KR20140056525A - Manufacturing method of strong antigen-presenting cells using hydrogel coating and electroporation - Google Patents

Abstract

The present invention relates to a method for producing a strong antigen-presenting cells using hydrogel coating and electroporation. More specifically, peripheral blood mononuclear cells (PBMC) are isolated from leukocytes in blood and monocytes are isolated in order to be differentiated into immature dendritic cells. The immature dendritic cells that contain antigens by using electroporation are fed and cultured in a culturing container in which hydrogel is coated so that the immature dendritic cells are differentiated into mature dendritic cells. According to the present invention, adhesion and spreading which are the problems of existing dendritic cell culturing methods are prevented so that changes in form of the dendritic cells are prevented and the yield of dendritic cells having a function as a strong antigen-presenting cell can be improved.

Description

Technical Field [0001] The present invention relates to a method of preparing a strong antigen-presenting cell using hydrogel coating and electroporation,

More particularly, the present invention relates to a method of preparing a strong antigen-presenting cell by hydrogel coating and electroporation, more specifically, separating peripheral blood mononuclear cells (PBMC) from white blood cells of blood, separating monocytes, Immature dendritic cells are differentiated. And then electroporating the immature dendritic cells with the antigen into a culture container coated with hydrogel to differentiate into mature dendritic cells. The mature dendritic cells, which are antigen-presenting cells, ≪ / RTI >

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) that bridge the innate and adaptive immune systems. Dendritic cells were first found in skin by Langerhans in 1868, and in 1973 Cohn and Steinmann became known as immune-assisted cells. In the 1990 's, it became known that it functions as a powerful antigen presenting cell (APC), and it plays an important role in initiating and inducing adaptive immune response. The dendritic cells in the human body are composed of heterogeneous populations with phenotypes distinct from macrophages, but only about 0.3% of total circulating white blood cells. Dendritic cells differ from B cells or macrophages with relatively weak antigen presentation ability in that they are powerful antigen presenting cells. Dendritic cells are known to be derived from a variety of lineages, but usually dendritic cells are derived from single cells or monocyte precursors (CD34 + cells) stimulated with IL-4 and GM-CSF and used in the vaccine protocol. When dendritic cells digest an antigen, MHC molecule expression is increased and matured with increased expression of co-stimulating molecules such as CD80 / CD86. Mature dendritic cells transfer antigenic information to CD4 + and CD8 + naive T lymphocytes, which can migrate to secondary lymphoid organs and induce antigen-specific immune responses (Banchereau et al., 1982; Banchereau et al., 2005; al., 2000). Dendritic cells are known to secrete cytokines such as IFN-y and IL-12 to activate a variety of immune cells. In particular, pro-inflammatory cytokine IL-12 produced by mature dendritic cells plays a pivotal role in inducing Th1 immune response by stimulating CD8 + T cells (Trinchieri et al., 2003). In addition to inducing immunological tolerance, dendritic cells play an important role in stimulating immune responses (Kapsenberg et al., 2003; Kalinski et al., 1999; Langenkamp et al., 2000) (Steinman et al., 2003; Zou et al., 2006).

When immature dendritic cells are phagocytosed in the presence of stimuli such as danger signals or inflammatory stimuli derived from pathogen associated molecular patterns (PAMPSs), they are differentiated into mature dendritic cells that induce antigen-specific immune responses (Gilboa et al., 2007; Matzinger et al., 2002). In contrast, when immature dendritic cells are phagocytosed under conditions that inhibit the maturation process, they express cell surface molecules that interfere with the adaptive immune response (Morelli et al., 2007; van Kooten et al., 2011).

Dendritic cell maturation is closely related to increased expression of CD40, CD58, CD80, CD86, CD83 and LAMP3 (lysosomal-associated membrane protein 3). Commonly stimulated molecules such as CD80 and CD86 play an important role in the activation of T cells (Banchereau et al., 1998; Gilboa et al., 2007; Lanzavecchia et al., 2001; O'Neill et al., 2004). In particular, CD83 is a marker of mature dendritic cells that is increased by maturation stimuli and stimulates T cells (Lechmann et al., 2002). In order to differentiate into mature dendritic cells, various biological agents such as cytokine cocktail (TNF, IL-6, IL-1β, and PGE2), CD40L, TNF-a, IFN-a, IFN- ) Have been used (Gilboa et al., 2007; Jonuleit et al., 1997).

