MICROCARRIER FOR CELL CULTURE, METHOD FOR PRODUCING THE SAME, AND CELL CULTURE METHOD USING THE SAME

Abstract

The present disclosure relates to a microcarrier that has excellent adhesion to cells and also is easily isolated from cells after culturing, a method for producing the same, and a cell culture method using the same.

Claims

1. A microcarrier for cell culture comprising: a foam core containing polystyrene; a primer polymer layer formed on a surface of the foam core; and a cell adhesion-inducing layer formed on the surface of the primer polymer layer, wherein the microcarrier has a density of 0.80 g/cm.sup.3 to 0.99 g/cm.sup.3.

2. The microcarrier for cell culture according to claim 1, wherein the foam core has a closed cell structure.

3. The microcarrier for cell culture according to claim 1, wherein the foam core has an internal porosity of 2% to 30%.

4. The microcarrier for cell culture according to claim 1, wherein the foam core has a culture medium permeation rate in the range of 0 vol % to 30 vol % of the internal pore volume at the time of culturing cells at 37° C. for 72 hours.

5. The microcarrier for cell culture according to claim 1, wherein the primer polymer layer comprises at least one selected from the group consisting of L-dihydroxy phenylalanine, dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

6. The microcarrier for cell culture according to claim 1, wherein the cell adhesion-inducing layer comprises at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly L-lysine, vitronectin, peptide containing RGD, lignin, cationic dextran, and derivatives thereof.

7. The microcarrier for cell culture according to claim 1, wherein the microcarrier has an average diameter of 50 um to 800 um.

8. The microcarrier for cell culture according to claim 1, wherein the microcarrier has a specific surface area of 50 cm.sup.2/g to 2000 cm.sup.2/g.

9. A method for producing a microcarrier for cell culture, the method comprising the steps of: heating an aqueous solution containing polystyrene containing a foaming agent; foaming the foaming agent to produce a foam core; and recovering and drying the produced foam core and then applying a primer polymer layer to a surface of the foam core.

10. The method for producing a microcarrier for cell culture according to claim 9, wherein the foam core has a closed cell structure.

11. The method for producing a microcarrier for cell culture according to claim 9, wherein the foam core has an internal porosity of 2% to 30%.

12. The method for producing a microcarrier for cell culture according to claim 9, wherein the primer polymer layer comprises at least one selected from the group consisting of L-dihydroxy phenylalanine, dopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

13. The method for producing a microcarrier for cell culture according to claim 9, further comprising a step of coating with a solution containing at least one or more selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly L-lysine, vitronectin, peptide containing RGD, lignin, cationic dextran, and derivatives thereof after the step of applying a primer polymer layer.

14. The method for producing a microcarrier for cell culture according to claim 9, wherein the foaming agent is selected from the group consisting of a physical foaming agent, a chemical foaming agent, an inorganic foaming agent, and mixtures thereof.

15. The method for producing a microcarrier for cell culture according to claim 9, wherein the step of heating the aqueous solution comprises a step of heat-treating at a temperature of 50° C. to 150° C. for 1 minute to 10 minutes.

16. The method for producing a microcarrier for cell culture according to claim 9, wherein the step of recovering and drying the produced foam core and then applying the primer polymer layer to the surface of the foam core comprises a step of immersing the foam core in the primer polymer solution for 1 hour to 10 hours.

17. The method for producing a microcarrier for cell culture according to claim 13, wherein the step of coating with the solution comprises: a step in which a material obtained from the step of recovering and drying the produced foam core and then applying a primer polymer layer to the surface of the foam core is immersed in the solution containing at least one selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly L-lysine, vitronectin, peptide containing RGD, lignin, cationic dextran, and derivatives thereof for 10 hours to 20 hours.

18. A cell culture method comprising a step of culturing a cell using the microcarrier of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0080] FIG. 1 is the optical microscope observation images of the microcarriers produced in Examples 1 to 3 of the present disclosure.

