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

Abstract

The present disclosure relates to a microcarrier for cell culture comprising: polystyrene-based particles containing at least one or more of hydrocarbon oil having 12 or more carbon atoms, or pores derived therefrom, a method for producing the same, and a cell culture composition using the same.

Claims

1. A microcarrier for cell culture comprising: polystyrene-based particles containing at least one of hydrocarbon oil having at least 12 carbon atoms, or pores derived therefrom.

2. The microcarrier for cell culture of claim 1, wherein: a ratio of perfect spherical particles without damage and breakage according to the following Equation 1 is more than 90% and 100% or less:
Ratio of perfect spherical particles without damage and breakage (%)=(Number of polystyrene-based particles having a perfect spherical shape without damage and breakage/Number of entire polystyrene-based particles)×100.  [Equation 1]

3. The microcarrier for cell culture of claim 1, wherein: the polystyrene-based particles have an apparent density of 0.95 g/cm.sup.3 or more and less than 1.00 g/cm.sup.3.

4. The microcarrier for cell culture of claim 1, wherein: the polystyrene-based particles have a porosity of 0% or more and less than 8% according to the following Equation 2:
Porosity (%)=[1−(Apparent density of polystyrene-based particles/True density of polystyrene-based particles)]×100.  [Equation 2]

5. The microcarrier for cell culture of claim 1, wherein: the hydrocarbon oil has a density of 0.75 g/cm.sup.3 or more and 0.80 g/cm.sup.3 or less.

6. The microcarrier for cell culture of claim 1, wherein: the hydrocarbon oil comprises a linear or branched saturated hydrocarbon compound having 12 or more and 50 or less carbon atoms.

7. The microcarrier for cell culture of claim 1, wherein: the polystyrene-based particles comprise a polystyrene-based polymer in which the at least one of hydrocarbon oil having at least 12 carbon atoms, or pores derived therefrom are dispersed therein.

8. The microcarrier for cell culture of claim 7, wherein: the polystyrene-based polymer comprise a reaction product of 1 part by weight of a styrene monomer; and more than 0.033 parts by weight and less than 3 parts by weight of an ethylenically unsaturated crosslinking agent.

9. The microcarrier for cell culture of claim 1, wherein: the polystyrene-based particles have an average diameter of 50 μm or more and 400 μm or less.

10. The microcarrier for cell culture of claim 1, wherein: the hydrocarbon oil is contained in an amount of 30% by weight or less based on the total weight of the polystyrene-based particles.

11. The microcarrier for cell culture of claim 1, wherein: based on the total surface area of the polystyrene-based particles, the ratio of the surface area of the polystyrene-based particles in contact with micropores existing on the surface of the polystyrene-based particles is less than 0.01%.

12. The microcarrier for cell culture of claim 1, wherein: the pores have a diameter of 0.1 μm to 5 μm.

13. The microcarrier for cell culture of claim 1, wherein: a primer polymer layer, a cell adhesion inducing layer, or a combination layer thereof is further included on the surface of the polystyrene-based particles.

14. A method for producing a microcarrier for cell culture, comprising the step of proceeding a suspension polymerization reaction of a monomer composition containing a styrene monomer in the presence of hydrocarbon oil having at least 12 carbon atoms.

15. The method of claim 14, wherein: a content of the hydrocarbon oil is 10% by weight or more and 30% by weight or less based on the weight of the monomer composition.

16. The method of claim 14, wherein: the monomer composition is a mixture of 1 part by weight of a styrene monomer and more than 0.033 parts by weight and less than 3 parts by weight of an ethylenically unsaturated crosslinking agent.

17. The method of claim 14, wherein: the suspension polymerization reaction of the monomer composition comprises a step of mixing the monomer composition with an aqueous dispersion and applying a shearing force to homogenize the monomer composition in the aqueous dispersion in the form of droplets; and a step of suspension-polymerizing the homogenized monomer composition at a stirring speed of 300 rpm or more and 1000 rpm or less.

