CN115404195A

CN115404195A - Microcarrier with continuous outer wall and scaffold structure for culturing cells

Info

Publication number: CN115404195A
Application number: CN202110587521.1A
Authority: CN
Inventors: 林鹏; 赵子元; 陈冠齐; 谢逸凡
Original assignee: Taiwan Advanced Nano Template Technology Inc
Current assignee: Taiwan Advanced Nano Template Technology Inc
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2022-11-29

Abstract

The invention relates to a microcarrier with a three-dimensional scaffold structure for culturing cells, which comprises a continuous medium consisting of a biocompatible polymer. The continuous medium constructs a three-dimensional support structure in the microcarrier to accommodate a plurality of spherical macro-pores which are stacked mutually, and forms a continuous outer wall on the peripheral surface of the microcarrier. Each spherical macro hole is surrounded by a plurality of adjacent spherical macro holes and communicated with each other through a communication hole. Some of the spherical macro-holes are in adjacent contact with the continuous outer wall, and exposed holes are formed on the continuous outer wall, respectively. The support structure inside these exposed holes can be in direct communication with the outside world. The microcarriers of the invention are molded via continuous media molding with a continuous outer wall, thereby retaining high porosity and maintaining good mechanical properties. The continuous medium is molded to substantially assume a substantially geometric configuration. The invention also relates to a die casting forming method of the three-dimensional bracket microcarrier.

Description

Microcarrier with continuous outer wall and scaffold structure for culturing cells

Technical Field

The present invention relates to a cell culture device, and more particularly, to a microcarrier for culturing cells, which has a continuous outer wall formed by mold casting (mold casting) and has a three-dimensional scaffold structure, thereby maintaining high porosity and maintaining good mechanical properties. The invention also relates to a die casting forming method of the three-dimensional bracket microcarrier.

Background

Traditionally, in vitro cell culture is performed by attaching cells to tissue culture plastic vessels or extracellular matrix attachment proteins and then administering appropriate liquid media to promote their growth and proliferation. However, such two-dimensional planar culture is far from the actual physiological environment in vivo, and cannot simulate the interaction between cells and extracellular matrix and between cells in vivo, and is not conducive to the reproduction of 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, and is not favorable for mass production of cells. Three-dimensional cell culture techniques are clearly the preferred solution to the above-mentioned industrial problems.

In the 1980 s, doctor Mina Bissell, lawrence berkeli national laboratory, usa, initiated a three-dimensional cell culture technique in the study of breast cancer (see Petersen o.w., et al, PNAS,89 (19): 9064-9068), which involved co-culturing cells in vitro with scaffolds having a three-dimensional structure, allowing the cells to grow and migrate in the three-dimensional spatial structure of the scaffold. In recent years, as the cell scaffold fabrication process has matured, three-dimensional cell culture techniques have been widely used in biomedical fields such as tissue engineering instead of conventional two-dimensional planar culture techniques. In application, the three-dimensional scaffold can be used for cell growth, tissue differentiation and remodeling in vitro or in vivo, and finally, a tissue with experimental application or further used for transplantation is generated. Structurally, it is a stacked structure with a large number of tiny holes for cell inoculation and attachment, and then guiding the cells to grow and differentiate in a planned three-dimensional direction, resulting in a pseudo-regenerated tissue or organ.

U.S. Pat. No. 8513014 describes a method for producing three-dimensional scaffold blocks, which involves using a bubble generator to introduce a gas stream into a matrix fluid to generate uniformly sized bubbles, gelling the bubbles, then breaking the bubbles under reduced pressure, and curing to form a three-dimensional scaffold material. U.S. patent publication No. 2019/091690A1 and U.S. patent No. 10,828,635 describe a device for generating a large amount of monodisperse bubbles using a microchannel device having a T-junction structure, and a separation device for separating bubbles from a liquid, so that the three-dimensional scaffold can be mass-produced by arranging the bubbles in a closest-packed state, followed by gelling, breaking and curing. U.S. patent application 17/184,276 describes a method of making a three-dimensional cell scaffold using a high internal phase emulsion templating procedure. Other types of conventional stent preparation techniques include salting-out (salting-out), freeze-drying (freeze drying), and solid freeform fabrication (solid freeform fabrication), among others.

