Method for mass production of extracellular composition by using cell growth promoting colloid for three-dimensional culture of cells

Abstract

The invention discloses a method for three-dimensionally culturing cells by using a colloid for promoting cell growth and mass production of a high-concentration extracellular composition by using the method. The method for three-dimensionally culturing cells by using the colloid for promoting cell growth comprises the following steps: providing a culture colloid to mix and coat the cells; performing cross-linking reaction on the culture colloid coated with the cells, so that the cross-linked culture colloid presents a three-dimensional structure, and the cells are suspended in the cross-linked culture colloid; and carrying out cell culture. Through the technical content of the invention, the damage of the cells in the three-dimensional culture process can be reduced, and the three-dimensional growth of the cells in the colloid is supported.

Description

Technical Field

[0001] The present invention relates to a method for culturing cells, in particular to a method for culturing cells in three dimensions using a colloid that promotes cell growth and mass production of a high-concentration extracellular composition using the method. Background Art

[0002] Exosomes are an important component of extracellular compositions with a diameter of about 30nm to 150nm released by eukaryotic cells of animals, plants, fungi, etc. They contain carbohydrates, lipids, proteins, messenger RNA (mRNA), deoxyribonucleic acid (DNA), etc., and play an important role in the transmission of information between cells. In addition, exosomes are also related to cell growth, inflammatory response, cancer cell metastasis and deterioration.

[0003] However, the secretion, yield and biological activity of exosomes may be affected by factors such as cell health and the existing two-dimensional culture environment, making it impossible to effectively mass-produce and reduce batch-to-batch differences.

[0004] Therefore, how to improve the cell culture method to increase cell survival rate and increase the yield and batch variability of extracellular compositions to overcome the above-mentioned defects has become one of the important issues that this business wants to solve. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for three-dimensionally culturing cells using a colloid that promotes cell growth and mass-produce a high-concentration extracellular composition using the method in view of the deficiencies in the prior art.

[0006] In order to solve the above-mentioned technical problems, one of the technical solutions adopted by the present invention is to provide a method for three-dimensionally culturing cells using a colloid that promotes cell growth, which includes: providing a culture colloid to mix and coat the cells; cross-linking the culture colloid coated with the cells so that the cross-linked culture colloid presents a three-dimensional structure, so that the cells are suspended in the cross-linked culture colloid; and performing cell culture.

[0007] In order to solve the above technical problems, another technical solution adopted by the present invention is to provide a method for mass-producing high-concentration extracellular compositions using the above-mentioned method of three-dimensionally culturing cells using colloids that promote cell growth, which includes: collecting conditioned culture fluid; and filtering the conditioned culture fluid.

[0008] In order to solve the above technical problems, another technical solution adopted by the present invention is to provide an extracellular composition, which is an extracellular composition obtained by the above method for mass production of high-concentration extracellular composition.

[0009] One of the beneficial effects of the present invention is that the method of culturing cells in three dimensions using cell growth-promoting colloids and the mass production of high-concentration extracellular compositions using the method provided by the present invention can reduce damage to cells during three-dimensional culture and support three-dimensional growth of cells in the colloid through the technical scheme of "cross-linking the culture colloid coating the cells so that the cross-linked culture colloid presents a three-dimensional structure, so that the cells are suspended in the cross-linked culture colloid."

[0010] Furthermore, the extracellular composition obtained by the method of culturing cells three-dimensionally using cell growth-promoting colloids of the present invention has a particle size distribution that is effectively reduced and a total protein concentration that is significantly increased, which not only effectively increases the production capacity of the extracellular composition but also has an excellent effect of promoting cell growth.

[0011] To further understand the features and technical contents of the present invention, please refer to the following detailed description and drawings of the present invention. However, the drawings provided are only for reference and description and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 FIG. 4 is a flow chart of a method for three-dimensionally culturing cells using cell growth-promoting colloids according to a first embodiment of the present invention.

[0013] Figure 2 This is a rheological curve diagram at different cross-linking times in three-dimensional culture using cell growth-promoting colloid according to the first embodiment of the present invention.

[0014] Figure 3 This is a diagram showing changes in cell proliferation using different cell culture methods according to the first embodiment of the present invention.

