KR20060048553A - Method of cell immobilization on a surface of a carrier - Google Patents

Abstract

The present invention relates to a method for immobilizing cells on the surface of an immobilized carrier which may contain an aqueous solution, wherein the cell or enzyme is brought into contact with the surface of the carrier by contacting an alginic acid solution containing the cells with an immobilized carrier moistened with a solution containing a polyvalent cation. Calcium-alginate gel comprising a method of forming a certain thickness. According to the method of the present invention, since the cell is applied to the surface of the carrier and gelation occurs, an even cell fixing layer is formed on the surface of the carrier, so that the fixing efficiency is excellent and the activity of the cell is maintained high even when the cell is fixed at a high concentration. The process can produce bioreactors with high efficiency and high cell activity.

Description

Method of immobilizing cells on the surface of the carrier {METHOD OF CELL IMMOBILIZATION ON A SURFACE OF A CARRIER}

FIG. 1 is a diagram illustrating the principle of forming calcium-alginate gel on the surface of an immobilized carrier containing a high concentration of calcium chloride solution in an alginic acid solution.

FIG. 2 is a micrograph of a gauze having a calcium-alginate gel formed on its surface, prepared by soaking the gauze in a 300 mM calcium solution and immersing it in a 1.5% alginic acid solution.

Figure 3 is a diagram illustrating the presence or absence of dead space in which cells are difficult to live in gauze surface fixation and gel bead immobilization.

Figure 4 shows the configuration of the hepatocyte fixation gauze that the surface is fixed with a hydrophobic silicon and the surface is immobilized only in a predetermined area.

5 is a photograph comparing the immobilization of mouse liver cells to the surface of the gauze and the immobilization in the form of gel beads.

6 is a schematic view showing the structure of a ceramic honeycomb for producing a bioreactor.

7 is a schematic cross-sectional view of a ceramic honeycomb having a calcium-alginate gel layer formed on its surface.

8 is a schematic cross-sectional view of the calcium-alginate gel layer formed on the inner surface of the hollow fiber membrane.

The present invention relates to a method of immobilizing cells or enzymes by forming an alginate gel on the surface of an immobilization carrier which may contain an aqueous solution.

Methods of immobilizing them as part of methods for exploiting biological activities such as animal cells, plant cells, microorganisms and enzymes have been developed (Birnbaum, S., et al., FEBS Letters , 122 , 393-404, 1981; And Brodelius, P., et al., FEBS Letters , 122 , 312-319, 1980). In general, synthetic and natural polymers such as alginic acid, chitosan, polyvinyl alcohol, collagen, carboxymethylcellulose, agar, agarose, gelatin and the like are widely used for such immobilization (Lambert, F., et al., BioChem. ., 759, 81-88, 1983).

As a method of using such a polymer, the method of immobilizing a cell in an alginic acid solution and dropping it in a divalent cation solution such as calcium chloride solution is usually immobilized in the form of a bead-like gel ("gel bead"). (D. Serp, et al., Biotechnology and Bioengineering , 70 (1), 41-53, 2000). In this method, various applications have been reported, such as increasing the strength and stability by varying the cation that acts as a crosslink or coating the surface of the alginate gel beads with a cationic polymer such as polylysine or chitosan (Lishan Wang, et al., Journal of Pharmaceutical Sciences , 90 (8), 1134-1142, 2001).

However, since the spherical beads formed in the immobilization of the gel bead form are generally 500 μm or more in diameter, cell necrosis often occurs due to depletion of oxygen or nutrients at the center of the gel bead (Schrezenmeir J). , et al., Transplantation , 57 (9), 1308-14, 1994). This problem can be solved to some extent by reducing the size of the gel beads, but it is difficult to manufacture gel beads having a diameter of 500 μm or less in general droplet formation methods, and a lot of cell damage occurs during the preparation of small size gel beads. Done.

