High-surface-area virtual sphere cell culture microcarrier and forming method thereof

Abstract

The invention relates to a polymer cell microcarrier which is edible or can be dissolved in a subsequent cell cultivation (or transplantation) process, and a plurality of pit structures are formed on the outer surface of the polymer cell microcarrier, so that an increased surface area is provided as an attachment surface of cells, the roughness of the outer surface of the microcarrier is increased, and stable attachment of the cells is promoted. The invention also relates to a method for manufacturing the cultured cell microcarrier. The methods involve producing a hydrogel particle containing an edible polymer and having a smooth outer surface, and forming a plurality of dimple structures on the outer surface of the hydrogel particle by a dimple production process.

Description

Technical Field

[0001] The present invention relates to a high molecular cell microcarrier which is edible or can be dissolved in the subsequent cell culture (or transplantation) process, and has a plurality of pit structures formed on its outer surface, which is conducive to cell attachment and promotes cell proliferation. The present invention also relates to a method for manufacturing the culture cell microcarrier. Background Art

[0002] Traditionally, in vitro cell culture is achieved by attaching cells to tissue culture plastic vessels or extracellular matrix attachment proteins, and then giving them appropriate liquid culture medium to promote their growth and proliferation. However, this two-dimensional planar culture method is far from the actual physiological environment in vivo, and cannot simulate the interaction between cells and extracellular matrix and cells in vivo, nor is it conducive to reproducing complex cell behaviors such as cell migration, apoptosis, transcriptional regulation and receptor expression. The two-dimensional culture method further limits the growth space of cells, which is not conducive to the mass production of cells. Three-dimensional cell culture technology is obviously a better solution to respond to the above industrial issues. Microcarriers or scaffolds are materials frequently used in three-dimensional cell culture technology, usually with non-toxic, non-rigid, and uniform density structures. It enables cells that rely on attachment to grow to grow on the surface of microcarrier particles during dynamic culture. Microcarriers usually have a large surface area / volume ratio, which makes the cell yield per unit culture medium higher than static, planar culture technology.

[0003] The microcarriers commonly used in laboratories and industry are produced by Cytiva. The main material of the series is a polystyrene core, with surface coatings such as DEAE-dextran making it suitable for cell attachment and growth. Figure 1The process of using this type of microcarrier to produce cells is shown, wherein the microcarrier is placed in a bioreactor and sterilized, then inoculated with the required cells, and the culture medium is injected and replaced regularly for cell culture. When the cell culture is to be terminated, the cells are separated from the microcarrier using a reagent, and then the microcarrier is completely separated from the cells by filtering, and the harvested cells can be used for subsequent cell transplantation and the like. However, this conventional microcarrier has important disadvantages in application, because the polystyrene microcarrier material is not edible or soluble in the tissue of the subsequent culture cell transplantation, so it is necessary to remove the cultured cells from the microcarrier in the later stage of the culture process before they can be used. Furthermore, when harvesting cells, enzymes such as trypsin are usually used to cut off the connection between the cells and the microcarrier material. In addition to increasing the cost of raw materials during production, such a culture process will also take up valuable time. Trypsin cannot separate the cells from the microcarrier 100%, and the cell activity after separation cannot survive 100%, so the overall cell recovery rate can only reach about 70%. Finally, when using filtration and other methods to separate microcarriers from cells, the filter often becomes clogged. In order to completely wash off the cells on the microcarriers, the filter must be replaced frequently and a large amount of buffer solution must be used for washing. This is a cumbersome operation and requires the use of a large amount of solvent. Due to the high cost and complexity of the procedure, the above conventional three-dimensional cell culture technology does not meet the needs of mass cell culture.

[0004] In particular, the artificial meat industry through cell culture has developed rapidly in recent years. In addition to solving the food problem, it is also an important technology to reduce environmental pollution and greenhouse gas emissions, while also taking into account the issue of not killing animals in certain religions. Artificial meat products give people who love animals and start a vegetarian lifestyle an opportunity and choice to start eating meat again. However, in the process of manufacturing cultured meat, common but inedible polystyrene cannot be used, nor can microcarriers or scaffolds made of animal-derived materials such as gelatin be used. Therefore, it is particularly necessary to develop microcarriers or scaffolds made of non-animal-derived materials. Summary of the invention