18. A cell culture composition comprising a cell and the microcarrier for cell culture of claim 1.

19. The cell culture composition of claim 18, wherein: the cell comprises at least one compound selected from the group consisting of fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, umbilical cord blood cells, mesenchymal stem cells, CHO cells, and kidney cells.

20. The cell culture composition of claim 18, wherein: a difference in apparent density between the microcarrier for cell culture and the cell is 0.02 g/cm.sup.3 or more and 0.20 g/cm.sup.3 or less.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0097] FIG. 1. shows an SEM image of the microcarrier for cell culture obtained in Example 1;

[0098] FIG. 2 shows an SEM image of the microcarrier for cell culture obtained in Reference Example 1;

[0099] FIG. 3 shows an SEM image of the microcarrier for cell culture obtained in Reference Example 5;

[0100] FIG. 4 is a SEM image showing the particle cross-sectional shape of the microcarrier for cell culture obtained in Example 2; and

[0101] FIG. 5 is a SEM image showing the particle cross-sectional shape of the microcarrier for cell culture obtained in Comparative Example 1.

[0102] Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited thereby.

Example: Production of Microcarriers for Cell Culture

Example 1

[0103] 2.5 g of polyvinyl alcohol was mixed with 250 g of water to prepare an aqueous dispersion, which was then stirred at room temperature for 20 minutes.

[0104] 25 g of the total sum of styrene as a monomer, divinylbenzene as a crosslinking agent, and the oil as shown in Table 1 (charging amount: based on the total amount of oil added) were mixed at the content ratio shown in Table 1 below, and then was sufficiently dissolved. 2.06 wt. % of a benzoyl peroxide initiator (Sigma Aldrich) and 0.3125 wt. % of a tert-butyl peroxybenzoate initiator (Sigma Aldrich) were added to the mixture (charging amount of initiator: based on the total amount of monomer, crosslinking agent, and oil), and the mixture was further stirred for 5 minutes to prepare a monomer composition.

[0105] Then, the monomer composition was added to the aqueous dispersion, a shear force was applied to the aqueous dispersion and monomer composition at a speed of 800 rpm, and the monomer composition was dispersed in the form of fine droplets in an aqueous dispersion and allowed to homogenize.

[0106] The homogenized mixture was reacted under nitrogen purge at 90° C. for 6 hours while stirring at the stirring speed described in Table 1 below to prepare polystyrene particles, and the particles washed with ethanol were dried at room temperature and recovered.

[0107] After drying, the recovered particles were immersed in tris buffer (pH 8.0) in which dopamine hydrochloride was dissolved at 1 mg/mL, and coated under stirring at room temperature for 2 hours. The excess coating material was washed with ethanol, the particles were filtered on a 70 μm sieve, dried at room temperature, and the dried particles were used as a microcarrier for cell culture.

Examples 2 to 8

[0108] As shown in Table 1 below, a microcarrier for cell culture was prepared in the same manner as in Example 1, except that the weight ratio of styrene and divinylbenzene, oil, or agitation speed were used differently.

Comparative Example: Production of Microcarriers for Cell Culture

Comparative Examples 1 to 3

[0109] As shown in Table 1 below, a microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of styrene and divinylbenzene, oil, and stirring speed were used differently.

Reference Example: Production of Microcarriers for Cell Culture

Reference Examples 1 to 8

[0110] As shown in Table 1 below, a microcarrier for cell culture was produced in the same manner as in Example 1, except that the weight ratio of styrene and divinylbenzene, and oil were used differently.