[0081] FIG. 2 shows the SEM analysis result of a cross section after foaming of a microcarrier produced in Example 3 of the present disclosure.

[0082] FIG. 3 shows the FT-IR spectrum results of a foam containing polystyrene.

[0083] FIG. 4 shows the SEM analysis results of the polystyrene surface before and after foaming of polystyrene containing a foaming agent and after coating a primer polymer layer.

[0084] FIG. 5 shows the results of observing the distribution of the microcarriers produced in Examples 1 to 3 of the present disclosure in the culture medium.

[0085] FIG. 6 shows the results of cell culture of 1×10.sup.4 chondrocytes for 3 days using 0.2 g of microcarriers produced in Example 3 and Comparative Example 2 of the present disclosure.

[0086] FIG. 7 shows the results of analyzing the cell adhesion shape of the microcarriers produced in Example 3 and Comparative Example 3 of the present disclosure.

[0087] Below, the present disclosure will be described in more detail by way of examples. However, these examples are provided for illustrative purposes only, and should not be construed as limiting the scope of the present disclosure to these examples.

EXAMPLE 1: PRODUCTION OF MICROCARRIERS FOR CELL CULTURE

[0088] An aqueous solution containing foamable polystyrene particles (provided by LG Chem) with an average diameter of 250 um or less was heated at 100° C. for 5 minutes, and then water at room temperature was added, and the foaming process was stopped to produce a foam core, which was then recovered, and dried at room temperature.

[0089] The dried foam core was immersed in a 1 mg/mL dopamine aqueous solution, and stirred for 4 hours to evenly introduce a polydopamine layer onto the surface of the foam, wherein the thickness of the primer polymer layer was 1 μm or less. The coated foam was recovered, washed with ethanol at least 3 times, and dried at room temperature for 18 hours. In order to purify particles with a density of 0.8 g/cm.sup.3 (0.78-0.83 g/cm.sup.3), separation based on density differences was performed using 75% aqueous ethanol solution and 100% ethanol, and then, placed in a sterile gelatin solution having a concentration of 0.2 w/v, coated for 18 hours, recovered, washed with ethanol, then dried and recovered. By the above method, a microcarrier for cell culture having an average particle size of 500 um and a specific surface area of 150 cm.sup.2/g was produced.

EXAMPLE 2: PRODUCTION OF MICROCARRIER FOR CELL CULTURE

[0090] An aqueous solution containing foamable polystyrene particles (supplied by LG Chem) with an average diameter of 250 um or less was heated at 100° C. for 4 minutes, and then water at room temperature was added, and the foaming process was stopped to produce a foam core, which was then recovered, and dried at room temperature.

[0091] The dried foam core was immersed in a 1 mg/mL dopamine aqueous solution, and stirred for 4 hours to evenly introduce a polydopamine layer onto the surface of the foam, wherein the thickness of the primer polymer layer was 1 μm or less. The coated foam was recovered, washed with ethanol at least 3 times, and dried at room temperature for 18 hours.

[0092] Subsequently, it was placed in a sterile gelatin solution having a concentration of 0.2 w/v, coated for 18 hours, recovered, washed with ethanol, then dried and recovered. In order to purify particles with a density of 0.9 g/cm.sup.3 (0.85-0.93 g/cm.sup.3), separation based on density differences was performed using 30% aqueous ethanol solution and 70% aqueous ethanol solution. By the above method, a microcarrier for cell culture having an average particle size of 350 um and a specific surface area of 190 cm.sup.2/g was produced.

EXAMPLE 3: PRODUCTION OF MICROCARRIER FOR CELL CULTURE

[0093] An aqueous solution containing foamable polystyrene particles (supplied by LG Chem) with an average diameter of 250 um or less was heated at 100° C. for 3 minutes, and then water at room temperature was added, and the foaming process was stopped to produce a foam core, which was then recovered, and dried at room temperature.