[0005] In order to solve the above problems and shortcomings, the present invention provides a microcarrier for culturing cells, which is made of edible polymers or materials that can be dissolved under mild conditions that do not harm cells during subsequent cell culture or transplantation, and is therefore suitable for various industrial applications that require mass production of cells, including the production of artificial meat. Furthermore, in the later stage of cell culture, the microcarrier of the present invention does not need to separate the cultured cells from the microcarrier, so that an almost 100% recovery rate can be achieved. More importantly, the microcarrier of the present invention presents a particle configuration with an irregular but smooth and non-angular outer surface, which is referred to as a virtual spherical microcarrier. This appearance configuration is generated by manufacturing hydrocolloid particles containing the above microcarrier material and having a smooth outer surface, and by a concave manufacturing process to produce a plurality of concave structures on the outer surface of the hydrocolloid particles. The concave structures on the outer surface of the microcarrier of the present invention not only provide an increased surface area as an attachment surface, making it easy for cells to approach and attach, but also these concave structures increase the roughness of the outer surface of the microcarrier, promoting the stable attachment of cells, thereby providing a culture environment that is conducive to cell growth and proliferation.

[0006] Therefore, the first aspect of the present invention provides a virtual spherical microcarrier for culturing cells, comprising:

[0007] A continuous medium composed of one or more edible polymers, which presents an irregular particle structure and has a particle size between 200 microns and 1000 microns, wherein the continuous medium has an outer surface, and a plurality of pit structures are formed on the outer surface, and the pit structures are made by freezing a solid sphere composed of the one or more edible polymers.

[0008] The second aspect of the present invention provides a method for producing the aforementioned microcarrier, which comprises the following steps:

[0009] A) micronizing an aqueous composition comprising one or more edible polymers and dispersing the micronized aqueous composition in a dispersion liquid to obtain a plurality of hydrocolloid droplets dispersed in the dispersion liquid and having a smooth outer surface;

[0010] B) solidifying the hydrogel droplets into hydrogel particles, and separating the hydrogel particles from the dispersion;

[0011] C) freezing the hydrogel particles at a temperature between -10°C and -60°C for a time between 8 and 24 hours, so that a plurality of pit structures are formed on the outer surface of the hydrogel particles, wherein each of the pit structures has a minimum geometric dimension in the range of 50 μm to 300 μm; and

[0012] D) drying the hydrocolloid particles with the pit structure to obtain microcarriers in the form of a continuous medium with irregular particle configuration and a particle size ranging from 200 μm to 1000 μm.

[0013] The third aspect of the present invention provides another method for producing the aforementioned microcarrier, which comprises the following steps:

[0014] A) in the presence of a pore-forming agent, micronizing an aqueous composition comprising one or more edible polymers and dispersing the micronized aqueous composition in a dispersion liquid to obtain a plurality of hydrocolloid droplets dispersed in the dispersion liquid and having a smooth outer surface;

[0015] B) solidifying the hydrogel droplets into hydrogel particles, and removing the pore former, so that a plurality of pit structures are formed on the outer surfaces of the hydrogel particles, wherein each of the pit structures has a minimum geometric dimension in the range of 50 μm to 300 μm, and separating the hydrogel particles from the dispersion; and

[0016] C) drying the hydrocolloid particles with the pit structure to obtain microcarriers in the form of a continuous medium with irregular particle configuration and a particle size ranging from 200 μm to 1000 μm. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Demonstrates the process of producing cells using conventional microcarriers.

[0018] Figure 2 is a schematic diagram of a specific microcarrier according to the present invention.

[0019] Figure 3 is a flow chart of a first method for producing microcarriers according to the present invention.

[0020] Figure 4 is an optical electrophotographic image according to a specific embodiment of the present invention, wherein Figure 4 a shows that the hydrocolloid droplets containing edible polymers dispersed in the dispersion are spherical; Figure 4 b shows the hydrocolloid particles forming a pit structure; Figure 4 c shows the microcarriers obtained after drying, the dotted line represents the original spherical configuration before drying, and the double arrows indicate the geometric dimensions measured under an electron microscope.

[0021] Figure 5 is a flow chart of a second method for manufacturing microcarriers according to the present invention; and

[0022] Figure 6 In order to utilize the microcarriers of the present invention, the irregular microcarriers after grinding and Cell growth curve obtained from cells cultured on microcarriers.

