subsequent shell modification of alginate microspheres.

[0169] Comparative Example 1 In vitro degradation of porous methacrylic anhydride modified gelatin (GelMA) microcarriers

[0170] 1. Preparation of methacrylic anhydride-modified gelatin (GelMA) microcarriers

[0171] Synthesis of GelMA: Dissolve 10g gelatin in 100mL PBS solution, stir at 50℃ until completely dissolved, then add 4-6mL methacrylic anhydride solution, and continue to react at 50℃ for 4-6h, then add 300-500mL PBS to terminate the reaction. The solution after the termination of the reaction is placed in a 3-10kDa dialysis bag and dialyzed for 7-14 days before freeze-drying. The whole process is carried out in the dark. The freeze-dried GelMA solution is dissolved in water to prepare a GelMA solution with a concentration of 8w / v%, and LAP (photoinitiator) is added at a concentration of 0.2%, and this is used as the aqueous phase. The oil phase is composed of fluorinated surfactants and fluorinated oils, and the surfactant concentration is 0.5-1%. Microspheres are prepared using the same chip and flow rate conditions as in Example 1, and after UV curing, 20%-50% PFO is used for cleaning. After cleaning, it is placed in a -80℃ refrigerator and freeze-dried to prepare porous GelMA porous microcarriers. The obtained porous microcarrier can be used for cell culture. The cell culture results are as follows: Fig. 22 As shown in the results, the surface porous GelMA microcarriers can be used for cell proliferation.

[0172] 2. GelMA microcarrier degradation experiment

[0173] The same mass of porous GelMA microcarriers were placed in an EP tube, and then a degradation solution under the same degradation conditions as alginate microcarriers (degradation solution in step 1 of Example 12) was added. After 15 minutes, the solution in the EP tube was poured into a six-well plate to observe the degradation of the GelMA microcarriers. Fig.23 It can be seen that the porous microcarriers did not undergo significant changes in morphology and structure 20 minutes after the addition of EDTA, and the microcarriers were not degraded. Comparative Example 2: Generation of Alginate Microspheres by Emulsion Method

[0174] Emulsion method for generating alginate microspheres: Sodium alginate is dissolved in deionized water, and the completely dissolved sodium alginate is added to an oil phase containing soybean oil and Span 80, and stirred at 200 and 300 rpm to generate microspheres. Fig.24 As shown in the figure, the size of the generated microspheres is uneven, with a distribution range of 200um-1000um. By changing the rotation speed, it can be seen that increasing the rotation speed can produce more small microspheres, but it cannot change the uneven size distribution.

[0175] Comparative Example 3 Commercial microcarrier cell culture

[0176] 1. Microcarrier culture protocol

[0177] The porous microcarriers of Example 7 of the present invention and the Huakan gelatin microcarriers (3D microcarriers), 125 mL reactor volume, inoculated with 1.5 × 10 6 cells, the initial culture volume was 50 mL, and the amount of the carrier was 200 mg. Intermittent stirring was performed 24 hours after inoculation, and after the intermittent stirring was completed, stirring was continued at 40 rpm until the end of the culture.

[0178] 2. After five days of culture, the number of cells in the microcarrier of the present invention was 5.88×10 7 The number of Huakan gelatin microcarriers cultured for five days was 3.22×10 7 The amplification factor was 20.8 times.

[0179] Comparative Example 4: Commercial Microcarrier Cell Degradation

[0180] Degradation of cells cultured with the microcarriers of the present invention in Comparative Example 3: After the culture is completed, degradation solution is added and stirred for 5 to 15 minutes at 37°C to complete the degradation;

[0181] Degradation of Huakan Gelatin Microcarriers:

[0182] Comparative Example 3: After the Chinese niche gelatin microcarrier culture was completed, degradation solution was added and stirred for 30 to 60 minutes at 37°C without degradation.

[0183] The degradation solutions are all the degradation solutions prepared in step 1 of Example 12.

Claims

1. A porous microsphere carrier for cell suspension adherent culture, characterized in that: The porous microsphere carrier has a porous core-shell structure, the core layer is formed by cross-linking water-soluble alginate to form a gel microsphere, the shell structure material is a composite material composed of one or a combination of gelatin, collagen, fibrinogen, albumin and fibrin, or the shell structure material is a composite material composed of one or a combination of gelatin, collagen, fibrinogen, albumin and fibrin and other biocompatible polymers; the cross-linking method is ionic cross-linking, photo-cross-linking, glutaraldehyde cross-linking or carbodiimide cross-linking; preferably, other biocompatible polymers are polyethylene glycol, polyvinyl alcohol, polylactic acid or polylysine; preferably, the water-soluble alginate is alginate, aldehyde-modified alginate, or a mixture of alginate and water-soluble polymers, and the water-soluble polymers include gelatin, methacrylic anhydride-modified gelatin or collagen.