[0094] The dried foam core was immersed in a 1 mg/mL dopamine aqueous solution, and stirred for 4 hours to evenly introduce a polydopamine layer onto the surface of the foam, wherein the thickness of the primer polymer layer was 1 μm or less. The coated foam was recovered, washed with ethanol at least 3 times, and dried at room temperature for 18 hours.

[0095] Subsequently, it was placed in a sterile gelatin solution having a concentration of 0.2 w/v, coated for 18 hours, recovered, washed with ethanol, dried and recovered. In order to purify particles with a density of 0.95 g/cm.sup.3 (0.93-0.98 g/cm.sup.3), separation based on density differences was performed using 20% aqueous ethanol solution and 5% aqueous ethanol solution. By the above method, a microcarrier for cell culture having an average particle size of 300 um and a specific surface area of 210 cm.sup.2/g was produced.

[0096] The density, internal porosity, average diameter, and specific surface area of the produced microcarriers are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Category Example 1 Example 2 Example 3 Density (g/cm.sup.3) 0.8 0.9 0.95 Internal porosity (%) 23 13 9 Average diameter (um) 500 350 300 Specific surface area (cm.sup.2/g) 150 190 210

COMPARATIVE EXAMPLE 1: PRODUCTION OF MICROCARRIER FOR CELL CULTURE

[0097] Non-foaming polystyrene particles having a diameter of 250 μm or less were immersed in water at room temperature for 3 minutes, which was then recovered and dried at room temperature. The dried non-foamed core was immersed in 1 mg/mL dopamine aqueous solution and stirred for 4 hours to evenly introduce a polydopamine layer onto the surface of the non-foamed one, wherein the thickness of the primer polymer layer was 1 μm or less. The coated foam was recovered, washed with ethanol at least 3 times, and dried at room temperature for 18 hours.

[0098] Subsequently, it was placed in a sterile gelatin solution having a concentration of 0.2 w/v, coated for 18 hours, recovered, washed with ethanol, then dried and recovered. By the above method, a microcarrier for cell culture having an average particle size of 250 um and a specific surface area of 230 cm.sup.2/g was produced.

COMPARATIVE EXAMPLE 2: PRODUCTION OF MICROCARRIER FOR CELL CULTURE

[0099] Low-density polyethylene particles with a diameter of 212-250 um and a density of 0.96 g/cm.sup.3 were immersed in water at room temperature for 3 minutes, which was then recovered and dried at room temperature. The dried low-density polyethylene core was immersed in 1 mg/mL dopamine aqueous solution, and stirred for 4 hours to introduce a polydopamine layer, wherein the thickness of the primer polymer layer was 1 μm or less. The coated particles were recovered, washed with ethanol at least 3 times, and dried at room temperature for 18 hours.

[0100] By the above method, a microcarrier for cell culture having an average particle size of 300 um and a specific surface area of 200 cm.sup.2/g was produced.

COMPARATIVE EXAMPLE 3: PRODUCTION OF MICROCARRIER FOR CELL CULTURE

[0101] An aqueous solution containing foamable polystyrene particles (supplied by LG Chem) with a diameter of 250 um or less was heated at 100° C. for 3 minutes, and then water at room temperature was added, and the foaming process was stopped to produce a foam core, which was then recovered, and dried at room temperature.

[0102] The dried foam core was placed in a sterile gelatin solution at a concentration of 0.2 w/v without introducing a primer polymer layer, coated for 18 hours, recovered, washed with ethanol, then dried and recovered. In order to purify particles with a density of 0.95 g/cm.sup.3 (0.93-0.98 g/cm.sup.3), separation based on density differences was performed using 20% aqueous ethanol solution and 5% aqueous ethanol solution. By the above method, a microcarrier for cell culture having an average particle size of 300 um and a specific surface area of 210 cm.sup.2/g was produced.

[0103] The density, internal porosity, average diameter, and specific surface area of the microcarriers produced in Comparative Examples 1 to 3 are summarized in Table 2 below.

