MICROCARRIER FOR CELL CULTURE AND METHOD FOR PREPARING THE SAME

Abstract

The present application relates to microcarrier particles for cell culture, a method for preparing the particles, and a cell culture medium composition including the particles. According to the present application, a microcarrier having a high degree of uniformity in shape or form, having porosity, and advantageous for cell attachment and isolation of cultured cells is provided.

Claims

1. A method for preparing microcarrier particles for cell culture, the method comprising the steps of: mixing a continuous phase composition (A) satisfying the following conditions 1 and 2; and a dispersive phase composition (B) containing a polymerizable monomer (b1) and then performing suspension polymerization: $\begin{array}{l}\text{45 mN/m}<\text{Surface Tension of the Continuous Phase}\\ \text{Composition}\le \text{54 mN/m}\end{array}$ $\text{Viscosity of the Continuous Phase Composition}\ge \text{2}\text{.0 cp}$ wherein, in the [condition 1], the surface tension is measured at a normal temperature according to a ring method, and in the [condition 2], the viscosity is measured under the conditions of a shear rate in the range of 66 to 264 ⅟s and a normal temperature.

2. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the continuous phase composition (A) comprises water and poly(vinyl alcohol) (PVA).

3. The method for preparing microcarrier particles for cell culture according to claim 2, wherein the polyvinyl alcohol (PVA) has a weight average molecular weight in the range of 80,000 to 190,000 and a hydrolyzed degree in the range of 80 to 99%.

4. The method for preparing microcarrier particles for cell culture according to claim 2, wherein the continuous phase composition (A) contains at least 1.0% by weight of the polyvinyl alcohol based on the total weight of the continuous phase composition.

5. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the dispersive phase composition (B) comprises a styrene monomer as the polymerizable monomer (b1).

6. The method for preparing microcarrier particles for cell culture according to claim 5, wherein the dispersive phase composition (B) further comprises a crosslinking agent (b2).

7. The method for preparing microcarrier particles for cell culture according to claim 6, wherein the dispersive phase composition (B) contains the crosslinking agent in an amount of 3 to 300 parts by weight based on 100 parts by weight of the styrene monomer.

8. The method for preparing microcarrier particles for cell culture according to claim 6, wherein the dispersive phase composition (B) further comprises a hydrocarbon oil (b3).

9. The method for preparing microcarrier particles for cell culture according to claim 8, wherein the hydrocarbon oil has a density in the range of 0.750 g/cm.sup.3 to 0.800 g/cm.sup.3.

10. The method for preparing microcarrier particles for cell culture according to claim 8, wherein the dispersive phase composition (B) contains the hydrocarbon oil in an amount of 10 to 30% by weight based on the total content of the dispersive phase composition.

11. The method for preparing microcarrier particles for cell culture according to claim 8, wherein the hydrocarbon oil contains a linear or branched saturated hydrocarbon compound having 12 to 50 carbon atoms.

12. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the suspension polymerization is performed under conditions of a temperature of 80 to 95° C. and at a speed of 300 to 900 rpm.

13. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the step of mixing the continuous phase composition (A) and the dispersive phase composition (B) followed by applying a shearing force to homogenize the dispersive phase composition (B) in the continuous phase composition (A) in the form of droplets; and the dispersive phase composition (B) is subjected to the suspension polymerization.

14. The method for preparing microcarrier particles for cell culture according to claim 1, further comprising a step of additionally adding the continuous phase composition (A) during the suspension polymerization.

15. The method for preparing microcarrier particles for cell culture according to claim 1, wherein at least 80% of the microcarrier particles have a single shape where satellite particles are not present on the surface of the particles.

16. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the microcarrier particles include porous microcarrier particles that are spherical and have pores of 5 .Math.m or less.

17. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the microcarrier particles include microcarrier particles having a diameter in the range of 90 to 250 .Math.m.

18. The method for preparing microcarrier particles for cell culture according to claim 1, wherein the microcarrier particles include microcarrier particles having a density in the range of 0.95 g/cm.sup.3 to 1.00 g/cm.sup.3.