Although the above method can produce the block material of the three-dimensional scaffold, in practical application, it is necessary to consider the diffusion rate of the fresh culture solution and the metabolite in the cell culture process so as to ensure that all the cells attached to the three-dimensional scaffold grow healthily. The bulk material must therefore be substantially subdivided into small particles for use, in order to be transported in time by diffusion back and forth from the interior of the microcarriers to the outside. It is known from past experience that suitable sizes of microcarriers can generally be selected in the range of 500 to 3000 microns. The specific size selected depends on the type of cells to be cultured and the culture conditions. Microcarriers in this size range are able to maintain a suspension flow at all times during continuous agitation of the culture.

The method of subdividing the bulk material into microcarriers generally employs conventional mechanical high-speed cutting. However, the internal stent of the microcarrier can bear overhigh instant mechanical stress during cutting, so that the stent has structural defects, and the appearance shape is irregular due to cutting, a large number of spherical macro-holes are exposed, and the stent does not have a continuous and tidy outer wall. Under the stirring state during cell culture, the microcarrier is easy to disintegrate and break due to insufficient mechanical strength, and the exposed structure of the pores can cause the cells attached to the microcarrier to excessively bear the shearing force caused by the flowing of the culture solution, thereby causing low cell productivity. In addition, the size distribution of the microcarriers obtained by high-speed cutting depends on the cutting method. If the required dimension uniformity is high, the cutting process is relatively complicated; the labor and time are very long. If the compression time is required to simplify the manufacturing processThe size distribution of the microcarrier is very large (about several microns to thousand microns). Then, the selection of those suitable for culturing cells (the ratio of the maximum size to the minimum size is not more than 1.5 times) through the screen shows that the yield (yield) is very low. If another conventional technique is adopted: cutting with very short wavelength laser. The method still needs to face the problems of complicated process, exposed holes on the surface of the product and expensive equipment. The width of the laser cut is about 0.1mm, and the final cutting of the microcarrier is set to 1mm ³ The yield benefit is roughly estimated to be equal to: (1/1.1) ³ =75%, with the result that 25% of the wear still remains. Therefore, there is still a high need in the art for microcarriers with high porosity without substantially decreasing their mechanical strength and for manufacturing methods that combine low cost, low man-hour, and low loss.

Disclosure of Invention

The present invention discloses a microcarrier which has high porosity and good mechanical properties, has a three-dimensional scaffold structure and can be used for culturing cells, and a novel process for preparing the microcarrier. The microcarrier is manufactured by die casting molding, and the method comprises the steps of injecting a foam body containing a biocompatible polymer into a proper mould, breaking the foam, curing and molding, and demoulding to obtain the microcarrier. The foam is substantially composed of a plurality of bubbles coexisting and stacked in a solution containing a biocompatible polymer. The bubbles occupy the original space after solidification to form spherical macro pores, and the polymer solution becomes a continuous medium with a three-dimensional bracket shape after solidification. The adjacent macro-hole contact area generates communicating holes due to the bubble breaking process. When the foam is injected into the mold and contacts the mold surface and the outside air, a film of solution is naturally formed to surround the foam due to the surface tension of the polymer solution. After solidification, the solution film becomes a continuous outer wall of the microcarrier. Some of the spherical macro-apertures will be in contiguous contact with the continuous outer wall, creating a plurality of exposed apertures in the continuous outer wall due to the bubble breaking process.

Thus, according to a basic aspect of the present invention, it is intended to provide a microcarrier, the size of which can be chosen between 500 and 3000 microns, depending on the type of cells to be cultured and on the culture conditions. Its overall appearance presents a certain simple basic geometry. It has the structural characteristics that the three-dimensional bracket constructed by the continuous medium, a plurality of spherical macro-holes stacked mutually, communication holes, exposure holes, continuous outer walls and the like form a micro-carrier. The periphery of each spherical macro hole can be contacted with a plurality of adjacent spherical macro holes and respectively communicated with the communication holes. The continuous outer wall is contacted with some spherical macro-holes to form exposed holes, and the solution can be communicated with the outside through the support structure inside the holes.