[0015] Figure 4 The flowchart of the method for mass-producing a high-concentration extracellular composition using a method for culturing cells in three-dimensional culture using a cell growth-promoting colloid according to the second embodiment of the present invention.

[0016] Figure 5 Another flow chart of the method for mass-producing a high-concentration extracellular composition using a method for three-dimensionally culturing cells using cell growth-promoting colloids according to the second embodiment of the present invention.

[0017] Figure 6 This is a bar graph showing the difference in total protein concentration using different cell culture methods according to the second embodiment of the present invention.

[0018] Figure 7 This is a particle size distribution diagram of the extracellular composition obtained by two-dimensional cultured cells according to the second embodiment of the present invention.

[0019] Figure 8 This is a particle size distribution diagram of the extracellular composition obtained from the three-dimensional cultured cells according to the second embodiment of the present invention.

[0020] Fig. 9 This is a bar graph showing the difference in total protein concentration of the extracellular composition using different cell culture methods according to the second embodiment of the present invention.

[0021] Fig.10 This is a diagram showing the changes in the effects of extracellular compositions on cell proliferation using different cell culture methods according to the second embodiment of the present invention. DETAILED DESCRIPTION

[0022] The following is an explanation of the implementation methods of the present invention regarding "a method for culturing cells in three dimensions using cell growth-promoting colloids and mass production of high-concentration extracellular compositions using the method" through specific specific examples. Those skilled in the art can understand the advantages and effects of the present invention from the contents disclosed in this specification. The present invention can be implemented or applied through other different specific embodiments, and the details in this specification can also be modified and changed in various ways based on different viewpoints and applications without departing from the concept of the present invention. In addition, the drawings of the present invention are only simple schematic illustrations and are not depicted in actual size. It is stated in advance. The following embodiments will further explain the relevant technical content of the present invention in detail, but the disclosed content is not intended to limit the scope of protection of the present invention. In addition, the term "or" used in this article may include any one or more combinations of the associated listed items depending on the actual situation.

[0023] [First embodiment]

[0024] See also Figure 1 As shown, the first embodiment of the present invention provides a method for three-dimensionally culturing cells using a cell growth-promoting colloid, which at least comprises the following steps:

[0025] Step S1: providing a culture colloid for mixing and coating the cells. In one embodiment, the culture colloid can be a group consisting of alginate, gelatin, gelatin methacrylamide, carboxymethyl cellulose, collagen, hyaluronic acid, tragacanth gum, gum arabic, xanthan gum, pectin, guar gum, carrageenan, and any combination thereof, and the concentration thereof is 0.1-10%, but the present invention is not limited thereto.

[0026] In addition, the culture colloid may further include a cell growth promoter to stimulate cell proliferation and increase the number of cell growth. In one embodiment, the cell growth promoter may be a group consisting of an extracellular matrix (ECM) composition, collagen, fibronectin, laminin, elastin, growth factor, extracellular vesicles, and any combination thereof, and the concentration thereof is 0.1-10%, but the present invention is not limited thereto.

[0027] In one embodiment, the method of culturing cells using a three-dimensional cell growth promoting colloid of the present invention can be used to culture mesenchymal stem cells (MSC), fibroblasts, epithelial cells, endothelial cells, epidermal stem cells, dental pulp / gingival stem cells, hair follicle papilla cells, keratinocytes or melanocytes of human and animal origin. It is worth noting that the cell types applicable to the method of culturing cells using a three-dimensional cell growth promoting colloid of the present invention can be adjusted according to actual applications, and the present invention is not limited thereto. In addition, the number of cells used in the method of culturing cells using a three-dimensional cell growth promoting colloid of the present invention can be 1×10 5 Up to 2×10 6 cells / mL, but the present invention is not limited thereto.

[0028] Step S2: Cross-link the culture colloid coated with cells so that the cross-linked culture colloid presents a three-dimensional structure, so that the cells are suspended in the cross-linked culture colloid. In one embodiment, three-dimensional cell culture can be carried out by three-dimensional planar layered colloid culture. For example, the cells can be mixed and coated with the culture colloid first and then the culture colloid is cross-linked so that the cells present a similar suspension state in the cross-linked planar layered culture colloid. In a preferred embodiment, the culture colloid mixed with cells is cross-linked and flattened on the surface of the culture dish until the height of the culture colloid is 10mm to 50mm, and then 0.1mL / cm 2 To 0.5mL / cm 2 It should be noted that the present invention only describes the differences from conventional cell culture techniques, and other cell culture methods not described are conventional techniques in the relevant technical field and can be adjusted according to actual applications, and will not be described in detail here.