2. The porous microsphere carrier according to claim 1, characterized in that: The diameter of the alginate gel microsphere is 10-600 μm; the porous structure of the porous microsphere carrier is composed of small pores with a diameter of 10-40 μm and large pores with a diameter of 40-60 μm; the average diameter of the porous structure of the porous microsphere carrier is 30 μm-50 μm.

3. The porous microsphere carrier according to claim 1 or 2, characterized in that: The porous microsphere carrier has a narrow particle size distribution with a coefficient of variation (CV) of less than 5%.

4. The porous microsphere carrier according to claim 1 or 2, characterized in that: In the porous microsphere carrier, the mass ratio of the core layer alginate gel microsphere to the shell layer material is 1:1 to 20:

1.

5. The porous microsphere carrier according to claim 1, characterized in that: The crosslinking method is ionic crosslinking or a mixture of ionic crosslinking and photocrosslinking; When the alginate microspheres are formed by ionic crosslinking, the water-soluble alginate is alginate, aldehyde-modified alginate or a mixture of alginate and a water-soluble polymer; the water-soluble polymer includes gelatin, methacrylic anhydride-modified gelatin or collagen; The crosslinking agent used in the ionic crosslinking method includes one or more of ethylenediaminetetraacetate, nitrilotriacetate, diethylenetriaminepentaacetate or water-soluble divalent cations; preferably, the ethylenediaminetetraacetate is calcium-ethylenediaminetetraacetic acid, magnesium-ethylenediaminetetraacetic acid, ferrous-ethylenediaminetetraacetic acid, manganese-ethylenediaminetetraacetic acid or copper-ethylenediaminetetraacetic acid; the nitrilotriacetate is calcium-nitrilotriacetic acid, magnesium-nitrilotriacetic acid, ferrous-nitrilotriacetic acid, manganese-nitrilotriacetic acid or copper-nitrilotriacetic acid; the diethylenetriaminepentaacetate is calcium-diethylenetriaminepentaacetic acid, magnesium-diethylenetriaminepentaacetic acid, ferrous-diethylenetriaminepentaacetic acid, manganese-diethylenetriaminepentaacetic acid or copper-diethylenetriaminepentaacetic acid; the water-soluble divalent metal salt is one or a combination of ferrous chloride, calcium chloride, cupric chloride, zinc chloride, strontium chloride, barium chloride, nickel chloride or magnesium chloride; Alginate microspheres formed by light crosslinking combined with ionic crosslinking, wherein the water-soluble alginate is a water-soluble photosensitive alginate, preferably, the alginate is one or a combination of methacrylic anhydride-modified alginate and methacryloyloxyethyl trimethylammonium chloride-modified alginate; the photoinitiator includes phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I-2959); The concentration of the water-soluble alginate or water-soluble light-sensitive alginate used is 0.1-8w / v%, the concentration of the photoinitiator is 0.1-2w / v%, and the concentration of the ion cross-linking agent is 10mM-1200mM.