To solve this problem, cell suspensions are mixed with porous inorganic carriers such as celite, ceramics, and activated carbon to infiltrate the cells into the pores of the carriers, which are immersed in organic polymers such as alginic acid, chitosan, and polyvinyl alcohol. Cell immobilization method (Korea Patent Publication No. 1999-021170), which forms a membrane on the surface and is then treated with a crosslinking solution again, or an alginate film using a plastic polymer having excellent moldability and mechanical properties instead of the inorganic carrier. (Korean Patent Publication No. 2003-0026943) and the like have been studied.

However, the above methods are difficult to form an even membrane on the surface of the carrier because the polymer solution treatment and gel formation process after the cell infiltration, the immobilization efficiency is inferior, and the activity and distribution of the cells are not constant, which may cause various problems. .

Accordingly, it is an object of the present invention to provide a cell immobilization method capable of maintaining cell activity evenly while fixing cells evenly on the surface of an immobilization carrier by a simple process.

In accordance with the above object, in the present invention 1) comprising a crosslinking solution in the immobilized carrier which may contain an aqueous solution; And 2) contacting the immobilized carrier containing the cross-linking solution with the immobilized polymer solution containing the cells in step 1 to form an immobilized gel layer containing the cells on the surface of the carrier. Provide a way to.

The term "cell" is used herein in a broad sense including animal cells, plant cells, microorganisms as well as various enzymes.

The following describes each step of each component and method used in the method of the present invention in more detail.

1. Immobilized Carrier

In the present invention, the immobilization carrier used for immobilization of the cell is characterized in that it is hydrophilic so as to contain an aqueous solution evenly, and various forms may be used depending on the type of the cell to be immobilized or the purpose of the reactor. Even in a carrier that does not contain an aqueous solution evenly on the surface, such as polyurethane foam, those given hydrophilicity through hydrophilic surface modification can be used as an immobilization carrier.

Examples of the immobilization carrier that can be used include various fibers such as gauze, hollow fiber membranes, ceramics, porous polymers, activated carbon, porous beads, and the like, but are not limited thereto. Any material capable of containing an aqueous solution may be used. In addition, immobilization carriers of various materials and shapes may be used depending on the intended use.

2. Crosslinking solution pretreatment

The immobilized carrier is immersed in an appropriate concentration of the crosslinking solution, or the crosslinked solution is sprayed on the immobilized carrier to contain the crosslinked solution.

As the crosslinking solution, an aqueous solution of a polyvalent cation (such as calcium chloride, barium chloride, aluminum chloride or strontium chloride) or a polyvalent anion (low molecular weight alginic acid, carboxymethyl cellulose or the like) can be used.

It is preferable to adjust the concentration of the crosslinking solution according to the capacity of the crosslinking solution containing the immobilized carrier, and the greater the amount of the crosslinking solution and the higher the concentration of the crosslinking solution, the thicker the gel is formed by the reaction with the immobilized polymer.

The concentration of the crosslinking solution may be varied depending on the type of cells desired and the capacity of the crosslinking solution containing the immobilized carrier. For example, when the target cells are hepatocytes, a medical gauze (area 4 cm 2) may be used as the immobilized carrier. A calcium chloride solution of 100 to 600 mM, preferably 300 mM, may be used, and a calcium chloride solution of 60 to 200 mM, preferably 100 mM, may be used when using ceramic honeycomb as an immobilization carrier.

3. Cell Immobilization

Immobilized carrier pretreated with a crosslinking solution is immersed in the immobilized polymer solution to which cells are added at a desired concentration. The crosslinking solution rapidly diffuses into the aqueous solution of the immobilized polymer and crosslinks the polymer to form a gel layer on the surface of the carrier.

The immobilized polymer which can be used in the present invention is, for example, an anionic polymer such as alginic acid or carboxymethyl cellulose when the crosslinking solution is an aqueous solution of a cation, and a cationic polymer such as chitosan when the crosslinking solution is an aqueous solution of anion. Do.