TABLE-US-00001 TABLE 1 Suspension polymerization conditions Weight ratio of Amount Stirring styrene:divinyl Type of Oil of oil speed Category benzene oil density added (rpm) Example 1 1:0.33 Isopar M 0.791 14 800 Example 2 1:0.33 Isopar M 0.791 20 800 Example 3 1:0.33 Isopar M 0.791 20 400 Example 4 1:1 Isopar M 0.791 20 800 Example 5 1:0.33 Dodecane 0.750 14 800 Example 6 1:0.33 Dodecane 0.750 20 800 Example 7 1:0.33 Hexadecane 0.773 14 800 Example 8 1:0.33 Hexadecane 0.773 20 800 Comparative 1:0 — — 0 800 Example 1 Comparative 1:0.33 n-heptane 0.684 14 800 Example 2 Comparative 1:0.33 Iso-octane 0.690 14 800 Example 3 Reference 1:0 Isopar M 0.791 20 800 Example 1 Reference 1:0 Isopar M 0.791 50 800 Example 2 Reference 1:0.0025 Isopar M 0.791 20 800 Example 3 Reference 1:0.033 Isopar M 0.791 20 800 Example 4 Reference 1:3 Isopar M 0.791 20 800 Example 5 Reference 1:0.33 Isopar M 0.791 7 800 Example 6 Reference 1:0.33 Dodecane 0.750 7 800 Example 7 Reference 1:0.33 Hexadecane 0.773 7 800 Example 8

Experimental Example: Measurement of Physical Properties of Microcarriers for Cell Culture

[0111] The physical properties of the cell culture microcarriers obtained in Examples and Comparative Examples were measured by the following method, and the results are shown in Table 2 below.

[0112] 1. Particle Size

[0113] For the microcarriers for cell culture obtained in Examples and Comparative Examples, the diameters of 100 particles were measured through an optical microscope, and an arithmetic mean value thereof was calculated.

[0114] 2. Apparent Density of Particles

[0115] For the microcarriers for cell culture obtained in Examples and Comparative Examples, a sample was prepared under the condition that the drying process has been completed. The sample was added to an ethanol aqueous solution having a density of 0.95 g/cm.sup.3, 0.97 g/cm.sup.3, 0.98 g/cm.sup.3 or 0.995 g/cm.sup.3, and a distilled water (DIW) having a density of 1 g/cm.sup.3 under the conditions of room temperature (25° C.) and atmospheric pressure (1 atm). It was confirmed whether the particles are floated or sedimented, and the apparent density was evaluated according to the following criteria.

[0116] 1) floated in an aqueous ethanol solution with a density of 0.95 g/cm.sup.3: less than 0.95 g/cm.sup.3

[0117] 2) sedimented in distilled water (DIW) with a density of 1 g/cm.sup.3: more than 1 g/cm.sup.3

[0118] 3) sedimented in ethanol aqueous solution with a density of 0.95 g/cm.sup.3 and floated in distilled water (DIW) with a density of 1 g/cm.sup.3: 0.95 g/cm.sup.3 or more and less than 1 g/cm.sup.3

[0119] 4) sedimented in an aqueous ethanol solution with a density of 0.95 g/cm.sup.3 and floated in an aqueous ethanol solution with a density of 0.97 g/cm.sup.3: 0.95 g/cm.sup.3 or more and less than 0.97 g/cm.sup.3

[0120] 5) sedimented in an aqueous ethanol solution with a density of 0.98 g/cm.sup.3 and floated in an aqueous ethanol solution with a density of 0.995 g/cm.sup.3: 0.98 g/cm.sup.3 or more and less than 0.995 g/cm.sup.3

[0121] 3. Ratio of Perfect Spherical Particles without Damage and Breakage

[0122] Using the microcarriers for cell culture obtained in Examples and Comparative Examples, a sample was prepared under conditions in which the drying process has been completed, and the number of spherical particles (complete spherical particles not broken) among all particles in the sample was measured through SEM, and the number percent ratio of spherical particles was measured according to the following Equation 1.

Ratio of perfect spherical particles without damage and breakage (%)=(Number of particles with perfect spherical shape without damage and breakage/Number of total particles)×100.  [Equation 1]

[0123] 4. Internal Porosity of Particles

[0124] Using the microcarriers for cell culture obtained in Examples and Comparative Examples, a sample was prepared under conditions in which the drying process has been completed, and the true density was measured from the sample by He pycnometry, and the porosity was measured according to the following Equation 2.