19. Non-foamed spherical microcarrier particles wherein the particles are porous with a pore size of 5 .Math.m or less inside thereof, and comprise polystyrene.

20. The non-foamed spherical microcarrier particles comprising polystyrene according to claim 19, wherein the particles have a diameter in the range of 90 to 250 .Math.m.

21. The non-foamed spherical microcarrier particles comprising polystyrene according to claim 19, wherein the particles have a density in the range of 0.95 g/cm.sup.3 to 1.00 g/cm.sup.3.

22. Non-foamed spherical microcarrier particles, wherein in which at least 80% of the total particles have a single shape where satellite particles are not present on the surface of the particles, and the particles comprise polystyrene.

23. Non-foamed spherical microcarrier particles comprising polystyrene according to claim 22, wherein the particles have a diameter of 90 to 250 .Math.m.

24. Non-foamed spherical microcarrier particles comprising polystyrene according to claim 22, wherein the particles have a density of 0.95 g/cm.sup.3 to 1.00 g/cm.sup.3.sup.-.

25. Non-foamed spherical microcarrier particles, wherein at least 80% of the total particles have a single shape where satellite particles are not present on the surface of the particles, the microcarrier particles are porous with a pore size of 5 .Math.m or less inside thereof, and comprise polystyrene.

26. Non-foamed spherical microcarrier particles comprising polystyrene according to claim 25, wherein the particles have a diameter of 90 to 250 .Math.m.

27. The non-foamed spherical microcarrier particles comprising polystyrene according to claim 25, wherein the particles have a density in the range of 0.95 g/cm.sup.3 to 1.00 g/cm.sup.3.sup.-.

28. A cell culture composition comprising the microcarrier particles prepared according to claim 19; cells; and a culture medium.

29. A cell culture composition comprising the microcarrier particles prepared according to claim 22; cells; and a culture medium.

30. A cell culture composition comprising the microcarrier particles prepared according to claim 25; cells; and a culture medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] FIGS. 1(a) to 1(c) are each an SEM image of a part of the particles prepared in each of Examples 1 to 3. The white bar at the right-side lower end of each image means a size of 150 .Math.m.

[0091] FIGS. 2(a) to 2(e) are each an SEM image of a part of the particles prepared in each of Comparative Examples 1 to 5. The white bar at the right-side lower end of each image means a size of 150 .Math.m. In the case of Comparative Example 4, since particles of 1 mm or larger were obtained as in Table 2, the particles were not compared through the image in which 150 .Math.m-sized white bars were shown.

[0092] FIGS. 3(a) and 3(b) are each a view showing the internal structure of the particles prepared according to Example 1. Specifically, it is confirmed that pores having a size of about 1 to 3 .Math.m are formed inside the particles prepared according to Example 1.

[0093] Hereinafter, the action and effect of the invention will be described in more detail with reference to specific examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of rights of the invention in any manner.

EXAMPLE AND COMPARATIVE EXAMPLE

Example 1

[0094] Preparation of Dispersive Phase

[0095] 8 g of a mixture of styrene monomer (st), divinylbezene (DVB) as a crosslinking agent and low-density hydrocarbon oil (density in the range of 0.750 to 0.800 g/cm.sup.3) in the content ratio shown in Table 1 below was stirred in a 100 ml vial. After that, benzoyl peroxide (BPO) and tert-butyl peroxybenzoate (t-BP), which are thermal initiators, were additionally added to a vial, and the mixture was stirred at normal temperature for about 5 minutes. The content between each component is shown in Table 1 below.

[0096] Preparation of Continuous Phase

[0097] 2.5 g of PVA having a weight average molecular weight in the range of 85,000 to 125,000 and a hydrolysis rate of 87 to 89% was dissolved in 250 g of distilled water. The detailed contents are shown in Table 1.