As the concentration of the immobilized polymer solution is higher, the thickness of the gel to be immobilized becomes thinner, so that the concentration of the immobilized polymer solution can be appropriately adjusted according to the desired gel thickness.

If there is information on the crosslinking solution content of the carrier, the average anionic or cationic valence of the immobilized polymer, the volume to be immobilized can be predicted. For example, in the case of 300 mM calcium solution and 1.5% alginic acid (Sigma Chem. Co.), It forms a calcium-alginic acid gel that is about 5 times the volume of the crosslinking solution.

Immobilized gel formed by the method of the present invention can be adjusted by adjusting the concentration of the cross-linking solution and the immobilized polymer solution according to the type of cells, 20 to 1,000 ㎛ for microorganisms, 20 to 300 for flora and fauna cells In the case of micrometer and enzyme, it can adjust arbitrarily in the range of about 20-100 micrometers. For example, when immobilizing human hepatocytes, single hepatocytes can form a gel with a thickness of 20-300 μm, in the case of hepatocyte spheroids, 100-500 μm, preferably 150-300 μm.

4. Post-treatment

In order to further increase the strength of the immobilized gel formed by the method of the present invention, a process of post-treatment of the immobilized gel with a solution of calcium chloride, polylysine or chitosan may be further included in the method of the present invention. At this time, the concentration of the solution can be adjusted in the range of 30 to 100 mM for calcium, 0.5 to 2% for polylysine or chitosan.

By using the method of the present invention, the immobilized carrier moistened with the crosslinking solution is placed inside the perfusion reactor, and then the immobilized polymer solution containing the cells is injected into the reactor to form a cell immobilized gel, which can be immediately used as a bioreactor. For example, when the immobilized cells are hepatocytes, a bioreactor having the immobilized cells can be used as a bioreactor for liver function research or a hepatocyte reactor of an extracorporeal circulating liver assist device.

In the cell immobilization method according to the present invention, since the cell is coated on the surface of the carrier and gelation occurs, a uniform cell fixation layer is formed on the surface of the carrier to provide a high level of fixation efficiency and high cell activity even when the cell is immobilized at a high concentration. Since it is kept high, bioreactors with high efficiency and high cell activity can be manufactured by a simple process.

The following examples are merely illustrative of the present invention, but the content of the present invention is not limited to these examples.

Example 1 Alginic Acid Immobilization Thickness

The medical gauze as the immobilization carrier was soaked in the 300 mM calcium chloride solution until the crosslinking solution was contained in the gauze carrier. The carrier containing the crosslinking solution was immersed in an aqueous 1.5% alginic acid solution for 10 seconds to form a gel, and then washed with phosphate buffer to obtain a gauze having an immobilized gel on the surface. The experiment was carried out using six gauze samples, and the crosslinking rate was calculated by measuring the mass of the gauze, the calcium chloride solution content of the gauze, and the crosslinked alginate mass at each step of the immobilization process, and the results are shown in Table 1.

As can be seen in Table 1, the gauze contained an average of 15.3 mg of calcium chloride solution, and an average of 74.7 mg of alginate was immobilized.

Figure 2 is a micrograph of the calcium-alginic acid gel formed on the surface of the gauze in the same manner as described above. The average thickness of the yarn gauze is 166 μm, and the length of the yarn constituting the gauze having an area of 4 cm 2 is 80 cm. This should be formed, as shown in FIG.

In the case of alginate spherical beads, when the concentration of immobilized cells is high or the size of the beads is large, nutrients or oxygen are depleted in the center, and the cells cannot survive, resulting in a dead space. However, in the case of surface immobilization using gauze, as shown in FIG. 2, all immobilized cells are present within 110 μm of the immobilization surface, and as shown in FIG. Necrosis of cells rarely occurs. Such conditions are the same as those of beads having a diameter of 220 µm in the case of bead-type gel. However, since actual alginate gel beads are difficult to manufacture with a diameter of 500 μm or less, surface immobilization with gauze is particularly useful when high concentrations of cells must be immobilized, for example, in hepatocyte reactors for liver aids.