Dendritic cells are present in small quantities in the blood and are difficult to separate because they are difficult to isolate. However, recently, a method of differentiating into dendritic cells using blood cells or stem cells in vitro has been developed, Clinical studies have been actively conducted. Recently, several studies have been conducted to increase the function and yield of dendritic cells, which are powerful antigen presenting cells. In previous studies, various tumor antigen pulsing protocols such as lipofection, passive pulse, or electroporation have been developed and used to derive functionally superior dendritic cells (O'Neill et al., 2004; van Tendeloo et al., 2001; Brossart et al., 2001; Kim et al., 2004). In particular, electroporation has shown that antigen can be loaded onto dendritic cells much more efficiently than other tumor antigen-based methods (Van Tendeloo et al., 2001; Kim et al., 2004). However, this method has obtained functional dendritic cells, but like other methods, there is a problem that the yield of dendritic cells to be obtained due to attachment and spreading of cells during the culture period is not high. When the cells are attached to the culture vessel during the culturing process and the morphology thereof is changed or spreading is not properly performed, it is difficult to obtain dendritic cells having such functions as powerful antigen presenting cells.

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art. As a result, they have found that when dendritic cells are cultured by combining a hydrogel coating culture method and an electroporation method, adhesion and spreading are prevented Thereby confirming the improvement of the function and yield of dendritic cells, thereby completing the present invention.

Accordingly, it is a main object of the present invention to provide a method of preparing a strong antigen-presenting cell using a hydrogel coating and an electroporation method, which have a dendritic cell function and yield improving effect by culturing dendritic cells by combining with a hydrogel coating culture method and an electroporation method And a manufacturing method thereof.

According to one aspect of the present invention, there is provided a method for producing dendritic cells, antigen-presenting cells, comprising the steps of:

a) separating peripheral blood mononuclear cells (PBMC) from white blood cells of blood;

b) isolating monocytes from said peripheral blood mononuclear cells;

c) culturing said single cells to differentiate into immature dendritic cells;

d) loading the immature dendritic cells with the antigen by electroporation; And

e) culturing the immature dendritic cells loaded with the antigen in a culture container coated with hydrogel to differentiate the mature dendritic cells into mature dendritic cells. do.

The leukocyte of step a) is diluted with PBS (or physiological saline) to obtain a leukocyte concentrate obtained through triple bag or apheresis.

The present invention provides a method for producing mature dendritic cells, which are antigen-presenting cells, characterized in that the separation of peripheral blood mononuclear cells in step a) is performed using centrifugation.

In the present invention, the separation of single cells in step b) may be performed using a plastic-adherent method, a MACS MicroBeads method, or a density gradient method. To provide a method for producing mature dendritic cells.

In accordance with the present invention, there is provided a method for producing mature dendritic cells which are antigen presenting cells, characterized in that the culture in step c) is cultured in an animal serum medium supplemented with rhIL-4 and rhGM-CSF.

In accordance with the present invention, there is provided a method for producing mature dendritic cells, which is an antigen-presenting cell, characterized in that an electroporation method of step d) comprises mixing immature dendritic cells with an antigen and then using a square wave pulse do.

In addition to the square wave pulse, there is an exponential pulse method. A square wave pulse is a method that can maintain the same voltage for a certain time, and an exponential pulse is a method of applying a shock only at a predetermined voltage.

Electroporation is a method of gene transfer using DNA that is introduced into a cell when a cell is suspended in a DNA solution and a direct current high voltage pulse is passed through it. A hole is formed in the cell membrane by electricity. At the same time, . It can be applied to various cell types (animals), plants and microorganisms, and it is widely used because it enables gene introduction with a considerably high efficiency when a proper condition is selected. It is also used for protein introduction.

In the present invention, immature dendritic cells and antigens are mixed with immature dendritic cells using the electroporation method, followed by electroporation using a square wave pulse waveform.

The present invention provides a method for producing mature dendritic cells which are antigen-presenting cells, characterized in that the hydrogel coating in step e) uses PolyHEMA (poly (2-hydroxyethyl methacrylate)) hydrogel coating material.