[0098] Preparation of Particles by Suspension Polymerization

[0099] 50 g of 1% PVA aqueous solution was mixed with the dispersive phase and stirred in an oil bath until a homogeneous dispersion was obtained. Specifically, the oil bath was gradually heated at normal temperature while stirring at 800 rpm, and suspension polymerization was performed under the conditions of a temperature of 85 to 88° C. and a speed of 600 to 800 rpm. The polymerization was carried out under nitrogen purging.

[0100] Obtaining Particles

[0101] After 6 hours of reaction, the prepared particles were recovered through a 100 .Math.m sieve, and washed 5 times with ethanol, and then dried at normal temperature.

Examples 2 to 3 and Comparative Examples 1 to 5

[0102] Particles were obtained through the same process as in Example 1, except that the composition of the dispersive phase and the continuous phase were adjusted as shown in Table 1 below.

Measuring Method

[0103] Surface Tension of Continuous Phase (unit: mN/m)

[0104] The surface tension was measured according to a ring method. Specifically, the surface tension was measured at normal temperature using a platinum ring and a surface tension meter (Surface Electro Optics).

[0105] Viscosity of Continuous Phase (unit: cp)

[0106] The viscosity was measured according to a shear rate. Specifically, the viscosity was measured using an LVDV2T instrument (Brookfield), which is a rotational viscometer, under the conditions of a shear rate range of 66 to 264 ⅟s and normal temperature (about 25° C.).

[0107] Weight Average Molecular Weight

[0108] The weight average molecular weight (in terms of standard polystyrene) of PVA (or PVP) was measured using gel permeation chromatography (GPC).

[0109] Hydrolyzed Degree

[0110] The hydrolyzed degree of the measured PVA was measured using 1H-NMR. For reference, as the hydrolysis proceeds, the hydrogen peak (H peak) at ethylene decreases, and the hydrogen peak at the hydroxyl group increases.

[0111] Oil/Water Fraction

[0112] When the dispersive phase is regarded as the oil phase and the continuous phase is regarded as the water phase, it means the ratio of the weight (W.sub.B) of the dispersive phase relative to the weight (W.sub.A) of the continuous phase.

TABLE-US-00001 Example 1 Example 2 Example 3 Compara tive Example 1 Compara tive Example 2 Compara tive Example 3 Compara tive Example 4 Compa rative Exampl e 5 Oil/Water fraction (W.sub.B/W.sub.A) 0.20 0.15 0.14 0.10 0.10 0.15 0.15 0.15 Dispersive phase St : DVB (weight ratio).sup.1.sup.) 1 : 0.33 1 : 0.33 1 : 0.33 1 : 0.33 1 : 0.33 1 : 0.33 1 : 0.33 1 : 0.33 Oil content (wt%).sup.2) 20 20 20 20 20 20 20 20 BPO content (wt%).sup.3) 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 t-BP content (Wt%).sup.4) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Continuous phase Water content (g) 50 50 50 50 50 50 50 50 Concentration (%) of PVA(or PVP).sup.5) PVA 1.01% PVA 2% PVA 2% (1% + 1%.sup.6)) PVA 0.6% PVA 0.8% PVA 1.01% PVP 1.01% PVP 1.01% Molecular weight /hydrolyzed degree of PVA(or PVP) 7) 8) 9) 10) Surface energy 54 54 50 55 54.5 49 65 66 Viscosity (cp) ≥ 2.0 4.0 4.0 ≤ 1 ≤ 1.5 1.6 1.5 3.0