Example 2: Rat Primary Hepatocyte Immobilization

The effects of alginate gauze surface immobilization and general alginate gel bead immobilization on hepatocyte viability under high concentration hepatocyte immobilization were investigated as follows.

Figure 4 is a device designed to be immobilized only in a predetermined area in order to precisely control the amount of alginic acid to be immobilized, the same amount because the edge of the gauze is finished with a very hydrophobic silicon material so that the Ca ²⁺ solution only bury the gauze It is possible to compare with alginate gel beads.

EDTA solution (NaCl 8 g / L, KCl 0.4 g / L, NaH ₂ PO ₄ 2H ₂ O 0.078 g / L, Na ₂ HPO ₄ 12H ₂ O 0.151 g / L) HEPES (4- (2-hydroxyethyl) -1-piperazineethane-sulfonic acid, Sigma Chem Co.) 2.38 g / l, EDTA (ethylenediaminetetraacetic acid, Gibco BRL Co.) 0.19 g / l, bicarbonate Sodium 0.35 g / l, glucose 0.9 g / l, penicillin 100 units / ml, streptomycin 10 mg / ml, amphotericin B 25 μg / ml) and collagen degrading enzyme solution (Gibco BRL) 0.5 g / l, trypsin inhibitor (Gibco BRL) 0.05 g / l, NaCl 8 g / l, KCl 0.4 g / l, CaCl ₂ 0.56 g / l, NaH ₂ PO ₄ 2H ₂ O 0.078 g / l, Na ₂ HPO ₄ 12H ₂ O 0.151 g / l, HEPES 2.381 g / l, sodium bicarbonate 0.35 g / l, penicillin 100 units / ml, streptomycin 10 mg / ml, amphotericin B 25 μg / ml) Hepatocytes were isolated and added to the alginic acid solution at a concentration of 4 × 10 ⁶ or 4 × 10 ⁷ cells / ml gel. All. The gauze surface immobilization was performed by soaking the silicone finishing gauze (approximately 1.46 mm in diameter of the immobilization site) of FIG. 4 in 300 mM calcium chloride solution and wiping with dry gauze so that only the gauze fiber was buried with calcium solution. Soak for about 10 seconds to allow the cells to be immobilized on the surface of the gauze. The volume of alginic acid immobilized on one silicone gauze was about 90 μl. Gauzes immobilized with hepatocytes were washed with Williams media (Sigma Chem. Co).

In the case of bead immobilization, the hepatocellular alginic acid solution of these two concentrations was dropped in a stirred 100 mM calcium chloride solution through a 24G needle. Beads about 2 mm in diameter were formed and washed as in the case of gauze immobilization. 5 is a photograph of immobilized hepatocytes by a gauze surface immobilization method and a gel bead immobilization method, respectively.

The gauze and beads immobilized as described above were put in one gauze and 15 beads in each well of a 6-well plate, and supplemented with epidermal growth factor (20 μg / L) and insulin (10 mg / L). Williams culture medium was added 3 ml per well and incubated in 37 ℃, 5% CO ₂ /95% air CO ₂ incubator.

Two days after the culture, cell viability was examined using the MTT assay. After removing the medium, 2 ml of MTT (3- [4,5-dimethylthiazole-2-yl] -2,5-diphenyl-tetrazolium bromide, Sigma Co.) 0.03% (w / v) solution per well The reaction was carried out at 37 ° C. for 4 hours. Then, after removing the MTT solution, colorimetric components were extracted for 2 hours at room temperature with isopropyl alcohol, and absorbance was measured with a UV-VIS spectrophotometer (Smart plus 2605, Young Hwa Co., Seoul, Korea) at 570 nm wavelength. Cell viability was measured.