The hydrogel component polyHEMA used in the present invention is a polymer named poly (2-hydroxyethyl methacrylate), which was developed by Wichterle in the 1960s. PolyHEMA is a material with a stable three-dimensional structure that absorbs water and expands, is soft and deformable, has water permeability and low surface tension. PolyHEMA is suitable as a coating material for cell culture because it is relatively inexpensive to produce, easy to bind to other monomers, very flexible, and stable to pH and temperature changes. PolyHEMA hydrogels, also known as porous materials, can stimulate cell proliferation and synthesize extracellular matrix components. PolyHEMA hydrogels are safe biomaterials that are used clinically in contact lenses, drug delivery, dental and orthopedic implants.

In accordance with the present invention, there is provided a method for producing mature dendritic cells, which is an antigen-presenting cell, characterized in that the culture in step (e) is cultured by adding a maturation inducing factor.

In the present invention, any inducer capable of matured non-aqueous cells into mature dendritic cells can be used as the maturation-inducing factor. For example, CD40L, TNF-a, IFN-a, IFN- The present invention provides a method for producing mature dendritic cells which are antigen presenting cells, characterized in that they are TNF- ?, IL-1 ?, IL-6 and PGE2.

The present invention provides a method for preparing mature dendritic cells, which are antigen-presenting cells, characterized in that the hydrogel coating improves the yield of mature dendritic cells compared to a hydrogel-free coating.

As shown in Fig. 1 for the improvement of yield of mature dendritic cells according to presence or absence of PolyHEMA hydrogel coating, it was found that the induction of mature dendritic cell differentiation by culturing in a culture container coated with hydrogel It is shown that the production of mature dendritic cells is significantly improved as compared with the case where mature dendritic cell differentiation is induced by culturing in a culture container. These results suggest that mature dendritic cells can be obtained at a high yield by culturing the attachment and spreading problems observed during dendritic cell culture in polyHEMA hydrogel coated culture vessels and that polyHEMA is suitable as a coating material for cell culture .

In addition, mature dendritic cell differentiation, which was cultured in the hydrogel-coated culture vessel without electroporation, was slightly lower than mature dendritic cell cultures grown using electroporation, suggesting that electroporation This is because the cell number tends to slightly decrease, with some damage.

The present invention provides a method for preparing mature dendritic cells, which are antigen-presenting cells, characterized in that the hydrogel coating improves the expression of the CD83 surface antigen, which is a maturation marker of dendritic cells, as compared with the hydrogel coating .

As shown in the results of surface phenotype of mature dendritic cells induced by presence or absence of PolyHEMA hydrogel coating, the co-stimulatory molecules CD80 and CD86 did not differ in all culture conditions. However, CD83, a dendritic cell maturation marker, was found to be significantly increased in the ployHEMA hydrogel coated condition than in the ployHEMA hydrogel coated condition. These results indicate that mature dendritic cells with increased T cell stimulating function can be obtained.

According to the present invention, the hydrogel coating is characterized in that the mature dendritic cells are longer than the hydrogel-coated ones, and the cells exhibit a round-shaped cell shape with dendritic dendrites. ≪ / RTI >

 As shown in the results of surface morphology of mature dendritic cells induced by polyHEMA hydrogel coating (Fig. 3), mature dendritic cells induced to differentiation under conditions coated with PolyHEMA hydrogel were not coated It can be seen that the morphology of rounded cells with dendrites is longer and stronger than mature dendritic cells induced by differentiation in culture conditions. This means that the cells are adhered to the culture container during the culturing process, so that the morphology thereof can be changed little and the differentiation can be properly performed, which means that dendritic cells having the function as powerful antigen presenting cells can be obtained.

In the present invention, the hydrogel coating improves the production of cytokines (IL-12 and IFN-r) showing the function of dendritic cells as opposed to hydrogel-coated matured dendritic cells Lt; / RTI > cells.

Results of cytokine production of mature dendritic cells induced by presence or absence of PolyHEMA hydrogel coating (Fig. 4) showed that IL-12 and IFN-r of mature dendritic cells induced in PolyHEMA hydrogel coated culture conditions Produced significantly higher amounts of IL-12 and IFN-r than mature dendritic cells induced in culture conditions without PolyHEMA hydrogel coating. This means that functionally superior mature dendritic cells can be obtained, which can increase immuno-inducing cytokines such as IL-12 and IFN-r.

In the present invention, the electroporation improves the expression of CD83 surface antigen, which is a maturation marker for dendritic cells, and suppresses the expression of cytokines (IL-12 and IFN-r) The present invention provides a method for producing mature dendritic cells which are antigen-presenting cells.