[0113] In Table 1, the reference numerals 1) to 10) are as follows. [0114] 1) Ratio of the weight ratio of St (styrene) to the weight of DVB (divinylbenzene) [0115] 2) Weight% occupied by the oil when the weight of the entire composition of the dispersive phase is 100, [0116] 3) Weight% occupied by BPO when the weight of the entire composition of the dispersive phase is 100, [0117] 4) Weight % occupied by t-BP when the weight of the entire composition of the dispersive phase is 100, [0118] 5) Weight % occupied by PVA (polyvinyl alcohol) or PVP (polyvinyl pyrrolidone) when the weight of the entire composition of the continuous phase is 100, [0119] 6) PVA is added to the continuous phase at a concentration of 1%, and a continuous phase composition having a concentration of PVA of 1% is additionally added during progress of suspension polymerization (a point of time when the polymerization rate is about 10%) [0120] 7) Use of PVA that satisfies the weight average molecular weight in the range of 85,000 to 125,000 and the hydrolyzed degree in the range of 87 to 89% [0121] 8) Use of PVA that satisfies the weight average molecular weight in the range of 13,000 to 50,000 and the hydrolyzed degree in the range of 87 to 89% [0122] 9) Use of PVP having a weight average molecular weight of about 55,000 [0123] 10) Use of PVP having a weight average molecular weight of about 360,000

Results of Experiments of Suspension Polymerization Product

[0124] The items shown in Table 2 were measured for the particles obtained in Examples and Comparative Examples. The measurement method for each item is as follows.

1. Recovery Rate of Particles (%)

[0125] In Examples, the ratio of the solid content (recovered polystyrene particles) to the substances (styrene and divinylbenzene) that actually participated in the polymerization of polystyrene particles was calculated according to Equation 3 below.

Equation 3

[0126] {(Weight of Recovered Polystyrene Particles (g))/(Weight of Styrene and Divinylbenzene (g))} × 100

[0127] The recovery rate of the particles is related to the stability of the dispersive phase in the suspension polymerization reaction system. Specifically, when the stability of the dispersive phase is ensured, the dispersive phase is not cracked during polymerization and can be polymerized into particles. Such stability may vary depending on the components forming the dispersive phase and the continuous phase and whether the conditions 1 and 2 are satisfied.

2. Confirmation of Particle Density (g/cm.SUP.3.)

[0128] Under the conditions of normal temperature (about 25° C.) and atmospheric pressure (1 atm), carrier particles obtained in Examples and Comparative Examples were added to an aqueous ethanol solution having a density of 0.95 g/cm.sup.3 and an aqueous ethanol solution having a density of 0.99 g/cm.sup.3, respectively. Then, it was confirmed whether the carrier particles floated or settled, and then the density was evaluated based on the following criteria. The density of the particles can be determined according to the presence or absence of the use of hydrocarbon oil, its content, and the residual of the hydrocarbon oil in the dispersive phase due to the satisfaction of the conditions 1 and 2.

[0129] 1) floats in an aqueous ethanol solution with a density of 0.95 g/cm.sup.3: the density of the carrier is less than 0.95 g/cm.sup.3.

[0130] 2) settles in an aqueous ethanol solution with a density of 0.99 g/cm.sup.3: the density of the carrier exceeds 0.99 g/cm.sup.3.

[0131] 3) settles in an aqueous ethanol solution with a density of 0.95 g/cm.sup.3 and floats in an aqueous ethanol solution with a density of 0.99 g/cm.sup.3: the density of the carrier is 0.95 g/cm.sup.3 or more and 0.99 g/cm.sup.3 or less.

3. Particle Size

[0132] The microcarrier particles obtained in Examples and Comparative Examples were prepared, and then the particle diameters of 100 particles were measured through an optical microscope. The arithmetic mean value of the measured diameters was calculated. Specifically, the diameters of individual particles was calculated by calculating the two-dimensional (2D) plane area of the particles through an optical microscope and then back calculating Equation (S=πr.sup.2) for the plane area.

[0133] The surface tension of the continuous phase affects the interfacial tension between the dispersive phase and the continuous phase and the shape or size of the dispersive phase. The surface tension under the condition 1 described above can prevent cracking of the dispersive phase or excessive decrease or increase in the size of the dispersive phase. In addition, the viscosity of the continuous phase affects the movement of the dispersive phase upon stirring associated with suspension polymerization. Appropriate levels of viscosity, such as the condition 2 described above reduces collisions and cracks between droplets or particles, and it also has a positive effect on the shape of the particles (flatness or single-shaped particles).