[0023] Description of the figure number:

[0024] Microcarrier 100

[0025] Continuous Media 120

[0026] The pit structure 140 . DETAILED DESCRIPTION

[0027] Figure 2 Schematic diagrams of two embodiments of the present application show that the microcarrier 100 mainly comprises a continuous medium 120. The "continuous medium" referred to in this specification refers to a single-piece medium material composed of one or more edible polymers, suitable for cells to attach to and grow, proliferate and migrate. The term "edible" here is used to describe materials that are suitable for animals, preferably mammals, and more preferably humans, to be orally ingested without causing damage to their biological systems such as cells, tissues and organs or causing adverse side effects. Edible polymers suitable for preparing the microcarriers of the present invention include, but are not limited to: plant polysaccharides, such as konjac glucomannan (KG), agarose, alginate, cellulose, gellan gum, carrageenan, dextran, guar gum, xanthan gum, starch, etc.; animal polysaccharides, such as hyaluronic acid, chitin, etc.; plant proteins, such as legume proteins such as pea protein (PP), soy protein, lentil protein and chickpea protein, nut proteins such as peanut protein, almond protein, hemp seed protein and chia seed protein, grain proteins such as quinoa protein, wheat protein, rice protein and corn protein, potato protein, vegetable and fruit crop protein, fungal protein, algae protein, etc.; animal proteins, such as gelatin, collagen, fibrins, etc.; and combinations thereof. In a preferred embodiment, the edible polymer is selected from polymers that can be dissolved in the subsequent cell culture or transplantation process, so that the microcarriers can be disintegrated by dissolving the polymer in the subsequent process, thereby releasing the cells attached to the microcarriers and harvesting them. These soluble polymers include but are not limited to alginate, cellulose, gellan gum, dextran, guar gum, xanthan gum and starch.

[0028] In a preferred embodiment, the microcarriers in this case are basically made of plant polysaccharides, such as konjac glucomannan (KG). KG is a high molecular weight polysaccharide formed by connecting glucose and mannose through β-1,4-glycosidic bonds, and can be extracted from konjac (Amorphophallus konjac). KG has good intermolecular forces and can easily form stable and strong solids. KG also has the characteristics of high hydrophilicity, containing a large number of active hydroxyl and carboxyl functional groups, and not undergoing non-specific adsorption with biological macromolecules, making it easy for cells to approach and attach, and is very suitable as a microcarrier material for culturing cells. The microcarrier made of KG as the main material has good processing capabilities to control the structure of the finished product, and its strength can be maintained in cell culture medium for one month of cell culture, indicating that it can withstand structural damage that may be caused by mechanical or fluid in the culture system. KG is one of the sources of dietary fiber in food, has food health safety, and low toxicity and health hazards. KG also has the characteristics of biodegradation. There are natural decomposition enzymes in nature, and it will not remain in the earth's environment for a long time.

[0029] In a more preferred embodiment, the microcarriers of the present invention are made of plant-based polysaccharides such as KG and plant-based proteins such as pea protein to provide appropriate surface chemical properties and enhance the ability of cells to adhere to the microcarrier surface. Taking pea protein as an example, its purification and mass production technologies are quite mature, and it can be used as the main protein source for whey and high-protein fitness formulas, and is quite reliable in terms of food safety.

[0030] In another more preferred embodiment, the microcarriers of the present invention are made of materials other than KG and also contain a small amount of alginate, so that the solution-gel conversion rate of KG can be adjusted by controlling the cross-linking rate of alginate, thereby preventing KG from gelling prematurely during the solution preparation process.

[0031] like Figure 2 As shown, the microcarrier 100 of the present invention is in a particle configuration and has an irregular but smooth outer surface without edges and corners. As described later, the appearance of the microcarrier of the present invention is originally a spherical configuration as shown by the dotted line, and the spherical configuration is not limited to a true sphere (such as Figure 2 Left), also includes approximately true spheres, ellipsoids (such as Figure 2The microcarrier 100 is a microcarrier having a shape of a plurality of concave structures 140 and a shape of a plurality of concave structures 140. The concave structures 140 are formed on the smooth outer surface of the original sphere (as shown by the dotted line) by a concave manufacturing process, thereby producing particles with irregular shapes. Therefore, the microcarrier in this case is called a virtual spherical microcarrier. The term "irregular" used in this case means that the microcarrier deviates from its original spherical configuration to a certain extent. According to the present invention, the particle size of the microcarrier 100 is within the range of 200 microns to 1000 microns, preferably within the range of 250 microns to 800 microns, for example, within the range of 300 microns to 700 microns, depending on the type of cells to be cultured and the culture conditions. The particle size of the microcarrier 100 can be defined by optical or electron microscopy technology (electron microscopy) or other measurement technology, which will not be repeated here. The distribution state of these concave structures 140 on the outer surface of the microcarrier can be an orderly arrangement, a disordered arrangement or a partially orderly arrangement, and the concave structures 140 can be interconnected, overlapped or exist independently of each other. The appearance of each pit structure 140 can be a smooth curved surface, an irregular rough surface, or a combination of the two. The minimum geometric dimension of each pit structure 140 is in the range of 50 microns to 300 microns, preferably in the range of 100 microns to 250 microns, for example, in the range of 100 microns to 200 microns. The "minimum geometric dimension" referred to herein means the minimum dimension used to describe the appearance of the individual pit structure, such as the smallest dimension of the side length, depth, diameter, etc. of the pit structure 140 measured using electron microscopy technology. As described below, the size and number of the pit structures can be adjusted by controlling the parameters and conditions of the pit manufacturing process.