[0029] In one embodiment, three-dimensional cell culture can be performed by three-dimensional spherical colloid culture. For example, cells can be mixed and coated with culture colloid before cross-linking reaction of culture colloid, and the culture colloid is formed into spherical culture colloid with a diameter of 0.1 mm to 1 mm by needle injection, dispensing machine, or three-dimensional (3D) printer, etc. (but the present invention is not limited thereto), so that the cells are in a state similar to suspension in the cross-linked spherical culture colloid.

[0030] In one embodiment, three-dimensional cell culture can be performed by three-dimensional printing colloid culture. For example, cells can be mixed and coated with culture colloid before cross-linking reaction of culture colloid, and the culture colloid mixed with cells is formed into a three-dimensional mesh culture colloid by 3D printer, so that the cells are in a state similar to suspension in the cross-linked three-dimensional mesh culture colloid. It is worth noting that the three-dimensional structure of the culture colloid formed by the 3D printer can be adjusted according to the actual application, and the present invention is not limited to this.

[0031] In one embodiment, the cross-linking reaction is carried out by mixing the culture colloid and the cross-linking agent, so that the culture colloid is transformed from the original colloidal state to a jelly-like solid state to obtain a cross-linked culture colloid. In the present invention, the cross-linking agent can be a metal ion containing divalent or more. For example, the cross-linking agent can be calcium chloride, barium chloride, zinc chloride, calcium carbonate, calcium sulfate or calcium lactate. In a preferred embodiment, the cross-linking agent is a calcium chloride solution with a volume percentage of 5 to 25, but the present invention is not limited thereto.

[0032] Step S3: Perform cell culture. Further, the three-dimensional cell culture is performed by culturing the cells in a culture medium. In one embodiment, the culture medium may be DMEM-HG, DMEM-F12, DMEM, MEM alpha, DMEM-LG, etc., which may be adjusted according to actual applications, and the present invention is not limited thereto. In addition, the conditions for culturing cells (e.g., temperature, carbon dioxide concentration, etc.) may be adjusted according to actual applications, and the present invention is not limited thereto.

[0033] In one embodiment, the rheological properties of the cross-linked cultured colloid are further analyzed by measuring the storage modulus (G') by performing dynamic frequency scanning analysis with a rheometer. Figure 2 As shown, the measured storage modulus (G') value increases with the increase of crosslinking time. In a preferred embodiment, the storage modulus of the culture colloid is 1,000Pa to 5,000Pa as a preferred characteristic range. If it is higher than this range, it is not conducive to cell growth. If it is lower than this range, the culture colloid cannot effectively coat the cells, causing the cells to easily penetrate the culture colloid.

[0034] As a comparative example, the difference between the method of culturing cells three-dimensionally using a cell growth promoting colloid of the present invention is that 1×10 5 Up to 2×10 6Cells / mL are inoculated in a culture dish for culture, so the cells are cultured in two dimensions by attaching to the culture dish. It should be noted that the culture medium and culture conditions of the comparative example are the same as those of the three-dimensional cell culture method of the present invention, and are not described in detail here.

[0035] like Figure 3 As shown, when the cells were cultured in three dimensions, cell proliferation gradually increased over time, especially between the 21st and 28th day of culture, which increased by more than two times. On the other hand, when the cells were cultured in two dimensions, the degree of cell proliferation was comparable to that of the cells cultured in three dimensions until the 21st day of culture, but it was significantly reduced by more than half between the 21st and 28th day of culture. That is, compared with the conventional two-dimensional cell culture method, the method of the present invention of culturing cells in three dimensions using cell growth-promoting colloids has a significant beneficial effect of expanding the number of cells.

[0036] However, the above example is only one feasible embodiment and is not intended to limit the present invention.