As shown in the results of surface phenotype of the induced mature dendritic cells when PolyHEMA hydrogel coating and electroporation were used (Fig. 2), the culture conditions using the electroporation method were not used It can be seen that it has increased significantly. In addition, as shown in the results of cytokine production of induced mature dendritic cells when PolyHEMA hydrogel coating and electroporation were used (Fig. 4), IL- 12 and IFN-r production, respectively.

In the present invention, there is provided a cancer immune cell therapeutic composition comprising mature dendritic cells differentiated by the above-mentioned method for producing mature dendritic cells as an active ingredient.

It is well known that the immune system is capable of recognizing and attacking tumors. Both humoral and cellular immunity are involved in tumor dissolution, but in most cases tumor regression through specific recognition of tumor antigens involves cellular immunity. In particular, it has been demonstrated in several models that CD8 + cytotoxic T lymphocytes (CTLs) can recognize and kill tumor cells (1. Boon, T., JC Cerottini, B. Van den Eynde, P. van der Bruggen, and A. Van 1996. Peptide-pulsed dendritic cells induce antigen-specific (T-cell) antigens identified by T lymphocytes. Annu Rev Immunol 12: 337. 2. Celluzzi, CM, JI Mayordomo, WJ Storkus, MT Lotze, and LD Falo, The CTL-mediated protective tumor immunity, J Exp Med 183, 1: 283. 3. Feltkamp, MC, HLSmits, MP Vierboom, RP Minnaar, BM de Jongh, JW Drijfhout, J. ter Schegget, CJ Melief, and WM Kast. 1993. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 23, 9: 2242.3).

Dendritic cells are the only antigen-presenting cells capable of inducing an immune response and have been widely used for antigen-specific immunotherapy of cancer and infectious diseases (Banchereau, J., F. Briere, C. Caux, J. Davout, S. Lebecque, YJ Liu, B. Pulendran, and K. Palucka 2000. Immunobiology of dendritic cells. Annu Rev Immunol 18: 767.). The ability of T cells to initiate dendritic cells to recognize and kill tumor cells in a tumor-specific manner has been reported in several animal models. Immunization based on dendritic cells also induces immunological memory, which can prevent further tumor development. Dendritic cells differ from B cells or macrophages with relatively weak antigen presentation ability in that they are powerful antigen presenting cells.

Recently, it has been demonstrated that dendritic cells are able to present exogenous antigens through MHC I molecules by proteasome and TAP - dependent mechanisms. Thus, dendritic cells can stimulate both T cells and B cells through a special process for external antigens. Because of these characteristics of dendritic cells, they are now widely used for cancer immunotherapy.

This means that a cancer immune cell therapeutic agent containing mature dendritic cells prepared by the above-described method as an active ingredient can be effective in cancer immune cell therapy.

The cancer immunotherapeutic agent of the present invention can be produced in the form of a formulation common in the art, for example, an injection, surgically implanted directly into a tumor site, or moved to a tumor site after intravenous administration. The cell immunotherapeutic agent of the present invention may be varied depending on the type of disease, the administration route, the age and sex of the patient, and the degree of the disease, but preferably 1 × 10 ⁶ to 10 ¹¹ cells are administered to the average adult.

This result implies that dendritic cells having excellent function can be obtained with high yield by combining the electroporation method and the hydrogel coating culture method, which are used to induce functionally excellent dendritic cells.

By combining a hydrogel-coated culture method that prevents attachment and spreading of cells in a culture vessel and an electroporation method in which dendritic cells are efficiently loaded with an antigen, a high yield and function of mature dendritic cells having a function as a strong antigen- Can be improved.

Figure 1 shows comparative cell numbers of mature dendritic cells derived from culture conditions using PolyHEMA hydrogel coated culture conditions, uncoated culture conditions and electroporation.
Figure 2 shows the surface antigens expression values of mature dendritic cells derived from culture conditions using PolyHEMA hydrogel coated culture conditions, uncoated culture conditions and electroporation.
Figure 3 shows the property comparison of mature dendritic cells induced in PolyHEMA hydrogel coated culture conditions and uncoated culture conditions.
Figure 4 shows IL-12 (figure above) and IFN-r (figure below) yield measurements by mature dendritic cells derived from culture conditions using PolyHEMA hydrogel coated culture conditions, uncoated culture conditions and electroporation .