[0032] By forming a pit structure, the microcarrier of the present invention can effectively increase its area / volume ratio, thereby effectively increasing the area of ​​cell attachment and the efficiency of growth. These pit structures also increase the surface roughness of the microcarrier, thereby increasing the adhesion of cells when they attach and grow, which makes it difficult for cells to fall off due to the rapid flow or stirring of the culture medium during dynamic culture. The above characteristics make the microcarrier of the present invention suitable for industrial applications such as artificial meat, in vitro organoid culture, artificial skin, artificial leather, cell therapy, gene therapy, etc., but not limited to them.

[0033] Figure 3The first method of the present invention for manufacturing microcarriers involves using freezing as a means of forming pits after the hydrogel droplets containing edible polymers are solidified to form a pit structure on their outer surface. The method comprises step A: miniaturizing an aqueous composition containing edible polymers and dispersing it in a dispersion to obtain a plurality of hydrogel droplets; step B: solidifying the hydrogel droplets into hydrogel particles; step C: freezing the hydrogel particles to form a pit structure on their outer surface; and step D: drying the hydrogel particles with the pit structure, etc., to obtain the virtual spherical microcarrier of the present invention.

[0034] In step A, the edible polymer and other components may be dissolved in an aqueous solvent to form an aqueous composition, and a dispersion may be prepared. The aqueous composition may then be micronized and dispersed in the dispersion. In the process of preparing the aqueous composition, dissolution may be promoted by heating or cooling, stirring, homogenizing, ultrasonic vibration, shaking, and the like. In a specific example where the edible polymer has a high viscosity, a high-speed homogenizer may be used to apply a high shear force to the aqueous composition to promote uniform mixing of the components in the aqueous composition.

[0035] In a preferred embodiment, the dispersion comprises an oily liquid that is immiscible with the aqueous composition, and the miniaturization and dispersion process comprises emulsifying the aqueous composition and the dispersion to prepare an oil-in-water emulsion. During the emulsification process, the aqueous composition is miniaturized and dispersed in the dispersion as a continuous phase, wherein the dispersed phase comprises a plurality of hydrogel droplets. In another preferred embodiment, the dispersion comprises an aqueous solvent, and the miniaturization and dispersion process comprises miniaturizing the aqueous composition into a plurality of hydrogel droplets and dispersing them in the aqueous solvent. The means of miniaturization and dispersion include, but are not limited to, dripping, spraying, transmembrane emulsification, mechanical stirring, high-speed shearing, and combinations thereof. The size of the hydrogel droplets can be adjusted by controlling the parameters and conditions of each dispersion means. For example, in the embodiment using mechanical stirring or high-speed shearing as the miniaturization and dispersion means, the size of the hydrogel droplets can be adjusted by regulating the disturbance rate and temperature of the mixer or homogenizer and / or the viscosity of the dispersion. In the specific example of using spraying or transmembrane emulsification as a means of miniaturization and dispersion, the size of the hydrogel droplets can be adjusted by selecting the size of the nozzle or the size of the through-holes of the emulsification membrane. In a more preferred specific example, the present invention uses high-speed shearing as a means of miniaturization and dispersion. The inventors of the present case have found that high-speed shearing can overcome the problem that high-viscosity aqueous compositions are not easy to pass through narrow through-holes when using dripping, spraying or transmembrane emulsification as a means of miniaturization and dispersion. Therefore, it can effectively increase the content of edible polymers in the aqueous composition, thereby enhancing the mechanical strength of the final microcarrier product, so that it has higher structural stability when immersed and stirred in the culture medium for a long time. The hydrogel droplets dispersed in the dispersion will spontaneously form into substantially true spheres, approximately true spheres or ellipsoids due to their own cohesive force, and thus have a smooth outer surface, such as Figure 4 As shown in a.

