TWI773101B - Microcarrier suitable for cell growth and method for microcarrier culture in microenvironment - Google Patents

Abstract

A microcarrier suitable for cell growth and a method for culturing the microcarrier in a microenvironment, comprising a plurality of cells; a loading solution for carrying the plurality of cells and mixing to form a plurality of inner cores; a shell of alginic acid layer, and cover the periphery of each inner core, and then mildly cross-link by mixing the alginic acid and calcium chloride solution to form a plurality of porous microcarriers. The carrier solution is placed in the culture medium in the outer shell layer, and cultured at 37 °C and 5% CO ₂ to promote the formation of cell clumps by cells, and then remove the outer shell layer by removing calcium ions to obtain pure clumps of cells.

Description

Microcarrier suitable for cell growth and method for microcarrier culture in microenvironment

The invention relates to a microcarrier suitable for cell growth and a method for culturing the microcarrier in a microenvironment, especially a capsule-type cell carrier, and selects biodegradable materials, which can be easily removed after the cells are cultured into clumps Agglomerate the outer material to obtain a cell mass with a cell matrix and apply it.

At present, the cell culture system plays a large part in the self-cell therapy, which can avoid large-scale and high-cost animal experiment resources. However, the traditional cell culture method makes the cells grow in a monolayer, which leads to the gradual loss of cells. The original characteristics of cells, such as cell type or protein secretion, are not the same as in vivo, which affects the expected results of subsequent animal experiments or human experiments. Therefore, the 3D cell culture system provides environmental conditions more similar to those in vivo. The best cell growth environment can have better and more accurate cell-level expected effects. 3D cell culture environment uses materials with three-dimensional structure to prepare scaffolds. Cell culture is carried out in vitro, so that cells can grow and migrate in the three-dimensional space structure of the carrier, which affects a wide range of levels, such as the interaction between tissue cells Integration and migration can also be seen in many literatures in the past, affecting cell morphology, such as cell growth patterns, protein secretion substances, etc. The purpose of the three-dimensionally structured cell culture environment is to create an environment similar to in vivo growth, which in turn helps the cell growth pattern and the intercellular cross-links to be closer to the in vivo model. and It is also seen now that the 3D cell culture system is expected to grow at a compound annual growth rate of 8.7% in the future. There are the following three models of the current 3D culture system, but they still have their own shortcomings: 1. Hanging drop (hanging drop) drop), it is impossible to cultivate larger cell clusters; 2. Scaffold, which will cause a large number of cells to be lost, and the removal of materials is quite difficult; 3. Hydrogel system (hydrogel system), the permeability of the material is poor , so the above three modes are not conducive to cell growth.

In addition, when using hydrogel to prepare the carrier, if the carrier and cell aggregates cannot be effectively separated, it is necessary to implant the cell aggregate together with the carrier into the patient, and in this case, the interaction between the cell aggregate and adjacent cells The effect will be increased by the carrier layer, so that after the cell mass is implanted in the affected area, it cannot have a good interaction with the adjacent cells to achieve the integration effect. In addition, if using a hydrogel, it is also necessary to face the impact on the cells after chemical cross-linking with a cross-linking agent.

However, traditional 3D cell culture mostly uses polymer materials to prepare scaffolds or carriers, which has poor effect on cell growth and attachment. In addition, after the cell clumps are formed in the carriers or scaffolds, they are prepared with polymer materials. The carrier is not easy to remove, and cannot be effectively separated from the cell mass. The cell mass must be implanted together with the carrier into the treatment affected area, resulting in poor cell contact with the cells near the affected area and poor cell integration, thereby reducing the effect of cell therapy.

Therefore, how to provide a material that can increase the adhesion of cells, so that cells can be completely attached and cultured inside the carrier, and develop in the direction of cell clumps, and unnecessary materials can be removed, and finally obtained with cell clumps of the cell matrix, and It is still a technical and problem to be overcome to make the cell aggregates integrate with adjacent cells after being injected into the affected area.

In view of this, the inventor of the present case has been engaged in the manufacturing and development of related products for many years, aiming at the above-mentioned goals, after detailed experiments and careful evaluation, finally came up with a practical invention.

The purpose of the present invention is to provide a microcarrier suitable for cell growth and a method for culturing the microcarrier in a microenvironment. The cells can be cultured in the carrier, and the materials that can increase the adhesion of the cells are selected inside the carrier, so that the cells can be cultured in the carrier. The inside of the carrier is completely attached and cultured, and develops in the direction of cell clumps, eventually forming a cell clump that can easily remove unnecessary materials and has a cell matrix, which can be used for subsequent treatment.