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Experimental Example  One: Peripheral blood mononuclear cells  ( PBMC , peripheral blood mononuclear cell ) detach

The leukocyte buffy coat obtained through triple bag or apheresis was diluted with PBS (or physiological saline) and placed on a density gradient solution of Histopaque-1077. The solution was then incubated at 400 × g for 30 minutes at room temperature To obtain peripheral blood mononuclear cells (PBMC).

Experimental Example  2: Peripheral blood mononuclear cells  ( PBMC ) To single cell ( monocyte ) detach

1) Plastic-adherent method

Separated peripheral blood mononuclear cells were washed 2 to 3 times with PBS, suspended in animal-free serum medium, transferred to a culture container, and cultured at 37 ° C and 5% CO ₂ for 60 to 90 minutes. Unattached cells were carefully removed by pipetting with PBS and single cells adhered to the culture vessel were obtained.

2) MACS MicroBeads method

Peripheral blood mononuclear cells were reacted with anti-body antibodies attached to magnetic microbeads such as CD14, CD3, CD19, etc. to obtain high-purity single cells through the column.

3) Density Gradient

The osmotic pressure and density of Histopaque-1077, a dense gradient solution, were adjusted to 335 mOsm and 1.072 g / ml for the preparation of single cell separation solution. (Histopaque-1077 9.35 ml + PBS 0.65 ml + 5 M NaCl 50 ul). 5 ml of this solution was added to peripheral blood mononuclear cells, mixed well and centrifuged at 600 Xg for 20 minutes at room temperature. The isolated single cell layer was collected to obtain single cell of high purity.

Experimental Example  3: Hydrogel  coating ( Hydrogel coating )

Prior to hydrogel coating, the culture vessel is washed 1-3 times with ethanol. PolyHEMA (hydroglycidyl methacrylate) hydrogel coating material is made to a final concentration of 0.5% and is sterilized by adding it to the culture container washed with ethanol to cover the surface. The dried coating culture container is washed with PBS (or physiological saline) 2-3 times.

Experimental Example  4: Dendritic cell ( Dendritic cells ) Culture

(1), (2), and (3) of Example 2 (culture container attachment method, MACS method, density gradient method) to mount the antigen on immature dendritic cells using electroporation Were cultured in an animal serum medium supplemented with rhIL-4 (1000 U / ml) and rhGM-CSF (1000 U / ml) (R & D System) at 37 ° C and 5% CO ₂ . On the third day of culture, fresh medium containing the same amount of rhIL-4 (1000 U / ml) and rhGM-CSF (1000 U / ml) was added. On day 6 of culture, the differentiated immature dendritic cells were carefully pipetted and collected from the culture vessel. For pulsing, immature dendritic cells (1 × 10 ⁶ cells / ml) were transferred to a culture vessel coated with polyHEMA hydrogel and uncoated culture, and antigen (100 μg / ml) extracted from K562 cells X 10 < ⁶ > cells). Then, maturation inducing factors (10 ng / ml of TNF-α, 10 ng / ml of IL-1β, 1000 U / ml of IL-6 and 1 μg / ml of PGE2) were added and further cultured for 24 hours to differentiate into mature dendritic cells.

Immunoreactive dendritic cells (5 × 10 ⁶ cells / cuvette) and antigens extracted from K562 cells (100 μg / cuvette) were added to the cuvette first by electroporation (+) to mount the antigen on immature dendritic cells. 1 × 10 ⁶ cells) are mixed and incubated at low temperature in ice for 10 minutes. Electric perforation uses a square wave pulse method. Immediately after electroporation, the cuvette is placed in ice and incubated at low temperature for 10 minutes. Immature dendritic cells that underwent electroporation were transferred to a culture vessel coated with polyHEMA hydrogel and an uncoated culture vessel and cultured in the presence of matured inducer (TNF-α 10 ng / ml, IL-1β 10 ng / ml, IL- 6 1000 U / ml and PGE2 1 μg / ml) were further added for 24 hours to differentiate into mature dendritic cells.