6. The porous microsphere carrier according to any one of claims 1 to 5, characterized in that: The steps include: (1) Preparation of alginate gel microsphere solution ①Preparation of aqueous solution: During ion crosslinking, water-soluble alginate is dissolved in water as a raw material to prepare an alginate aqueous solution as an aqueous phase solution; During photo-crosslinking, photo-crosslinked alginate is dissolved in water as a raw material to prepare an alginate aqueous solution, and a photo-crosslinking initiator is added as an aqueous phase solution; When ionic crosslinking and photocrosslinking are combined, water-soluble alginate and photocrosslinking alginate are mixed as raw materials and dissolved in water, and a photocrosslinking initiator is added as an aqueous phase solution; in: The ion-crosslinked water-soluble alginate is alginate, aldehyde-modified alginate or a mixture of alginate and a water-soluble polymer; the water-soluble polymer includes gelatin, methacrylic anhydride-modified gelatin or collagen; The ionic crosslinking reaction initiator is selected from one or more combinations of calcium-ethylenediaminetetraacetic acid, magnesium-ethylenediaminetetraacetic acid, ferrous-ethylenediaminetetraacetic acid, manganese-ethylenediaminetetraacetic acid, copper-ethylenediaminetetraacetic acid, calcium-nitrilotriacetic acid, magnesium-nitrilotriacetic acid, ferrous-nitrilotriacetic acid, manganese-nitrilotriacetic acid, copper-nitrilotriacetic acid, calcium-diethylenetriaminepentaacetic acid, magnesium-diethylenetriaminepentaacetic acid, ferrous-diethylenetriaminepentaacetic acid, manganese-diethylenetriaminepentaacetic acid, and copper-diethylenetriaminepentaacetic acid chelate aqueous solution; The concentration of the ion-crosslinked water-soluble alginate is 0.1%-8w / v%, and the final concentration of the ion-crosslinking initiator is 30mM-400mM in terms of divalent metal ions; The photo-crosslinked alginate is one or a combination of methacrylic anhydride-modified alginate and methacryloyloxyethyltrimethylammonium chloride-modified alginate; The photocrosslinking initiator is one or a combination of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone; The concentration of the photo-crosslinked alginic acid is 0.1% to 8w / v%, and the concentration of the photo-crosslinking initiator is 0.1% to 2w / v%; The final concentration of the water-soluble alginate solution and the photo-crosslinked alginate solution crosslinked by the two crosslinking methods is 0.1% to 8w / v%, the final concentration of the ionic crosslinking initiator is 30mM to 400mM in terms of divalent metal ions, and the concentration of the photo-crosslinking initiator is 0.1% to 2w / v%; ② Oil phase configuration: fluorinated oil, fluorinated surfactant, acidic substance are blended, or one or two of mineral oil and soybean oil are blended with non-ionic surfactant; the acidic substance is acetic acid, citric acid, oxalic acid or lactic acid; the non-ionic surfactant is Span-80 or Span-20; ③ Collection phase configuration: one or more of fluorinated oil, perfluoroalcohol, and perfluoro acid are mixed with an acidic substance; or one or more of soluble divalent metal salts such as ferrous chloride, calcium chloride, cupric chloride, magnesium chloride, zinc chloride, strontium chloride, barium chloride, and nickel chloride solution; the acidic substance is acetic acid, citric acid, oxalic acid, or lactic acid; ④ Cleaning phase configuration: mixing fluorinated oil with one or more of perfluoroalcohol and perfluoroacid; or one or more of ethanol, acetone and isopropanol; (2) Formation of alginate droplets The aqueous solution obtained in step ① is injected into the aqueous channel of the microfluidic chip at a low flow rate, and the oil phase solution is injected into the oil phase channel of the microfluidic chip at a high flow rate. The aqueous solution and the oil phase solution are mixed through the emulsification channel to form water-in-oil emulsion droplets; preferably, the low flow rate is 0.1-5 mL / h, and the high flow rate is 1-30 mL / h; The flow rate ratio of the water phase to the oil phase is 1:1 to 1:30, preferably 1:1 to 1:20; the microfluidic chip emulsification channel has a cross structure, a co-flow type, a T-shaped mixing structure or a fluid focusing structure, and has at least two-phase liquid input ports, an emulsification channel and an output channel; (3) forming alginate microgels, including ion-crosslinked alginate microgels, photo-crosslinked alginate microgels, or ion-crosslinked combined with photo-crosslinked alginate microgels; When forming ion-crosslinked alginate microgels, there are three ways to form them: Method 1: The acidic substance reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous solution to release the soluble divalent metal ions, thereby completing the cross-linking in the collection pipeline of the microfluidic chip, and obtaining the alginate gel microspheres with the surfactant removed after washing with a washing phase; the collection phase solution is a mixture of one or more of fluorinated oil, perfluoroalcohol and perfluoro acid and the acidic substance; Method 2: inputting the water-in-oil emulsion droplets prepared in step (2) into a collection phase solution to form alginate gel microspheres, and then washing with a washing phase to obtain alginate gel microspheres with the surfactant removed; the collection phase solution is a soluble divalent metal salt solution; Method 3: The acidic substance reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous phase solution to release soluble divalent metal ions, thereby completing partial crosslinking in the collecting pipeline, and then the water-in-oil emulsion droplets prepared in step (2) are input into the collecting phase solution to form alginate gel microspheres, and the alginate gel microspheres with the surfactant removed are obtained after