4. Percentage of Single-Shaped Particles

[0134] The microcarrier particles obtained in Examples and Comparative Examples were prepared, scanning electron microscopy (SEM) images were photographed (using a magnification of x 250). A plurality of images, in which 30 or more particles were visually confirmed from the photographed image, were randomly selected, and the ratio of the number of single-shaped particles without satellite particles on the surface to the total number of the particles observed in each image was calculated.

[0135] The proportion of single-shaped particles being high is associated with less aggregation of particles due to particle collision during suspension polymerization. In particular, a viscosity of the condition 2 of the continuous phase 2 relates to the movement of the dispersive phase particles.

[0136] In the case of having a viscosity that satisfies the condition 2, the movement of the dispersive phase particles is reduced, particle aggregation and agglomeration due to particle collisions are reduced, and the proportion of single-shaped particles can be increased. At this time, when the condition 1 is satisfied, excessive decrease in particle size due to low surface tension and decrease in interfacial tension caused thereby is suppressed. Therefore, when the condition 1 and the condition 2 are satisfied, the proportion of single-shaped particles having an appropriate size can be increased.

5. Internal Structure of Particles

[0137] For the cell culture microcarrier obtained in Example 1, the internal structure of the particles was confirmed through SEM. Specifically, after embedding the particles into the epoxy, the cross section was prepared through ion milling, and the shape of the particle cross section was confirmed through SEM.

TABLE-US-00002 Example 1 Example 2 Example 3 Comparat ive Example 1 Comparat ive Example 2 Comparat ive Example 3 Comparat ive Example 4 Comparat ive Example 5 Particle recovery rate (%) 75.3 74.2 88.0 48.5 50.0 66.8 N.A. 94.8 Particle density (g/cm.sup.3) within the range of 0.95 -0.99 within the range of 0.95 -0.99 within the range of 0.95 -0.99 within the range of 0.95 -0.99 within the range of 0.95 -0.99 within the range of 0.95 -0.99 N.A. within the range of 0.95 -0.99 Particle size (.Math.m)(based on single particle shape) 130 ± 33 197 ± 28 182 ± 27 115 ± 16 114 ± 16 84 ± 16 1 mm or more less than 100 .Math.m, more than 300 .Math.m Proportion of single-shaped particles (%) 93.55 80.65 100.0 86.11 93.55 69.49 87.69 80.65

[0138] Comparing Comparative Examples 1 to 3 with Examples through Table 1 and Table 2, it can be seen that the particle recovery rate of Examples according to the present application is superior to that of Comparative Examples. This means that the method of the present application induces the formation and polymerization of a stable dispersed phase, and as a result, a large amount of carrier particles having low density and large surface area properties can be improved. (process efficiency or yield is improved).

[0139] In addition, comparing Comparative Examples 4 and 5 with Examples through Tables 1 and 2, it can be seen that the Examples according to the present application provide micro-sized particles with a narrow particle size distribution and an appropriate size as compared with Comparative Examples 4 and 5. Further, it is confirmed that in the case of Examples, the proportion of single-shaped particles (of appropriate size) is generally higher than in Comparative Examples 4 and 5. This means that the method of the present application can provide a large surface area suitable for cell culture.

[0140] Moreover, comparing Comparative Examples 4 and 5 with Examples, it can be seen that in the case of using PVP, the particle size of the particles prepared by suspension polymerization becomes excessively large or the particle size distribution becomes excessively wide. The weight of the particles is taken into account when calculating the recovery rate, and the recovery rate of Comparative Example 5 being high is because the weight of the large particles (size of more than 300 .Math.m) prepared in Comparative Example 5 is relatively large. On the other hand, the molecular weight of the PVP used in Comparative Example 4 is very small compared to the molecular weight of the PVP used in Comparative Example 5, whereby in Comparative Example 4, the steric effect between PVPs was weak, and aggregation between particles was excessively occurred, so that the size of the particles was very large, and it was difficult to confirm the individual particle density or the recovery rate.

[0141] In summary, the present application can provide porous carrier particles having a single shape, spherical shape, low density properties and excellent flatness with a high recovery rate.