After the cells form a cell mass with a cell matrix in the carrier, use the method of removing unnecessary shells to obtain a cell mass with a cell matrix and implant it into the affected part to be treated, so that the cell mass can be injected into the affected part. After successfully integrating with adjacent cells, this method can also help cell aggregates to interact better with adjacent cells to achieve good cell integration. Therefore, the following factors need to be considered when conducting cell culture environment:

1. High biological activity: When the material inside the carrier is a biological material that cells are relatively fond of attaching to, it can effectively stimulate the interaction between cells, thereby helping the proliferation and differentiation of cells, so that the cells grow well inside the carrier. , which facilitates the formation of cell clumps by cells.

2. Stability: the current degradation and other conditions affect the formation of cell clumps inside the carrier.

3. The material cells are separable: after the formation of cell clumps with cell matrix, when the cell clumps are taken out and implanted in the affected part of the body, the interaction between the cell clumps and adjacent cells becomes a key factor, and the use can be easily used. The step of separating the carrier from the cell mass can effectively increase the interaction between the cell mass and adjacent cells.

According to the above purpose, the present application proposes a capsule-type cell carrier with a shell-core structure suitable for three-dimensional cell culture. The material of which is selected from biodegradable materials, which can facilitate the easy removal of unnecessary external components after the cells are cultured into clumps. materials, so as to obtain cell aggregates with cell matrix and apply them directly. At the same time, the materials located in the core can effectively promote the better growth and differentiation of cells, and the capsule-type cell carrier of this putamen structure is used in three-dimensional cell culture. Clinically, it will be able to show better results.

A microcarrier suitable for cell growth, comprising a plurality of cells; one is a carrier solution for multiplying the plurality of cells, and can be mixed to form a plurality of inner cores, and each inner core has a plurality of cells; one is alginic acid The outer shell layer is coated on the periphery of each inner core, and then the alginic acid and the calcium chloride solution are mixed for mild cross-linking to form a plurality of porous microcarriers.

In an embodiment of the present invention, the carrier solution is a biodegradable material, and can be gelatin, collagen, hyaluronic acid, chitin, fibrin, poly(Glycolic Acid) , PGA), glycolic acid copolymer (Poly (Lactic Acid-co-Glycolic Acid), PLGA), hydroxymethyl chitin.

In one embodiment of the present invention, the alginic acid can be another extension of gardenia, or other biopolymers and chemical cross-linking agents.

In one embodiment of the present invention, the degradable biomaterial is an extension of natural polymers and synthetic polymers.

A method for culturing microcarriers in a microenvironment, comprising step 1, placing a loading solution into a first syringe, adding a plurality of cells and mixing them, so that the plurality of cells can be loaded on the loading solution , to form a plurality of inner cores; Step 2, place the plurality of inner cores in a first container, and place an alginic acid in the container at the same time, so that the alginic acid can be respectively coated on the plurality of inner cores, to form a plurality of microcarriers; step 3, placing the plurality of microcarriers in a second container with calcium chloride, to obtain a plurality of cell microcarriers after the calcium chloride and the alginic acid are used for cross-linking ; Step 4, place the cell microcarriers in a culture medium for culturing, so that the cells in each of the cell microcarriers grow to form clumps; The outer layer of calcium and alginic acid is removed to obtain a pure cell mass.

In an embodiment of the present invention, the carrier solution is a biodegradable material, and can be gelatin, collagen, hyaluronic acid, chitin, fibrin, poly(Glycolic Acid) , PGA), glycolic acid copolymer (Poly (Lactic Acid-co-Glycolic Acid), PLGA), hydroxymethyl chitin.

In one embodiment of the present invention, the alginic acid can be another extension of gardenia, or other biopolymers and chemical cross-linking agents.

In one embodiment of the present invention, the culture medium is placed in a culture medium, and the reaction temperature of the culture environment is 25°C to 39°C, wherein the optimum temperature is 37°C.

In one embodiment of the present invention, the culture medium is placed in a culture medium, and the culture environment is a carbon dioxide (CO2) environment of 5%.

In one embodiment of the present invention, the degradable biomaterial is an extension of natural polymers and synthetic polymers.

In an embodiment of the present invention, the calcium ion removal method is to add an anticoagulant and sodium citrate to remove the outer shell layer.

(110): Loading solution

(120): a plurality of cells

(130): multiple cores

(140): Shell layer

(150): calcium chloride solution

(160): Microcarriers

(S210-S250): Process

Figure 1 is a schematic diagram of a microcarrier suitable for cell growth of the present invention.

FIG. 2 is a flow chart of the method for culturing the microcarriers in the microenvironment of the present invention.

Figure 3 is a schematic diagram of the culture of the microcarriers suitable for cell growth of the present invention.

FIG. 4 is an analytical view of the culture microscope of the microcarrier suitable for cell growth of the present invention.

Fig. 5 is a cell growth state diagram of the microcarrier suitable for cell growth of the present invention.