The diffusion of the culture solution and the metabolite from the center of the microcarrier to the boundary is via a plurality of tortuous paths formed by structures such as spherical macro-pores, communication pores, exposed pores and the like. These structures all constitute a diffusion bottleneck. In order to achieve a diffusion rate that is sufficient for cell growth in microcarriers, the diffusion bottleneck should not be too great. According to the experimental experience of the inventor, if the diameter of the spherical macro pores is taken as 1 unit, the diffusion distance is preferably about 3 to 5 units. Thus, the ratio of the characteristic dimension of the microcarrier to the diameter of the spherical macropores is between about 6:1 to about 10:1.

according to another aspect of the present invention, there is provided a method for manufacturing the microcarrier, comprising the steps of:

A. preparing a polymeric foam comprising a continuous phase and a dispersed phase immiscible with the continuous phase and comprised of a plurality of mutually isolated units dispersed in the continuous phase, wherein the continuous phase comprises a member selected from the group consisting of a biocompatible polymer, a monomer thereof, an oligomer thereof, and combinations thereof;

B. filling the polymeric foam into a porous slab mold, and curing the polymeric foam to obtain a continuous medium, wherein the porous slab mold defines a plurality of micro-through-holes connecting two major surfaces of the porous slab mold, and each micro-through-hole is configured to have a basic geometric configuration and has a characteristic dimension of between 500 microns and 3000 microns, and wherein each of the mutually isolated cells in the dispersed phase has a ratio of diameter to the characteristic dimension of between about 1:6 to about 1:10; and

C. and (3) separating the continuous medium from the porous flat plate mould to obtain the microcarrier with the three-dimensional fine scaffold and the continuous outer wall for culturing cells.

In a preferred aspect, the diameter of the exposed pores of the microcarriers is substantially smaller than the diameter of the adjacent spherical macro-pores.

In a preferred aspect, at least 50% of the spherical macropores in the microcarrier are arranged in a closest packed form.

In a preferred aspect, the microcarrier is shaped with a basic geometry selected from the group consisting of cylinders, spheres, cones, cubes, cuboids, prisms and pyramids. In a more preferred aspect, the basic geometry is selected from a cylinder.

In a preferred aspect, the biocompatible polymer is selected from the group consisting of proteins, polysaccharides, artificial polymers, and combinations thereof. More preferably, the biocompatible polymer is selected from the group consisting of gelatin, collagen, fibrin (fibrins), agarose, hyaluronic acid, chitin, alginate, cellulose, gellan gum.

The microcarrier and the manufacturing process thereof disclosed by the scheme have the beneficial effects that:

1. benefits of microcarrier internal structure: the spherical macro pores inside are communicated with each other to form a continuous network structure body with connected holes, and the continuous network structure body has a very large specific surface area and is suitable for a large number of cells to enter the spherical macro pores and attach and grow on the walls of the macro pores. The fresh culture solution and the metabolite can rapidly diffuse back and forth between the interior of the microcarrier and the outside through the communicating holes and the exposed holes, thereby promoting the growth of cells.

2. Benefits of microcarrier outer continuous wall: the turbulence generated by the continuous stirring of the culture solution during the cell culture will cause frequent impact on the exposed scaffold or cells. Having a substantially flat continuous outer wall provides a layer of protection for the inner scaffolding and cells, reducing damage from turbulent flow. The continuous outer wall has structural linking and integrating functions on the support in the microcarrier, and can uniformly disperse the shearing force of external fluid, thereby strengthening the mechanical strength of the three-dimensional cell support and avoiding the disintegration of the microcarrier under the cell culture condition.

3. Simplified benefits of the die casting process: the foam is injected into a proper mould as above, and then is used as the microcarrier for culturing cells directly after breaking the foam, solidifying and demoulding. The process can avoid the following processes and disadvantages: (1) Preparing a manufacturing process of the three-dimensional support block material in the early stage; (2) High-cost mechanical or laser cutting block material process at high speed; (3) high-proportion loss of the block materials caused by cutting; and (4) damage to the surface of the microcarrier due to cleavage, which leads to decrease in mechanical strength of the microcarrier and cell yield. Therefore, the product efficiency can be greatly improved.