[0036] The term "aqueous solvent" used in this case means a solvent system mainly composed of water. Applicable aqueous solvents are any known aqueous solvents that can achieve the purpose of dissolving edible polymers and will not produce substantial chemical reactions with edible polymers or other components in the manufacturing process. It is preferred that the aqueous solvent itself is edible, and it is more preferred that the aqueous solvent does not contain ingredients of animal origin, including but not limited to water, such as deionized water, pure water; aqueous solutions of C1-4 alcohols, such as 50-95% by volume ethanol aqueous solutions; and aqueous solutions of salts or acids and bases, such as aqueous solutions of sodium chloride, calcium chloride, acetic acid, sodium acetate, sodium bicarbonate, sodium carbonate, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium trimetaphosphate, and combinations thereof; Aqueous solutions of carbohydrates include aqueous solutions of linear or cyclic monosaccharides, disaccharides, and polysaccharides, such as aqueous solutions of glyceraldehyde, erythrose, arabinose, xylose, fructose, glucose, galactose, mannose, sedoheptulose, lactose, sucrose, maltose, trehalose, starch, glycogen, cellulose, and chitin, and combinations thereof; aqueous solutions of sugar alcohols, such as aqueous solutions of glucitol, xylitol, sorbitol, maltitol, erythritol, lactitol, mannitol, maltitol, and lactitol, and combinations thereof; aqueous solutions of polyols, such as aqueous solutions of maple syrup, trehalose, corn sugar, pomegranate sugar, and glycerol; and aqueous solutions of polyethers, such as aqueous solutions of polyethylene glycol. Preferably, the aqueous solvent is selected from the group consisting of water and salt solutions thereof.

[0037] The aforementioned "oily liquid" is preferably edible itself, and more preferably the oily liquid is oil of non-animal origin, including but not limited to silicone oil, sunflower oil, canola oil, salad oil, olive oil and combinations thereof.

[0038] The aqueous composition and / or the dispersion may selectively contain a surfactant for promoting dispersion of the material and / or for stabilizing the water-in-oil emulsion to prevent the hydrocolloid droplets dispersed in the continuous phase from merging with each other. The surfactants suitable for the present invention include, but are not limited to, cationic surfactants, such as fatty ammonium salts, ethanol ammonium salts, and polyethylene polyammonium salts; anionic surfactants, such as alkylbenzene sulfonates, alkyl sulfonate salts, alkyl sulfonates, alkyl sulfates, olefin sulfonates, cyclopentaneates, and stearates; zwitterionic surfactants, such as alkyl amino acids, carboxy betaines, sulfonic acid betaines, phosphate betaines, alkyl tetrahydroimidazoles, and polyacrylamides; and nonionic surfactants, such as fatty alcohol polyoxyethylene ethers, alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene esters, alkyl polyoxyethylene amines, alkyl polyoxyethylene amides, and polyethers. Preferably, the surfactant accounts for about 0 to 30% by weight in the emulsion.

[0039] In the specific example where the dispersion is an oily liquid, the aqueous composition and / or the dispersion may selectively contain solid particles that can promote the stabilization of the emulsion, so that the water-in-oil emulsion prepared in step A becomes a Pickering emulsion to prevent the hydrocolloid droplets dispersed in the continuous phase from merging with each other. Solid particles suitable for the present invention include, but are not limited to, silicon dioxide and polystyrene.

[0040] Step B involves solidifying the hydrogel droplets dispersed in the dispersion into hydrogel particles, so that the structure has sufficient mechanical strength to withstand the subsequent pit manufacturing process without disintegration. The term "solidification" used in this case means the process of applying physical or chemical bridging means to the hydrogel droplets in a fluid state to transform them into hydrogel particles with a stable configuration. The reaction conditions for solidification vary depending on the type of edible polymer, but they are all familiar to the industry. For example, in the specific example of using konjac glucomannan (KG) as an edible polymer, the temperature can be increased to gelatinize the hydrogel droplets. In the specific example of using collagen or gelatin as an edible polymer, the hydrogel droplets can be dehydrated at low temperature to gelatinize and shape. In the specific example of using alginate as an edible polymer, salts that can dissociate divalent metal ions such as calcium ions or magnesium ions can be added to cause cross-linking reactions between alginate molecules in the hydrogel droplets, thereby solidifying and shaping.

[0041] Step B further separates the solidified hydrogel particles from the dispersion. Applicable separation processes include all conventional solid-liquid separation processes. In a preferred embodiment, the hydrogel particles can be separated by filtering, such as using a screen, a membrane, vacuum extraction, wet filtration or a sieve shaker. The separated hydrogel particles can be washed several times with water (e.g., deionized water, pure water) and / or an alcohol solvent (e.g., ethanol, isopropanol) to remove the residual dispersion.