FIG. 6 is an analytic view of the environment microscope of the microcarrier suitable for cell growth of the present invention.

Fig. 7 is a graph showing the difference of the environment growth of the microcarriers suitable for cell growth of the present invention.

In order to facilitate the examiners to understand the technical features, content and advantages of the present invention and the effects that can be achieved, the present invention is hereby described in detail with the accompanying drawings and in the form of embodiments as follows. The subject matter is only for illustration and auxiliary description, and is not necessarily the real scale and precise configuration after the implementation of the present invention. Therefore, the ratio and configuration relationship of the attached drawings should not be interpreted or limited to the scope of rights of the present invention in actual implementation. Together first to describe.

First, please refer to FIG. 1 and FIG. 3 , which are schematic diagrams of a microcarrier suitable for cell growth and a schematic diagram of culture according to the present invention. A microcarrier suitable for cell growth includes a plurality of cells (120); A carrier solution (110) for cells (120) and mixed to form a plurality of inner cores (130), wherein the carrier solution (110) is a degradable biomaterial, and can be gelatin, collagen, hyaluronate Acid, chitin, fibrin, poly(Glycolic Acid, PGA), poly(Lactic Acid-co-Glycolic Acid, PLGA), hydroxymethyl chitin, etc. , wherein, the degradable biological material is an extension of natural polymers and synthetic polymers; one is an outer shell layer (140) of alginic acid, which is wrapped around the periphery of each of the inner cores (130), and then through The alginic acid and the calcium chloride solution (150) are mixed for mild cross-linking to form a plurality of porous microcarriers (160). Extensions of cross-linking agents, etc.

As can be seen from the above, please refer to FIG. 4 at the same time, which is a culture microscope analysis diagram of the microcarrier suitable for cell growth of the present invention. A microcarrier suitable for cell growth proposed by the present invention is analyzed by scanning electron microscope (SEM). For the structure of the microcarriers on the cross section, the microcarriers at 37°C compared with the microcarriers at 25°C, because the gelatin in the inner core will gradually melt, and the microcarriers that successfully form the shell-core structure can verify the potential of cell encapsulation. At the same time, because it is a cell carrier with high biocompatibility and can be used for three-dimensional cell culture, its microcarriers The body is a porous carrier to facilitate cell growth. And use alginic acid as the outer shell layer of the inner core, and use the culture medium and gelatin as a material for the inner core to facilitate cell attachment, and then contact and mix the alginic acid and the calcium chloride solution. Gentle cross-linking can provide a compartmentalized environment outside the cell culture, which is conducive to the cultivation of cells in the microcarrier. The cells will grow in the microcarrier and gradually aggregate into agglomerates. Finally, the alginic acid is removed by removing calcium. In order to obtain the cell mass with cell matrix inside, and then implant it on the affected part to assist in the cell therapy of the affected part.

Please refer to FIG. 2 again, which is a flow chart of the method for culturing microcarriers in a microenvironment according to the present invention, which includes step 1. (S210) Put the loading solution into a first syringe, and simultaneously add a plurality of cells and mix them. , so that the plurality of cells can be loaded on the loading solution to form a plurality of inner cores; step 2, (S220) the plurality of inner cores are placed in a first container, and an alginic acid is placed in the container at the same time In, the alginic acid can be respectively coated on the plurality of inner cores to form a plurality of microcarriers; Step 3, (S230) place the plurality of microcarriers in a second container with calcium chloride, After the calcium chloride and the alginic acid are used for cross-linking, a plurality of cell microcarriers are obtained; step 4, (S240) the cell microcarriers are placed in a medium for culturing, so that the cell microcarriers in each cell microcarrier are The cells grow to form clumps; step 5, (S250) removes the outer shell layer with calcium chloride and alginic acid from the clumps by calcium ion removal to obtain pure cell clumps.

According to step 1, wherein the carrier solution is a biodegradable material, and can be gelatin, collagen, hyaluronic acid, chitin, fibrin, poly (Glycolic Acid), PGA ), glycolic acid copolymer (Poly (Lactic Acid-co-Glycolic Acid), PLGA), hydroxymethyl chitin, and its degradable biomaterials are extensions of natural polymers and synthetic polymers and the alginic acid described in step 2 can be another extension of gardenia, or other biopolymers and chemical cross-linking agents, and the alginic acid described in step 4 is cultured in a medium, and the cultured The reaction temperature of the environment is 37°C plus or minus 0.5°C, and the optimum temperature is 37°C, and the culture environment is an environment where carbon dioxide (CO2) is 5%. The calcium ion removal method described in step 5 is added by adding Anticoagulant and sodium citrate, thereby removing the outer shell with calcium chloride and alginic acid.