[0037] [Second embodiment]

[0038] See also Figure 4 and Figure 5 As shown, the second embodiment of the present invention provides a method for mass producing a high-concentration extracellular composition by using a method for culturing cells in three dimensions using a cell growth-promoting colloid, which includes at least the following steps in addition to steps S1 to S3 of the first embodiment described above:

[0039] Step S4: Collect the conditioned medium (i.e., the culture medium in which the cells have been cultured). In one embodiment, after the cells have been cultured for at least 7 to 14 days, the culture medium is removed and washed with PBS 2 to 3 times, and then a culture medium without nutrients (e.g., fetal bovine serum (FBS), culture supplements, etc.) is added to culture the cells. The conditioned medium is collected every 1 to 3 days, and new culture medium without nutrients is added to culture the cells, and this step is repeated 3 to 5 times to pool the collected conditioned medium. It is worth noting that the cell culture time and the number of times the conditioned medium is collected can be adjusted according to actual applications, and the present invention is not limited thereto. In addition, the collected conditioned medium can be used directly, but the present invention is not limited thereto.

[0040] Step S5: Filter the conditioned medium. In one embodiment, the collected conditioned medium can be further mixed with 500 mM to 1000 mM of a cross-linking agent, and after being reacted at 4°C for 8 to 16 hours, centrifuged at 1000 g for 20 to 30 minutes, and then filtered through a 0.22 μm filter to filter out cell debris and colloids to obtain a conditioned medium rich in extracellular components. In addition, if Figure 4As shown, before performing the following step S6, the conditioned medium rich in the extracellular composition can be used directly, but the present invention is not limited thereto.

[0041] Step S6: Extracting the extracellular composition. In one embodiment, the conditioned medium rich in the extracellular composition obtained after the implementation of step S5 can be further concentrated 10 to 50 times by a hollow fiber membrane (polyethersulfone filter material, molecular weight cut-off (MWCO) is 1 kDa to 500 kDa) of a tangential flow filtration system to obtain a high concentration of the extracellular composition. It should be noted that the extraction method of the extracellular composition can also be adjusted according to the actual application. For example, the high concentration of the extracellular composition can be obtained by separation or concentration by ultra-high speed centrifugation, polymer precipitation, size exclusion chromatography, antigen-antibody specific separation, etc., and the present invention is not limited thereto.

[0042] Similarly, as a comparative example, except for culturing cells in two dimensions, the other steps are the same as the method of the present invention for mass production of high-concentration extracellular composition by culturing cells in three dimensions using cell growth-promoting colloids. Figure 6 As shown, the total protein concentration was measured by the Bio-Rad protein assay kit. The three-dimensional culture cells of the present invention (such as Figure 6 As shown, the total protein concentration of the three-dimensional planar layered colloidal culture, three-dimensional spherical colloidal culture and 3D printed colloidal culture) is much higher than the total protein concentration of two-dimensional cultured cells, indicating that the method of mass production of high-concentration extracellular compositions of the present invention has higher production capacity than the known methods.

[0043] like Figure 7 and Figure 8 As shown in Figure 2, further analysis of the particle size distribution and concentration of the obtained extracellular composition (exosomes) revealed that the main particle size distribution of the extracellular composition (exosomes) obtained by two-dimensional culture cells was between 50 nm and 200 nm, while that of three-dimensional culture cells (such as Figure 8 As shown in Figure 2, the main particle size distribution of the extracellular composition (exosomes) obtained by three-dimensional planar layered colloidal culture is between 0nm and 50nm, and its concentration is much greater than the concentration of the extracellular composition obtained by two-dimensional culture cells, with a main particle size of 50nm to 200nm. Fig. 9 As shown, the particle concentration of extracellular compositions (exosomes) was measured by Nanoparticle Tracking Analysis (NTA), and three-dimensional cultured cells (such as Fig. 9As shown, the concentration of the extracellular composition (exosomes) obtained by three-dimensional planar layered colloidal culture) is much higher than the concentration of exosomes in the extracellular composition obtained by two-dimensional culture cells. Therefore, the method of mass production of high-concentration extracellular composition of the present invention can effectively improve the production capacity of extracellular composition (exosomes) compared with the known method.