[0042] Step C involves a pit manufacturing process in which the separated hydrocolloid particles are frozen. The inventors of the present case have found that the freezing temperature is a key condition for determining the size of the pit structure, and the length of the freezing time will affect the integrity of the pit structure. If the freezing temperature or freezing time is insufficient, not only will the pit structure be incomplete, but its average size will also be out of control, and the thickness of the partition wall of the pit structure is uneven, which may constitute a structural weakness of the microcarrier, which is not conducive to long-term immersion in the culture medium to withstand the shear force caused by the flow of the culture medium under cell culture conditions. Therefore, the freezing temperature is selected to be between -10 and -60°C, and the freezing time is between 8 and 24 hours, preferably the absolute value of the freezing temperature × freezing time ≧ 250°C hours, for example ≧ 300°C hours. Thereby, the minimum geometric dimension of each pit structure is within the range of 50 microns to 300 microns, more preferably within the range of 100 microns to 250 microns, for example, within the range of 100 microns to 200 microns. In a specific example using KG as an edible polymer, a freezing temperature of about -20°C and a freezing time of about 16 hours can be used to form a pit structure with a diameter in the range of 150 to 200 microns on the outer surface of the hydrogel particles. In another specific example using KG as an edible polymer, a freezing temperature of about -50°C and a freezing time of about 10 hours can be used to form a pit structure with a diameter in the range of 30 to 80 microns on the outer surface of the hydrogel particles. An example of the hydrogel particles obtained after performing step C is shown in Figure 4 b.

[0043] The term "freezing" as used herein may also include "freeze drying", which includes a freezing process to freeze the water in the hydrocolloid particles into ice crystals, and a drying process to sublime the ice crystals into water vapor under reduced pressure, which is then removed by vacuum.

[0044] In step D, the hydrocolloid particles with the pit structure are dried to achieve the purpose of removing moisture. In addition to being convenient for storage, they are also less likely to grow dirty, and are easy to weigh, measure volume, and perform other required packaging operations. The applicable drying method is any known drying method that can achieve the above-mentioned purpose of removing moisture, including but not limited to hot air drying and freeze drying. The volume of the dried hydrocolloid particles may be slightly reduced, causing their outer surface to be slightly deformed, presenting a continuous medium with irregular particle configurations, such as Figure 4 As shown in c, Figure 4 The white dotted line shown in c is the spherical shape before the pit is formed. Compared with the hot air drying method, the microcarriers dehydrated by freeze drying method have no volume reduction and are relatively advantageous.

[0045] The microcarriers prepared by the above method have a particle size between 200 microns and 1000 microns. The dried microcarriers can be pre-soaked in a culture medium for rehydration before being used for cell culture, and then can be used for inoculating cells.

[0046] Figure 5 The present invention is a flow chart of a second method for manufacturing microcarriers, which comprises step A: in the presence of a pore-forming agent, miniaturizing an aqueous composition comprising an edible polymer and dispersing it in a dispersion to obtain a plurality of hydrogel droplets; step B: solidifying the hydrogel droplets into hydrogel particles so that a pit structure is formed on the outer surface thereof; and step C: drying the hydrogel particles having the pit structure, etc., to obtain the virtual spherical microcarriers of the present invention.

[0047] The definitions of chemical materials and processing methods contained in the above-mentioned second method are the same as those defined for the first method above. The main difference between the second method and the first method in this case is the timing and means of forming the pit structure; that is, the second method in this case forms the pit structure by adding a pore-forming agent before the hydrogel droplets are solidified, while the first method in this case forms the pit structure by freezing the hydrogel droplets after they are solidified. According to the present invention, the pore-forming agent can be added to the aqueous composition or the dispersion in step A. Preferably, the pore-forming agent is mixed in the aqueous composition by aeration, stirring, homogenization, shaking, thin film emulsification, etc. after the edible polymer is dissolved in an aqueous solvent to form an aqueous composition. Subsequently, as the aqueous composition is miniaturized, it is distributed in the hydrogel droplets that are still in a fluid state. At this time, many pore-forming agent molecules or gases derived therefrom will exist near the outer surface of the hydrogel droplet and occupy space, causing the outer surface of the hydrogel droplet to be concave inward, thereby forming a pit structure as the hydrogel droplet is solidified into hydrogel particles in step B. The pore-forming agent includes but is not limited to gas, gas blowing agents, oil liquids and aqueous solvents containing surfactants.