Therefore, according to the above-mentioned microenvironmental culture method, please also refer to Figure 5, where the cell microcarriers are placed in the culture medium and cultured at a fixed temperature of 37°C and the corresponding cell culture medium is replaced for different cells at the same time. The cells in each cell microcarrier gradually grew to form clumps, as shown in Figure 5 on the 3rd day, the 7th day, and the 14th day of cell growth, and continued to culture until the 14th day to form a cell matrix. Cell clumps.

Compared with the temperature of the culture environment, the cell microcarriers also have differences in temperature during the process of transporting the environment. Please refer to FIG. 6 and FIG. 7 , which are the environmental microscope analysis diagrams of the microcarriers suitable for cell growth according to the present invention and The difference map of environmental growth. With the current cell carrying method, when the frozen tube in the control group carries cells, the problem is that when reaching the destination, it needs to be defrosted and thawed before being injected into the affected part, and the problem of frozen ice crystals It will lead to a certain degree of cell death (as shown in Figure 6 and Figure 7 cryovial), so the survival rate of cells is not good, and in the same way, in another control group in the refrigerator at a low temperature of 4 ℃, due to cells are unable to enter Energy-consuming behavior, unable to carry out endocytosis, will obviously cause cell death after 3 days (as shown in Figure 6 and Figure 7 in the refrigerator), while the microcarrier of the present invention is at room temperature of 25 ℃. Under this condition, cells not only will not die, but can also proliferate in an appropriate culture medium environment. Therefore, between 25°C and 39°C of general room temperature, and 37°C is the optimum temperature, the microcarriers described in the present invention Optimum cell viability is obtained.

To sum up, the material used in the present invention is a biodegradable material, and the main purpose is to make the material of the inner core provide a good growth environment for cells, so that it can proliferate and differentiate at the same time close to the real physiological state of the human body. The material of the outer shell layer is to provide a good protective layer for the capsule-type cell carrier to avoid the loss and death of the cell material in the inner core. At the same time, the present invention can better provide the growth conditions of cells in a three-dimensional culture environment, and reduce or even avoid the loss and loss of cell materials during the culture process, so that the cell aggregates can be adjusted later to achieve better performance during application. effect and success rate.

As can be seen from the above-mentioned implementation description, compared with the prior art and products, the present invention has the following advantages:

1. Use mild physical crosslinking of alginic acid and calcium chloride to form a stable cell carrier.

2. The biodegradable material described in the inner core can enhance the cell adhesion ability and assist the formation of cell aggregates.

3. The outer layer of the cell carrier can be effectively separated from the cell mass by removing calcium, so as to obtain a pure cell mass with cell matrix.

The above descriptions are only the best specific embodiments of the present invention, but the structural features of the present invention are not limited thereto. Any changes or modifications that can be easily conceived by those skilled in the art in the field of the present invention can be covered. In the following patent scope of this case.

To sum up, the present invention does have an unprecedented innovative structure, which has not been seen in any publications, nor has there been any similar products on the market, so there should be no doubts about its novelty. In addition, the unique features and functions of the present invention are far from comparable to conventional ones, so it is indeed more progressive than conventional ones.

(110): Loading solution

(120): a plurality of cells

(130): multiple cores

(140): Shell layer

(150): calcium chloride solution

(160): Microcarriers

Claims (3)

A method for culturing microcarriers in a microenvironment, comprising: step 1, placing a loading solution into a first syringe, adding a plurality of cells and mixing them, so that the plurality of cells are loaded on the loading solution , to form a plurality of inner cores, wherein the loading solution is a degradable biomaterial, and is gelatin, collagen, hyaluronic acid, chitin, fibrin, polyglycolic acid (Poly (Glycolic Acid), PGA), Glycolic acid copolymer (Poly (Lactic Acid-co-Glycolic Acid), PLGA), or hydroxymethyl chitin; step 2, placing the plurality of inner cores in a first container, and simultaneously adding an alginic acid Put into this first container, make this alginic acid coat on this plurality of inner cores respectively to form outer shell layer, to form a plurality of microcarriers; Step 3, put the plurality of microcarriers into a calcium chloride In a second container, the calcium chloride and the alginic acid are used for cross-linking to obtain a plurality of cell microcarriers; step 4, the plurality of cell microcarriers are placed in a medium for culturing, so that each cell microcarrier is The cells in the carrier grow to form clumps; step 5, add anticoagulant and sodium citrate to remove the outer shell layer with the calcium chloride and the alginic acid to obtain a pure cell clump; in the step 4 Between step 5 and step 5, it further includes: step 4', delivering the plurality of cell microcarriers at 25°C. The method for culturing microcarriers in a microenvironment according to claim 1, wherein the culturing temperature in step 4 is 37°C plus or minus 0.5°C. The method for culturing microcarriers in a microenvironment according to claim 1, wherein the culturing environment in step 4 is a carbon dioxide (CO ₂ ) environment of 5%.

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