4. Systematic benefits of the new molding process: the shape and size of the die used by the new die casting process can be optimally designed, and a large number of dies can be accurately copied on the planar template. Numerous microcarriers were produced with a narrow size distribution. Is favorable for controllability, predictability and analyzability of cell culture.

Drawings

FIG. 1 is a schematic diagram of a microcarrier according to one embodiment of the invention, showing the cross-sectional two-dimensional structure of a cylindrical microcarrier, wherein the continuous medium and the continuous outer wall are a continuous integral structure after the polymeric foam has been cured, and are deliberately divided into two components for illustrative purposes;

FIGS. 2A and 2B are an electron micrograph and a schematic representation, respectively, of a microcarrier made according to one embodiment of the present invention, showing the top surface of the cylindrical microcarrier;

FIGS. 3A and 3B are, respectively, an electron micrograph and a schematic representation of a microcarrier made according to an embodiment of the present invention showing the side of the cylindrical microcarrier;

FIGS. 4A and 4B are two other electron micrographs of a microcarrier made according to one embodiment of the present invention showing the pore structure of the microcarrier;

FIG. 5 is a flow chart of a method for making microcarriers according to the invention;

FIG. 6 is an optical micrograph showing a foam that has been freeze-dried in a mold, according to one embodiment of the present invention; and

FIG. 7 is an electron micrograph of a microcarrier made by a conventional cutting process.

Description of the figure numbers:

100 … microcarriers

120 … continuous media

121 … spherical macro-pore

122 … communicating hole

123 … continuous outer wall

124 … exposing holes

125 … dispersed phase

126 … continuous phase liquid

200 … die.

Detailed Description

FIG. 1 is a schematic representation of one embodiment of the present application, showing a microcarrier 100 that contains primarily a continuous medium 120. The term "continuous medium" as used herein refers to a single piece (monolithic) mediator material of biocompatible polymers suitable for cell attachment and growth, proliferation and migration. "biocompatible" is used herein to describe a material that does not cause undesirable side effects on biological systems such as cells, tissues and organs. Biocompatible polymers suitable for use in the preparation of microcarriers are well known in the art and include, but are not limited to: proteins such as gelatin, collagen, fibrin (fibrins), and the like; polysaccharides such as agarose, hyaluronic acid, chitin, alginate, cellulose, gellan gum (gellan gum), and the like; artificial polymers such as polyesteramides, polycaprolactone Polyol (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and the like, and polymers such as Polydimethylsiloxane (PDMS), thermoplastic polyurethane, polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polystyrene, and the like, which have no biodegradability; and combinations thereof. In the preferred embodiment shown in fig. 2A to 2B, fig. 3A to 3B, fig. 4A to 4B and fig. 6, the microcarriers 100 are all made of collagen.

As shown in fig. 1, 2A-2B, and 3A-3B, microcarrier 100 has a configuration that substantially exhibits a basic geometry. As used herein, "basic geometry" means a three-dimensional structure composed of simple curved and/or planar surfaces, which includes: curved surface geometric bodies such as cylinders, spheres and cones, and plane geometric bodies such as cubes, cuboids, prisms and pyramids. Microcarrier 100 may be constructed by molding to have any basic geometry configuration that facilitates molding and demolding operations. In a preferred embodiment, microcarriers 100 are configured in a cylindrical configuration. As described later, the microcarrier 100 is molded through a die having a fixed size and thus has a narrow size distribution. According to the present invention, microcarrier 100 has a characteristic size of between 500 microns and 3000 microns, depending on the type of cells to be cultured and the culture conditions. The term "characteristic dimension" as used herein means the largest dimension used to describe the appearance of microcarrier 100, such as the length, width, height, diameter, etc. of microcarrier 100. For example, the characteristic dimension of a cylindrical microcarrier may refer to its height or diameter. In the present application, "feature size" is also used to describe the maximum dimensions of the micro-vias of the mold, such as the aperture and height of the micro-vias. Therefore, the present invention allows the microcarrier 100 to be manufactured with a characteristic dimension of between 500 microns and 3000 microns by sizing the mold to be easily suspended in the liquid cell culture medium. In one embodiment, the microcarrier 100 has a feature size between 500 microns and 880 microns, such that the feature size of the microcarrier 100 is smaller than the diameter of the industrial and laboratory burette, thereby allowing for the ready access of a quantitative sample through a conventional pipette, facilitating rapid detection of cell growth and regulation of growth conditions. The characteristic dimensions of the microcarrier 100 can be measured by electron microscopy, as shown in fig. 3A. The characteristic size can also be measured by sieving, i.e., the microcarrier 100 is able to pass through a6 mesh screen (mesh size 3350 microns) according to the Tyler standard sieve (Tyler standard screen scale), even through an 18 mesh screen (mesh size 880 microns), but not through a 32 mesh screen (mesh size 500 microns).