Porosity (%)=[1−(Apparent density/True density)]×100  [Equation 2]

[0125] 5. Particle Internal Structure and Pore Diameter

[0126] (1) Particle Internal Structure

[0127] For the microcarriers for cell culture obtained in Examples and Comparative Examples, the internal structure of particles was confirmed through SEM. Specifically, the particles were embedded in epoxy, and then subjected to milling to prepare a cross section, and then the shape of the particle cross section was confirmed through SEM.

[0128] (2) Pore Diameter

[0129] The particles were embedded in an epoxy, and subjected to ion milling to prepare a cross section, and then the shape of the particle cross section was confirmed with SEM to obtain the minimum and maximum diameters among the pores inside the particles.

[0130] 6. Evaluation of the Recovery Efficiency of Microcarriers

[0131] The microcarriers for cell culture obtained in Examples and Comparative Examples were immersed in tris buffer (pH 8.0) in which dopamine hydrochloride was dissolved at 1 mg/mL, and coated for 2 hours at room temperature under stirring. The excess coating material was washed with ethanol, the particles were filtered through a 70 μm sieve and the dried at room temperature.

[0132] A culture medium containing mesenchymal stem cells (density: 1.05 g/cm.sup.3) was filled in 100 mL vertical wheel bioreactor (PBS), and a polydopamine-coated microcarrier for cell culture was injected into the culture medium and stirred. After culturing at room temperature for 14 days, it was treated with 0.25% trypsin, then the culture solution was centrifuged, and the microcarriers floating in the supernatant were recovered. This was dried and then the weight was measured to evaluate the recovery efficiency.

[0133] 7. Analysis of Pore Component

[0134] In order to analyze the components contained in the internal pores for the microcarriers for cell culture obtained in the Examples and Comparative Examples, the microcarriers for cell culture were freeze-pulverized and dissolved in chloroform to extract unreacted residual compounds. Detailed components were qualitatively and quantitatively analyzed through GC/FID (Gas Chromatography-flame ionization detector).

[0135] Specifically, a standard sample of styrene ( 1/4000 mg/mL˜1 mg/mL), divinylbenzene ( 1/10000 mg/mL˜1 mg/mL), Isopar M ( 1/400 mg/mL to 10 mg/mL) was prepared by concentration in a mixed solvent of chloroform and methanol in a 1:2 volume ratio. 1 uL of a standard sample was injected into a GC/FID instrument and a calibration curve was prepared. 1 uL of the unreacted residual compound solution extracted from the sample in Table 1 was injected, and the content was calculated using a calibration curve. For GC/FID measurement, a column on Rtx (styrene, Isopar M) or wax (divinylbenzene) with an inner diameter of 0.53 mm, a length of 30 m, and a film thickness of 5 μm was used. The initial oven temperature was started from 50° C. and the temperature was raised to 250° C. at a rate of 10° C./min. As the mobile phase gas, 15 mL/min of helium gas was used. At this time, it was marked as “0” if oil or a component derived therefrom was detected, and it was marked as “X” if oil or a component derived therefrom was not detected.