[0048] In step B of the second method of the present case, a process of removing the pore-forming agent may be included after the curing process. In one embodiment, the pore-forming agent may be removed in conjunction with the drying process, and the pore-forming agent may be removed by means of exhaustion, blowing, vacuuming, etc., leaving a pit structure. In the specific example of using a foaming pore-forming agent such as ammonium bicarbonate to generate gas, the pore-forming agent may be made to generate gas by physical or chemical methods such as heating and changing the pH value during or after the curing of the hydrogel droplets to form a pit structure. The generated gas escapes on its own or is removed by exhaustion, blowing, vacuuming, etc. In the specific example of using a homogeneous pore-forming agent, after the pit structure is generated during the curing process of the hydrogel droplets, the pore-forming agent is removed by exhaustion, blowing, vacuuming, etc., leaving a pit structure. The microcarrier prepared by this method has a particle size between 200 microns and 1000 microns.

[0049] The following examples are only provided to illustrate the present invention, but not to limit the scope of the present invention.

[0050] Example 1: Preparation of hydrocolloid particles

[0051] A high-speed homogenizer is used to apply shear force to dissolve konjac glucomannan (KG), calcium alginate (ALG) and an edible hardness-increasing additive in deionized water to prepare an aqueous composition. The aqueous composition is then dispersed in a canola oil dispersion using a high-speed homogenizer to form an oil-in-water emulsion. During the dispersion, the shear force applied by the high-speed homogenizer turns the aqueous composition containing konjac glucomannan into droplets, wherein the droplet size and dispersion state can be controlled by adjusting parameters such as the speed of the homogenizer and the viscosity of the dispersion. At this time, the aqueous composition dispersed into droplets presents a smooth curved surface appearance, such as a spherical appearance, in the dispersion due to surface tension. After dispersion, the obtained oil-in-water emulsion is heated at 80°C for 1 hour to solidify the droplets into particles to strengthen its structure. During the entire heating process, the particles are stirred at an appropriate speed to maintain a dispersed state. Next, these particles are removed from the dispersion by filtration. A screen of a specific size can be used to further remove oversized or undersized particles, with an overall recovery rate of 30-95%. During the filtration process, water and alcohol solvents can be used for washing to remove substances such as canola oil and surfactants, and finally water is used for washing.

[0052] Example 2: Preparation of hydrocolloid particles

[0053] The powders of KG, ALG and pea protein (PP) are mixed in deionized water and shear force is applied to dissolve them to make an aqueous composition. The aqueous composition is then quickly added to the canola oil being stirred in a homogenizer assembly and continuously stirred to form an oil-in-water emulsion, wherein the particle size of the dispersed phase droplets is about 300 microns ± 200 microns. Then, the calcium chloride aqueous solution is quickly added to the oil-in-water emulsion, and the temperature is increased and continuously stirred to make the micronized particles produce cross-linking strength. If a positively charged cross-linking agent is required, it can be added to the calcium chloride aqueous solution and added to the oil-in-water emulsion together. The cross-linked oil-in-water emulsion is passed through a sieve of the required size to filter out hydrocolloid particles of the required size. During the filtration process, water and alcohol solvents can be used for washing to remove the canola oil, and finally washed with water.

[0054] Example 3: Preparation of hydrocolloid particles

[0055] The powders of KG and ALG are mixed in deionized water and dissolved by applying shear force at 80°C to make an aqueous composition. Then, the mixture is dispersed into water containing calcium ions by syringe dropwise addition, so that the droplets of the aqueous composition are converted from liquid to colloidal state, thereby forming spherical hydrocolloid particles containing KG. The hydrocolloid particles of the required size are filtered out by sieving.

[0056] Example 4: Preparation of microcarriers

[0057] The KG-containing hydrocolloid particles prepared in Example 1 were frozen to form a pit structure on the outer surface, and then the hydrocolloid particles were dried to obtain microcarriers.

[0058] Example 5: Cell growth efficiency of microcarriers

[0059] Equal amounts of cells were inoculated on the microcarriers prepared in Example 4, irregular microcarriers made of KG material in large blocks and then crushed into powder, and gelatin microcarriers produced by Taiwan Innovative Materials Co., Ltd. The cells were cultured on microcarriers under conditions suitable for cell growth and the cell numbers were counted on the first and fourth days after inoculation. Figure 6 The cell growth curves obtained using the above three microcarriers are shown in Figure 4. The virtual spherical microcarriers prepared in Example 4 can grow to 14 times the original number of cells after four days of culture. The irregular microcarriers made of the same material can grow 10 times after four days of culture. The microcarriers have a cell growth rate of about 3 times after four days. The experimental results of this embodiment indicate that the surface structure of the virtual spherical microcarriers in this case provides more surface area and better surface roughness, which is conducive to the stable attachment of cells, thus providing an environment more suitable for cell growth and also suitable for dynamic culture.