As shown in fig. 1 and fig. 4A to 4B, the microcarrier 100 is formed with a plurality of spherical macro-pores 121. As described later, the size of the spherical macro pores 121 can be adjusted by adjusting the parameter conditions of the manufacturing process of the polymer foam and adjusting the size of the mutually isolated units (such as bubbles or micro-droplets) in the dispersed phase, so that the ratio of the diameter of the spherical macro pores 121 to the characteristic size of the microcarrier 100 is between about 1:6 to about 1:10 to facilitate the transport of the substances inside the microcarrier and outside by diffusion. For example, a microcarrier with a characteristic size of about 1000 microns contains spherical macropores with a diameter of about 100 to 170 microns, while a microcarrier with a characteristic size of about 3000 microns contains spherical macropores with a diameter of about 300 to 500 microns. In one embodiment, the spherical macro-holes 121 are fabricated to have a diameter between 5 microns and 500 microns, preferably between 50 microns and 200 microns. The spherical macro-holes 121 are adjacent to each other. The term "adjacent to each other" as used herein means that one of the spherical macro-pores in the microcarrier is usually in communication with at least one other spherical macro-pore via at least one communication hole. The size of the communication holes 122 can be controlled by adjusting the contact area between adjacent mutually isolated cells (e.g., bubbles or micro-droplets) in the dispersed phase in the process of manufacturing the above-described foam. In some embodiments, all of the spherical macro-pores 121 have a substantially uniform diameter, while in other embodiments, the size distribution of the spherical macro-pores 121 is broader depending on whether the bubbles or micro-droplets have monodispersity. In one embodiment, at least a portion of the spherical macro-pores 121 in the microcarrier 100 are arranged in an ordered, e.g., closest packed, arrangement. Preferably, at least 50% of the spherical macro-pores 121, more preferably at least 60% of the spherical macro-pores 121, most preferably at least 70% of the spherical macro-pores 121, e.g., at least 80% of the spherical macro-pores 121, are arranged in the closest packed form in the microcarrier 100.

Microcarrier 100 has a continuous outer wall 123. For example, microcarrier 100 is configured as a cylindrical configuration with continuous outer wall 123 consisting of a top flat surface, a bottom flat surface and a side curved surface, as shown in fig. 1, 2A-2B and 3A-3B. The term "continuous outer wall" as used herein means that all points in the outer wall are directly contiguous without interruption. The continuous outer wall 123 completes the structure of the continuous medium 120 and may strengthen the mechanical strength of the microcarrier 100 to avoid disintegration under cell culture conditions. The continuous outer wall 123 also prevents the spherical macro-pores 121 from being completely exposed, and can protect cells attached to the inside of the three-dimensional cell scaffold 100 from being subjected to shear force caused by the flow of culture solution under cell culture conditions, thereby providing stability of the cell growth environment.

As shown in fig. 1, 2A to 2B and 3A to 3B, the continuous outer wall 123 is formed with a plurality of exposing holes 124 communicating with the outside to partially expose the spherical macro-holes 121, i.e., the diameter of the exposing holes 124 is substantially smaller than that of the adjacent spherical macro-holes 121, so that the spherical macro-holes 121 are not completely exposed. The cells being cultured may enter the interior of microcarrier 100 via exposed pores 124.