washing with the washing phase; the collecting phase solution is a soluble divalent metal salt solution; The acidic substance includes citric acid, acetic acid, oxalic acid or lactic acid; When forming a photo-crosslinked alginate microgel, the forming method is: The photoinitiator generates free radicals under ultraviolet light irradiation, which triggers the free radical polymerization reaction of the photo-crosslinked alginate, so that the photo-crosslinked alginate is gelled in the collection pipeline of the microfluidic chip. After being cleaned with a cleaning phase, the photo-crosslinked alginate gel microspheres with the surfactant removed can be obtained. When forming ion-crosslinked combined with photo-crosslinked alginate microgels, there are three formation methods: Method 1: Acid reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in an aqueous solution to release soluble divalent metal ions, and ultraviolet light is used to irradiate the collecting pipeline, thereby completing ion crosslinking and photocrosslinking in the collecting pipeline, and alginate gel microspheres with the surfactant removed are obtained after cleaning with a cleaning phase; the collecting phase solution used is a mixture of one or more of fluorinated oil, perfluoroalcohol, and perfluoro acid and an acidic substance; Method 2: using ultraviolet light to irradiate the collection pipeline to make the alginate droplets first complete photo-crosslinking, inputting the water-in-oil emulsion droplets prepared in step (2) into the collection phase solution to form alginate ion-crosslinked and photo-crosslinked gel microspheres, and obtaining the alginate gel microspheres with the surfactant removed after washing by the washing phase; the collection phase solution is a soluble divalent metal salt solution; Method 3: Acid reacts with ethylenediaminetetraacetate, nitrilotriacetate and diethylenetriaminepentaacetate in the aqueous phase solution to release soluble divalent metal ions, and ultraviolet light is used to irradiate the collecting pipeline to complete ion crosslinking and photocrosslinking in the collecting pipeline, and then the water-in-oil emulsion droplets prepared in step (2) are input into the collecting phase solution to further completely crosslink the alginate gel microspheres, and after washing with a washing phase, the alginate gel microspheres with the surfactant removed are obtained; the collecting phase solution is a soluble divalent metal salt solution; (4) Surface modification of alginate microgels: The washed alginate gel microspheres are coated with a protein shell layer. There are two preparation methods: Method 1: Soak the prepared alginate gel microspheres doped with aldehyde-modified alginate in a shell protein aqueous solution, and then immerse them in a crosslinker aqueous solution for crosslinking after the shell protein is coated, and then centrifuge the above mixture at a speed of 1000-2000 rpm, remove the supernatant, and wash three times with deionized water; the mixing mass ratio of the aldehyde-modified alginate and the alginate gel microspheres is 0.1:10-10:1; the concentration of the crosslinker aqueous solution is 0.1-8w / v%, preferably 0.1-4w / v%; the crosslinker includes one or more combinations of glutaraldehyde, thrombin, and ethylenediamine; the shell protein aqueous solution includes one of gelatin, collagen, fibrinogen, albumin and fibrin, or the above proteins and other biocompatible polymers, and the concentration is 0.1-6%; Method 2: The prepared alginate gel microspheres are immersed in an aqueous solution composed of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), the carboxyl groups on the alginate microspheres are activated, and then immersed in a shell protein aqueous solution for cross-linking, and then the mixture is centrifuged at a speed of 1000-2000 rpm, and after removing the supernatant, it is washed three times with deionized water; the coating aqueous solution includes one of gelatin, collagen, fibrinogen, albumin and fibrin, or the above proteins and other biocompatible polymers, and the concentration is 0.1-6%; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (NHS) is 0.1-5mM, preferably 0.1-2mM; other biocompatible polymers include polyethylene glycol, polyvinyl alcohol or polylactic acid and polylysine; (5) Formation of porous ion-crosslinked alginate microgels The alginate microspheres modified with shell proteins formed above are fully frozen at -20°C to -196°C, and then transferred to a freeze dryer for freeze drying to obtain porous microsphere carriers.

7. Use of the controllably degradable porous microcarrier according to any one of claims 1 to 6 in the field of large-scale suspension amplification culture of adherent cells.

8. The use according to claim 7, characterized in that: The porous microcarrier is mixed with a cell suspension to allow the cells to adhere to the surface of the porous carrier. After large-scale culture, the cells are detached from the porous microcarrier to complete cell harvesting.

9. The use according to claim 8, characterized in that: After the cells are cultured on a large scale, a degradation solution is added, and the cells are quickly separated from the porous microcarriers; the degradation solution includes a metal chelator and a protein degrading enzyme; the concentration of the metal ion chelator is 0.01-0.5w / v%, preferably 0.02%-0.3w / v%; the concentration of the protein degrading enzyme is 0.5-20mg / mL, preferably 1-10mg / mL.

10. The use according to claim 9, characterized in that: The metal chelator comprises one or more of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid (DTPA); the protein degrading enzyme is an enzyme that has the function of degrading extracellular matrix (ECM) proteins; preferably, it comprises one or more of collagenase, trypsin, papain, gelatinase, or matrix lysin.

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