TABLE-US-00002 TABLE 2 Experimental Example Measurement Results of Examples and Comparative Examples Ratio of perfect Apparent spherical particles Particle density of Pore without damage Particle Recovery Analysis size particles Porosity diameter and breakage internal efficiency of pore Category (μm) (g/cm.sup.3) (%) (μm) (%) structure (%) components Example 1 104(±24)  0.98 or more 3.9 Minimum: 1 99 Porous 90 ◯ 0.995 or less Maximum: 4 (FIG. 1) Example 2 92(±22) 0.95 or more 7.1 Minimum: 1 99 Porous 90 ◯ 0.97 or less Maximum: 4 (FIG., 4) Example 3 322(±51)  0.95 or more 7.5 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Example 4 99(±21) 0.95 or more 6.7 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Example 5 96(±30) 0.95 or more 6.9 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Example 6 105(±27)  0.95 or more 7.3 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Example 7 107(±23)  0.95 or more 7.2 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Example 8 102(±26)  0.95 or more 6.5 Minimum: 1 99 Porous 90 ◯ 1 or less Maximum: 4 Comparative 198(±99)  More than 1 0   — 100  No pores Cell-microcarriers X Example 1 (FIG. 5) can be separated by centrifugation Comparative 93(±20) 0.95 or less — Minimum: 1 90 Porous ◯ Example 2 Maximum: 4 Comparative 99(±15) 0.95 or less — Minimum: 1 90 Porous Cell culture ◯ Example 3 Maximum: 4 is not possible Reference 92(±66) Greater — Minimum: 1  0 Porous Cell culture ◯ Example 1 than 1 Maximum: 4 (FIG. 2) is not possible Reference Particle production — — — — — Cell culture — Example 2 is not possible is not possible Reference 80(±16) More than 1 — Minimum: 1 50 Porous Cell culture ◯ Example 3 Maximum: 4 is not possible Reference 97(±24) 0.95 or more — Minimum: 1 50 Porous Cell culture ◯ Example 4 1 or less Maximum: 4 is not possible Reference 112(±22)  0.95 or more — Minimum: 1 70 Porous Cell culture ◯ Example 5 1 or less Maximum: 4 (FIG. 3) is not possible Reference 96(±26) More than 1 — Minimum: 1 90 Porous Cell culture ◯ Example 6 Maximum: 4 is not possible Reference 120(±26)  More than 1 — Minimum: 1 90 Porous Cell culture ◯ Example 7 Maximum: 4 is not possible Reference 98(±24) More than 1 — Minimum: 1 90 Porous Cell culture ◯ Example 8 Maximum: 4 is not possible

[0136] As shown in Table 2 above, the microcarriers for cell culture of Examples exhibit a porosity of 3.9% or more and 7.5% or less inside the particles, have a porous structure and satisfies a low density of 0.95 g/cm.sup.3 or more and less than 1 g/cm.sup.3, and at the same time, the ratio of perfect spherical particles without damage and breakage is 99% which is very high and thus, most of the particles could have homogeneously spherical shapes. In addition, it was confirmed that in the microcarriers for cell culture of Examples, carrier culture is effectively carried out under cell culture conditions, and the recovery efficiency of the microcarriers after culture was also as high as 90%. On the other hand, the microcarrier for cell culture of Comparative Example 1 has a non-porous structure having no pores with an internal particle porosity of 0%, where the density increased to more than 1 g/cm.sup.3, which caused a problem that the cells and microcarriers could not be isolated through centrifugal separation after cell culture and removal.

[0137] In addition, the microcarriers for cell culture of Comparative Examples 2 and 3 have an excessively low particle density of less than 0.95 g/cm.sup.3, and under stirring conditions, the particles are suspended on the surface of the culture medium, and cell adhesion is reduced, so there was a problem that cell culture is not possible. Further, in the microcarriers for cell culture of Comparative Examples 2 and 3, the ratio of perfect spherical particles without damage and breakage was measured to be 90%, and also the particle surface is not uniform and recessed particles are generated in a large amount compared to Examples, so that the cell culture efficiency is lowered to such an extent that the cell culture is not possible.

[0138] On the other hand, the microcarriers for cell culture of Reference Examples 1 to 5 have a perfect spherical particle ratio of 0% or more and 70% or less without damage or breakage, which is lower than Examples, and there is a limitation in that the shape of the particles is relatively non-uniform compared to Examples. Further, the microcarriers for cell culture of Reference Examples 6 to 8 have an apparent density of more than 1 g/cm.sup.3 during cell culture, so that during culture, precipitation occurs during low-speed stirring, or the suspension of the culture solution is increased due to non-spherical particles. Thus, there was a problem in that cell culture efficiency is lowered to such an extent that cell culture is not possible due to physical impact in the future.