FIG. 5 is a flow chart of a method for making microcarriers according to the invention, comprising step A: preparing a polymeric foam; and B: molding the polymer foam into a continuous medium; and step C: the continuous medium is demolded.

Step A comprises preparing a polymeric foam comprising a continuous phase and a dispersed phase immiscible with the continuous phase and comprised of a plurality of isolated cells dispersed in the continuous phase. According to the present invention, the continuous phase is a phase that undergoes a curing reaction to form a continuous medium, and comprises a component selected from the group consisting of the aforementioned biocompatible polymers, monomers thereof, oligomers thereof, and combinations thereof. The continuous phase may also contain other ingredients necessary for the curing reaction, such as cross-linking agents, polymerization initiators, emulsion stabilizers, surfactants, salts, solvents, and the like. The continuous phase is typically a viscous fluid at ambient temperature. As used herein, "solidification" refers to the process of applying a physical or chemical bridging means to a continuous phase in a fluid state to convert it into a continuous medium having a stable solid conformation. In certain embodiments, the dispersed phase is a gas and the continuous phase is an oily or aqueous solution or suspension. In another embodiment, the dispersed phase is an aqueous solution and the polymeric foam is in the form of a water-in-oil emulsion.

Means for making such foams are disclosed in us 8513014 and us 2019/091690A1, which relate to the use of multiphase flow methods to introduce a gas or liquid stream into a continuous phase via a microchannel device, and to generate gas bubbles or micro-droplets dispersed in the continuous phase through the specific design of the microchannel device and control of the fluid flow rate. By using the method disclosed in the above patent document, monodisperse bubbles or micro droplets can be produced in large quantities by changing the size and geometry of the micro flow channel, the properties (such as viscosity, surface tension) and flow rate of the fluid, and they can be further arranged in a close-packed state to produce microcarriers with spherical macro pores of uniform size. The above patents and patent applications are all incorporated in their entirety by reference into this specification.

Another means for making the foam involves vigorously stirring the continuous phase composition and the immiscible dispersed phase composition in a homogenizer at a high rotational speed to uniformly disperse the dispersed phase in the continuous phase to obtain a water-in-oil emulsion. The water-in-oil emulsion can be optionally subjected to further external force to precipitate so as to increase the volume ratio of the dispersed phase to the continuous phase in the emulsion to obtain a high internal phase emulsion, thereby increasing the porosity of the prepared microcarrier and increasing the size of the communicating pores. As is well known to those of ordinary skill in the relevant art, the size and uniformity of the microdroplets in the dispersed phase can be adjusted by varying the volume ratio of the dispersed phase to the continuous phase in the emulsion, as well as the rate and temperature of the perturbation.

Other methods are available for making foams and are suitable for use in the present invention.

In step B, a porous plate mold is prepared, which defines a plurality of micro through holes connecting two major surfaces of the porous plate mold, preferably, the micro through holes are arranged in an array. The through holes have a substantially geometric configuration, i.e. they have a configuration selected from the group consisting of cylinders, spheres, cones, cubes, cuboids, prisms and pyramids. In the preferred embodiment shown in fig. 6, the through-holes are configured to have a cylindrical configuration. Each micro-via has a feature size in the range of 500 to 3000 microns, i.e., the height of the micro-via (corresponding to the thickness of the porous plate mold) is in the range of 500 to 3000 microns, and/or the pore size of the micro-via is in the range of 500 to 3000 microns. In one embodiment, each micro-via is configured to have a feature size in a range of 500 microns to 880 microns. The porous plate mold may be made of any inert material that does not physically and chemically react with the polymer foam, such as carbon fiber, ceramic, glass, quartz, or polyvinyl chloride (PVC), polyoxymethylene (POM), polycarbonate (PC), polyphenylene oxide (PPO), PA6/66 nylon plastic, polycarbonate/acrylonitrile-butadiene-styrene copolymer (PC/ABS) composite plastic, polyethylene terephthalate (PET), polyethyleneimine (PEI), polymethyl methacrylate (PMMA), polyphenylene sulfide (PPS), polyethylene (PE), polypropylene (PP), polystyrene (PS), ethylene/vinyl acetate copolymer (EVA), or metal materials such as stainless steel, titanium, aluminum, and aluminum-magnesium alloy.