Claims

1. A virtual spherical microcarrier for culturing cells, characterized in that: It contains: A continuous medium composed of one or more edible polymers, which presents an irregular particle structure and has a particle size between 200 microns and 1000 microns, wherein the continuous medium has an outer surface, and a plurality of pit structures are formed on the outer surface, and the pit structures are made by freezing a solid sphere composed of the one or more edible polymers.

2. The microcarrier according to claim 1, characterized in that The continuous medium has a particle size ranging from 250 microns to 800 microns.

3. The microcarrier according to claim 2, characterized in that The continuous medium has a particle size ranging from 300 microns to 700 microns.

4. The microcarrier according to claim 1, characterized in that Each of the pit structures has a minimum geometric dimension ranging from 50 micrometers to 300 micrometers.

5. The microcarrier according to claim 1, characterized in that Each of the pit structures has a minimum geometric dimension ranging from 100 micrometers to 250 micrometers.

6. The microcarrier according to claim 1, characterized in that Each of the pit structures has a minimum geometric dimension ranging from 100 micrometers to 200 micrometers.

7. The microcarrier according to claim 1, characterized in that The edible polymer is selected from the group consisting of plant polysaccharides, animal polysaccharides, plant proteins, animal proteins and combinations thereof.

8. A method for producing a microcarrier for culturing cells, characterized in that: It includes the following steps: A) micronizing an aqueous composition comprising one or more edible polymers and dispersing the micronized aqueous composition in a dispersion liquid to obtain a plurality of hydrocolloid droplets dispersed in the dispersion liquid and having a smooth outer surface; B) solidifying the hydrogel droplets into hydrogel particles, and separating the hydrogel particles from the dispersion; C) freezing the hydrogel particles at a temperature between -10°C and -60°C for a time between 8 and 24 hours, so that a plurality of pit structures are formed on the outer surface of the hydrogel particles, wherein each of the pit structures has a minimum geometric dimension in the range of 50 μm to 300 μm; as well as D) drying the hydrocolloid particles with the pit structure to obtain microcarriers in the form of a continuous medium with irregular particle configuration and a particle size ranging from 200 μm to 1000 μm.

9. A method for producing a microcarrier for culturing cells, characterized in that: It includes the following steps: A) in the presence of a pore-forming agent, micronizing an aqueous composition comprising one or more edible polymers and dispersing the micronized aqueous composition in a dispersion liquid to obtain a plurality of hydrocolloid droplets dispersed in the dispersion liquid and having a smooth outer surface; B) solidifying the hydrogel droplets into hydrogel particles, and removing the pore-forming agent to form a plurality of pit structures on the outer surfaces of the hydrogel particles, wherein each of the pit structures has a minimum geometric dimension in the range of 50 μm to 300 μm, and separating the hydrogel particles from the dispersion; as well as C) drying the hydrocolloid particles with the pit structure to obtain microcarriers in the form of a continuous medium with irregular particle configuration and a particle size ranging from 200 μm to 1000 μm.

10. The method according to claim 8 or 9, characterized in that Each of the pit structures has a minimum geometric dimension ranging from 50 micrometers to 300 micrometers.

11. The method according to claim 8 or 9, characterized in that The miniaturization and dispersion in step A are selected from the group consisting of dripping, spraying, transmembrane emulsification, mechanical stirring, high-speed shearing and combinations thereof.

12. The method according to claim 8, characterized in that The freezing has the following conditions: the absolute value of the freezing temperature×the freezing time≧250°C hours.

13. The method according to claim 8, characterized in that The freezing has the following conditions: the absolute value of the freezing temperature×the freezing time≧300°C hours.

14. The method according to claim 9, characterized in that The pore-forming agent is selected from the group consisting of gas, foaming agent, oil liquid, and aqueous solvent containing surfactant.

15. The method according to claim 8 or 9, characterized in that The edible polymer is selected from the group consisting of plant polysaccharides, animal polysaccharides, plant proteins, animal proteins and combinations thereof.

16. The method according to claim 8 or 9, characterized in that The drying method is selected from the group consisting of hot air drying and freeze drying.

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