Table 2 shows the viability of surface immobilized and gel bead immobilized hepatocytes when immobilized at low and high cell concentrations.

As can be seen in Table 2, the viability of gel beads at high concentrations dropped sharply after 2 days of culture, while the gauze surface immobilization showed relatively much less survival.

Example 3: Immobilization of Ceramic Honeycomb Hepatocyte Reactors

Fig. 6 is a cylindrical honeycomb for gas catalysts, in which a square channel having a diameter of 2 mm is divided into a ceramic membrane having a thickness of 0.5 mm and arranged in the longitudinal direction. Since the ceramic membrane had a crosslinking solution content of about three times higher than that of gauze, the ceramic honeycomb was soaked in a calcium chloride solution having a concentration of 100 mM and then fixed in a perfusion reactor having the same diameter. 15 ml of alginic acid solution containing a high concentration of hepatocytes was slowly injected into the lower inlet of the reactor, followed immediately by 15 ml of cell-free alginic acid solution. By perfusion with phosphate buffer solution, a calcium-alginate gel layer with a thickness of 200 μm was formed on the inner surface of the ceramic rectangular channel as shown in FIG. 7. This ceramic honeycomb reactor can be used as a bioreactor for liver function research or as a hepatocyte reactor for in vitro circulatory liver auxiliaries.

Example 4 Hollow Fiber Membrane Hepatocellular Reactor Immobilization

FIG. 8 is a schematic diagram showing a calcium-alginate gel layer formed by injecting an alginate solution containing cells into the hollow fiber membrane as in Example 3 after wetting the hydrophilic hollow fiber membrane with a 200 mM calcium solution. Thus, in the form in which the cell immobilization layer is formed inside the hollow fiber membrane, the channel flows out of the hollow fiber membrane and the channel is formed inside the gel layer formed inside the hollow fiber membrane. This type is particularly useful when a plasma channel and an oxygen or nutrient supply channel are required at the same time, such as a hepatocyte bioreactor of a liver assistive device. Conversely, when the alginic acid solution is injected out of the hollow fiber membrane, a calcium-alginate gel layer is formed on the outer surface of the hollow fiber membrane.

As described above, using the cell immobilization method of the present invention can simultaneously perform the process of applying and gelling the cell solution on the surface and the oxygen and nutrient delivery environment for the immobilized cells is very excellent, the activity of the immobilized cells Since this is kept high, the carrier to which cells are fixed by the method of the present invention can be applied to a cell immobilization reactor for various uses.

Claims (7)

1) incorporating the crosslinking solution into an immobilized carrier which may contain an aqueous solution; And 2) immobilizing the cells on the surface of the immobilized carrier, comprising contacting the immobilized carrier containing the crosslinking solution with the immobilized polymer solution including the cells in step 1 to form an immobilized gel layer containing the cells on the surface of the carrier. Way. The method of claim 1, Wherein the immobilized carrier is selected from fibers, hollow fiber membranes, ceramics, porous polymers, activated carbon, and porous beads, which may contain aqueous solutions. The method of claim 1, The crosslinking solution is an aqueous solution of a polyvalent cation selected from calcium chloride, barium chloride, aluminum chloride and strontium chloride, or an aqueous solution of a polyvalent anion selected from low molecular weight alginic acid and carboxymethylcellulose. The method of claim 1, Wherein the immobilized polymer is selected from alginic acid, carboxymethylcellulose and chitosan. The method of claim 1, The cell is selected from animal cells, plant cells, microorganisms and enzymes. The method of claim 1, If the cells to be immobilized are animal cells, the thickness of the immobilized gel layer is characterized in that 20 to 500 ㎛. The method of claim 1, In step 2, the contacting of the immobilized carrier with the immobilized polymer solution is carried out in a perfusion reactor.

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