According to the invention, the diameter of each of the mutually isolated cells in the dispersed phase of the foam is adjusted to a ratio of the characteristic dimensions of the micro-vias of about 1:6 to about 1:10. in one embodiment, each of the isolated cells has a diameter in the range of 5 microns to 500 microns, preferably in the range of 150 microns to 200 microns.

The manufacturing process of the porous flat mold is familiar to those skilled in the relevant art, and can be adjusted according to the material selected. For example, when the mold is made of a plastic material, suitable manufacturing processes include, but are not limited to, plastic processing processes such as injection molding, compression molding, thermoforming, etc., and the micro-vias can be manufactured subsequently by using conventional punching or drilling processes. When the mold is made of a metal material, it can be manufactured by a conventional metal processing process such as stamping, rolling, die forming, forging, etc., and then optionally a conventional punching or drilling process can be used to manufacture the micro-via.

And D, pouring the polymer foam prepared in the step A onto a porous flat plate die, and scraping the surface of the die by using a plastic scraper at a proper speed to enable the foam to be extruded to enter and fill the micro through holes. If the foam body overflows from the lower part of the mould, the foam body can be further scraped by a scraper. Because of the very low density of the foam, its adhesion to the inside walls of the micro-vias is sufficient to maintain itself within the vias without running off before curing. The composition is then cured. As used herein, "solidification" refers to the process of applying physical or chemical bridging means to a continuous phase in a fluid state to transform it into a continuous medium having a stable solid configuration. The reaction conditions for curing vary depending on the type of polymer, but are well known in the relevant arts. For example, in the case of using collagen or gelatin as the biocompatible polymer, the composition filled in the mold may be dehydrated at a low temperature to be gel-shaped. In a specific example of using alginate as the biocompatible polymer, a solution containing divalent metal ions such as calcium ions or magnesium ions may be added to cause a crosslinking reaction between alginate molecules, thereby gelling and setting. In a specific example of using polystyrene as the biocompatible polymer, a styrene monomer in the continuous phase is promoted to undergo radical polymerization and cure. The freeze-drying of the solidified continuous medium may be continued, preferably under vacuum, to help break up the gas bubbles or micro-droplets in the dispersed phase to create interconnected pores and exposed pores.

Fig. 6 shows a freeze-dried collagen foam in a mold 200, wherein the spaces left by the dispersed phase 125 are spherical macro-pores in the continuous medium. It is noted that, as shown in FIG. 6, a thin layer of the continuous phase liquid 126 remains between the dispersed phase 125 and the mold 200 due to the cohesion of the continuous phase liquid 126, and the thin layer forms the continuous outer wall 123 after solidification. The continuous outer wall 123 thus formed is substantially complementary in shape to the mold 200, such that the continuous medium 120 is structurally intact, enhances its mechanical strength, and avoids disintegration under cell culture conditions. During freeze-drying, the weak portions of the spherical macro-holes, where they meet the continuous outer wall 123, will rupture due to the imbalance of the internal and external pressures of the continuous medium 120, thereby forming exposed holes. The diameter of the exposed hole is substantially smaller than that of the adjacent spherical macro-hole, so as not to expose the spherical macro-hole completely. After the curing reaction is completed, a sponge-like or honeycomb-like continuous medium having a large number of spherical macro pores inside for cell attachment and growth can be obtained.

Step C may use any demolding process that allows the continuous medium 120 to exit the mold without substantial damage to its structure, for example, high pressure air may be used to blow the continuous medium 120. In the specific case of using collagen and gelatin as the biocompatible polymer, the continuous medium 120 after the mold release may be heated at a temperature higher than 37 ℃ to be dried and thermally bridged. For example, a continuous medium 120 made of collagen can be placed in an oven (DENG YNG DO60 type), vacuum dried at 50 ℃ for 1 hour, and then heated at 150 ℃ for 12 to 48 hours to obtain a large number of microcarriers 100 with a narrow size distribution and a continuous outer wall.