[0044] The effect of the extracellular composition on promoting cell growth was further analyzed. MTT analysis of two-dimensional cultured cells and three-dimensional cultured cells (such as Fig.10 As shown, the extracellular composition (exosomes) obtained by three-dimensional planar layered colloidal culture) is used to culture cells for the effect on cell proliferation. Fig.10 As shown, two-dimensional culture cells and three-dimensional culture cells (such as Fig.10 As shown in the figure, the extracellular compositions (exosomes) obtained from three-dimensional planar layered colloidal culture) can promote cell growth. However, in comparison, the doubling time of the extracellular compositions (exosomes) obtained from three-dimensional cultured cells is 73.01 hours, while the doubling time of the extracellular compositions (exosomes) obtained from two-dimensional cultured cells is 74.8 hours, and the slope of the curve of the extracellular compositions (exosomes) obtained from three-dimensional cultured cells promoting cell growth is greater than that of the curve of the extracellular compositions (exosomes) obtained from two-dimensional cultured cells promoting cell growth (as shown in the figure). Fig.10 As shown, the linear part), it shows that the extracellular composition (exosomes) obtained by the method for mass production of high-concentration extracellular composition of the present invention has excellent efficacy in promoting cell growth.

[0045] However, the above example is only one feasible embodiment and is not intended to limit the present invention.

[0046] [Beneficial Effects of Embodiments]

[0047] One of the beneficial effects of the present invention is that the method of culturing cells in three dimensions using cell growth-promoting colloids and the mass production of high-concentration extracellular compositions using the method provided by the present invention can reduce damage to cells during three-dimensional culture and support three-dimensional growth of cells in the colloid through the technical scheme of "cross-linking the culture colloid coated with cells so that the cross-linked culture colloid presents a three-dimensional structure, so that the cells are suspended in the cross-linked culture colloid."

[0048] Furthermore, the extracellular composition obtained by the method of culturing cells three-dimensionally using cell growth-promoting colloids of the present invention has a particle size distribution that is effectively reduced and a total protein concentration that is significantly increased, which not only effectively increases the production capacity of the extracellular composition but also has an excellent effect of promoting cell growth.

[0049] The contents disclosed above are only preferred feasible embodiments of the present invention, and are not intended to limit the protection scope of the claims of the present invention. Therefore, all equivalent technical changes made using the contents of the present invention description and drawings are included in the protection scope of the claims of the present invention.

Claims

1. A method for three-dimensionally culturing cells using a cell growth-promoting colloid, characterized in that: The method for three-dimensionally culturing cells using a cell growth-promoting colloid comprises: Providing culture colloid to mix and coat the cells; Performing a cross-linking reaction on the culture colloid coated with the cells, so that the cross-linked culture colloid presents a three-dimensional structure, so that the cells are suspended in the cross-linked culture colloid; and Perform cell culture.

2. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 1, characterized in that: The culture colloid comprises a group consisting of alginate, gelatin, gelatin methacrylate hydrogel, carboxymethyl cellulose, collagen, hyaluronic acid, tragacanth gum, gum arabic, xanthan gum, pectin, guar gum, carrageenan and any combination thereof.

3. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 2, characterized in that: The culture colloid further comprises a cell growth promoting agent.

4. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 3, characterized in that: The cell growth promoting agent comprises a group consisting of an extracellular matrix composition, collagen, fibronectin, laminin, elastin, growth factors, extracellular vesicles and any combination thereof.

5. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 1, characterized in that: The cells are mesenchymal stem cells, fibroblasts, epithelial cells, endothelial cells, epidermal stem cells, dental pulp / gingival stem cells, hair follicle papilla cells, keratinocytes or melanocytes of human or animal origin.

6. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 1, characterized in that: The cross-linking reaction is performed by mixing 5 to 25 volume percent of calcium chloride into the culture colloid mixed with the cells.

7. The method for three-dimensionally culturing cells using a cell growth-promoting colloid according to claim 1, characterized in that: The storage modulus of the culture colloid is 1,000Pa to 5,000Pa.

8. A method for mass-producing a high-concentration extracellular composition using the method for three-dimensionally culturing cells using a cell growth-promoting colloid according to any one of claims 1 to 7, characterized in that: The method for mass-producing a high-concentration extracellular composition comprises: collecting the conditioned medium; and The conditioned medium was filtered out.

9. The method for mass production of a high-concentration extracellular composition according to claim 8, characterized in that: The method for mass-producing a high-concentration extracellular composition further comprises: Extraction of extracellular components. 10 . An extracellular composition obtained by using the method for mass-producing a high-concentration extracellular composition according to claim 8 or 9 .

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