TABLE-US-00002 TABLE 2 Comparative Comparative Comparative Category Example 1 Example 2 Example 3 Density (g/cm.sup.3) 1.05 0.96 0.95 Internal porosity(%) 0 0 9 Average diameter (um) 250 300 300 Specific surface area 230 200 210 (cm.sup.2/g) Remarks — No cell No primer adhesion- polymer inducing layer layer

EXPERIMENTAL EXAMPLE: MEASUREMENT OF PHYSICAL PROPERTIES OF MICROCARRIER FOR CELL CULTURE

[0104] For the microcarriers for cell culture produced in Examples and Comparative Examples, the cell culture rate of the microcarriers, the behavior of the microcarriers in the culture medium, and the recovery efficiency of the microcarriers were evaluated by the following methods.

[0105] 1. Evaluation of Cell Culture Rate of Microcarriers

[0106] The culture medium was filled in a 250 ml spinner flask, and 0.1 g of a microcarrier was added, and the mixture was stirred at 30 rpm. At this time, the temperature of the culture medium was maintained at 37° C., and cultured for 3 days to confirm the cell culture rate of the microcarrier.

[0107] 2. Evaluation of the Behavior of Microcarrier in the Culture Medium

[0108] The culture medium was filled in a 250 ml spinner flask, and 0.1 g of a microcarrier was added, and the mixture was stirred at 30 rpm. At this time, the temperature of the culture medium was maintained at 37° C. and the behavior of the microcarrier was confirmed. The degree of sedimentation of the microcarriers according to the culture time was evaluated.

[0109] 3. Evaluation of Recovery Efficiency of Microcarrier

[0110] The cells (chondrocytes) in the culture medium and the microcarriers for cell culture produced in Examples and Comparative Examples were injected and stirred, and the cells were adhered to the microcarriers. After culturing at 37° C. for 3 days, the microcarriers to which the cells were adhered were recovered, and then subjected to trypsin treatment to remove the adhered cells, and then, re-dispersed in the culture medium, and the adhered cells were separated in a centrifuge at a rotation speed of 1000 rpm for 5 minutes. Thereafter, the supernatant of the precipitated culture medium was filtered through a 0.2 μm filter to recover microcarriers, which were dried, and then the weight was measured to evaluate the recovery efficiency.

TABLE-US-00003 TABLE 3 Example Example Example Comparative Comparative Comparative 1 2 3 Example 1 Example 2 Example 3 Cell culture rate ~200% ~300% ~400% ~400% ~150% ~200% Degree of — — —   100% — — sedimentation Recovery    75%    90%    95%    0%    95%    95% efficiency

[0111] As shown in Table 3, it was confirmed that when the microcarriers produced in Examples 1 to 3 were used, the cells could be easily isolated from the microcarriers after culturing, while maintaining a high cell culture rate compared to Comparative Examples.

[0112] On the other hand, FIG. 2 shows an SEM analysis result of a cross section after foaming of a microcarrier produced in Example 3 of the present disclosure. Through FIG. 2, the cross section of the microcarrier with increased internal porosity after foaming could be confirmed. FIG. 5 shows the results of observing the distribution of the microcarriers produced in Examples 1 to 3 of the present disclosure in the culture medium. Through FIG. 5, it was confirmed that when the density of the microcarrier falls within the range of 0.90-0.98 g/cm.sup.3, it is evenly floated in the culture medium, which is advantageous for the cell culture.

[0113] In addition, FIG. 6 shows the results of cell culture of 1×10.sup.4 chondrocytes for 3 days using 0.2 g of microcarriers produced in Example 3 and Comparative Example 2 of the present disclosure. Through Comparative Example 2, it was confirmed that the cell culture efficiency was significantly reduced when the cell adhesion-inducing layer was not introduced.

[0114] FIG. 7 shows the results of analyzing the cell adhesion shape of the microcarriers produced in Example 3 and Comparative Example 3 of the present disclosure. In the case of Comparative Example 3, it was confirmed that the cell adhesion-inducing layer into which the primer coating layer was not introduced was unstable as compared with Example 3 under the same conditions, so that the cells were not evenly adhered, and the cells could not be efficiently cultured.