FIG. 7 shows microcarriers that have been cut by conventional machining processes and are broken in shape and varying in size, while FIGS. 2A-2B and 3A-3B show microcarriers according to the invention having a complete basic geometry and exhibiting a narrow size distribution. The microcarrier according to the invention has excellent mechanical strength. Through verification under actual cell culture strips, the microcarrier provided by the invention is not decomposed or broken after being stirred for more than 14 days in a stirring bioreactor system.

The microcarrier of the invention has wide application in tissue engineering, oncology, regenerative medicine, drug screening test and stem cell biology. The microcarrier of the invention has the advantages of high mechanical strength, high specific surface area and high pore connectivity, and is suitable for co-culture with various cells in vitro to produce cells in large quantities or for growth of implanted cells in vivo to remodel damaged tissues. In the case of using proteins or polysaccharides as the scaffold material, the microcarriers may be lysed using an appropriate enzyme such as trypsin to recover the cells.

Claims

1. A microcarrier having a three-dimensional scaffold structure for culturing cells, comprising:

a continuous medium of biocompatible polymer having a substantially geometric configuration and a feature size of between 500 microns and 3000 microns;

wherein the micro-carrier is formed with a plurality of spherical macro-holes adjacent to each other, and the spherical macro-holes are communicated with each other through one or more communication holes, wherein the ratio of the diameter of the spherical macro-holes to the characteristic size is about 1:6 to about 1:10 is between; and

wherein the micro-carrier has a continuous outer wall, and the spherical macro-holes are formed with an exposed hole respectively at the connection with the continuous outer wall.

2. The microcarrier of claim 1, wherein the exposed pores have a pore size substantially smaller than the pore size of the adjacent globular macropores.

3. The microcarrier of claim 2, wherein the base geometry is selected from the group consisting of a cylinder, a sphere, a cone, a cube, a cuboid, a prism, and a pyramid.

4. The microcarrier of claim 3, wherein the continuous medium has a feature size between 500 microns and 880 microns.

5. The microcarrier of claim 4, wherein the biocompatible polymer is selected from the group consisting of proteins, polysaccharides, artificial polymers, and combinations thereof.

6. The microcarrier of claim 5, wherein the biocompatible polymer is selected from the group consisting of gelatin, collagen, fibrin (fibrins), agarose, hyaluronic acid, chitin, alginate, cellulose, gellan gum (gellan gum).

7. The microcarrier of claim 6, wherein the spherical macro-pores have a diameter between 50 microns and 200 microns.

8. The microcarrier of claim 7, wherein at least 50% of the spherical macropores in the continuous medium are in a closest packed arrangement.

9. A method for making a microcarrier, comprising the steps of:

A. preparing a polymeric foam comprising a continuous phase and a dispersed phase immiscible with the continuous phase and comprised of a plurality of isolated cells dispersed within the continuous phase, wherein the continuous phase comprises a member selected from the group consisting of biocompatible polymers, monomers thereof, oligomers thereof, and combinations thereof;

C. and (3) separating the continuous medium from the porous flat plate mould to obtain the microcarrier with the three-dimensional cell scaffold and a continuous outer wall.

10. The method of claim 9, wherein the basic geometry is selected from the group consisting of a cylinder, a sphere, a cone, a cube, a cuboid, a prism, and a pyramid.

11. The method of claim 10, wherein the continuous medium has a feature size of between 500 microns and 880 microns.

12. The method of claim 11, wherein the biocompatible polymer is selected from the group consisting of proteins, polysaccharides, artificial polymers, and combinations thereof.

13. The method of claim 12, wherein the biocompatible polymer is selected from the group consisting of gelatin, collagen, fibrin (fibrins), agarose, hyaluronic acid, chitin, alginate, cellulose, gellan gum (gellan gum).

14. The method of claim 13, wherein step a comprises introducing a gas into the continuous phase to create bubbles, whereby the dispersed phase is the gas.