Experimental Example  5: Mature dendritic cells  Manufacturing yield measurement

To determine if the PolyHEMA hydrogel coating affects the production of mature dendritic cells, immature dendritic cells were induced to differentiate into mature dendritic cells for 24 hours in a culture vessel coated with polyHEMA hydrogel and in an uncoated culture vessel. PolyHEMA hydrogel coated culture conditions [Electroporation (-); (A) 3.64 ± 0.38 × 10 ⁶ cells, (B) 3.80 ± 0.39 × 10 ⁶ cells, (C) 3.98 ± 0.36 × 10 ⁶ cells, Electroporation (+); (A) 3.46 ± 0.39 x 10 6 cells, (B) 3.54 ± 0.34 x 10 6 cells, (C) 3.56 ± 0.37 x 10 6 cells] and uncoated culture conditions [(A) 2.62 ± 0.46 x 10 6 cells , (B) 2.74 ± 0.45 × 10 ⁶ cells, (C) 2.98 ± 0.54 × 10 ⁶ cells].

Single cells were isolated from peripheral blood mononuclear cells (PBMC) through culture vessel attachment method, MACS method and density gradient method, respectively. Pulsing or electroporation was used for antigen immobilization in immature dendritic cells.

As a result, as shown in Fig. 1, the production of mature dendritic cells was remarkably improved in a culture condition in which a polyHEMA hydrogel was coated [(A) hydrogel-coated. non-coated, P &lt;0.05; (B) hydrogel-coated vs. non-coated, P &lt;0.05; (C) hydrogel-coated. non-coated, P <0.05].

Experimental Example  6: Dendritic cell  Properties and Surface antigen  analysis

To confirm the characteristics of dendritic cells, the morphology of dendritic cells was observed with an inverted microscope at a ratio of 400 times.

As shown in FIG. 3, the mature dendritic cells derived from the uncoated culture condition (A) have a large number of cells attached to the surface of the culture container and an oval shape with few dendrites. There are various types of cells. However, mature dendritic cells derived from polyHEMA hydrogel coated culture conditions (B) are characterized by large, rounded morphologies with many dendrites. There was no significant difference in dendritic cell morphology between electroporation (-) and electroporation (+) under polyHEMA hydrogel coating.

 For surface antigen analysis, monoclonal antibodies such as FITC-, PE- or PC5- conjugated isotype control, anti-CD80, anti-CD83 and anti-CD86 (Beckman Coulter) were added and incubated at 4 ° C for 20 minutes. Lt; / RTI &gt;

Surface antigen analysis revealed that CD83, a maturation marker for dendritic cells, was detected in polyHEMA coating culture conditions [Electroporation (-); (A) 55.08 7.48%, (B) 63.78 6.82%, (C) 60.58 4.66%, Electroporation (+); (A) 72.14 ± 5.53%, (B) 74.02 ± 3.71%, (C) 69.44 ± 6.90%) and uncoated culture conditions (A) 40.14 ± 3.91%, (B) 43.30 ± 5.34% 42.22 ± 4.28%).

As a result of the surface antigen analysis, as shown in FIG. 2, the degrees of expression of the co-stimulatory molecules CD80 and CD86 were not statistically different in the polyHEMA hydrogel coating culture conditions (electroporation; - / +) and uncoated culture conditions (P > 0.05). However, expression of CD83, a dendritic cell maturation marker, was significantly increased in polyHEMA hydrogel coated culture conditions (electroporation; - / +). (A) hydrogel-coated vs. non-coated, P &lt;0.05; (B) hydrogel-coated vs. non-coated, P &lt;0.05; (C) hydrogel-coated. non-coated, P < 0.05.

Experimental Example  7: Dendritic  Cytokine Secretory function

To determine the secretion of IL-12 from mature dendritic cells, cultures of mature dendritic cells cultured in culture media coated with polyHEMA hydrogel and uncoated culture media were collected and quantitatively analyzed by ELISA. In order to confirm IFN-r releasing ability of T lymphocytes activated by dendritic cells, each mature dendritic cell (5 × 10 ⁵ cells / well) cultured in polyHEMA hydrogel-coated culture vessel and uncoated culture vessel ) And allogeneic lymphocytes (1 × 10 ⁶ cells) were cultured for 3 days, and then cultured and quantitated by ELISA.

As a result, IL-12 production [Electroporation (-)] of mature dendritic cells induced differentiation in PolyHEMA hydrogel coated culture conditions (electroporation; - / +) (a) 824.6 ± 76.7 pg / ml, (b) 894.4 ± 91.3 pg / ml, (c) 869.6 ± 71.3 pg / ml, Electroporation (+); (a) 959.0 ± 65.8 pg / ml, (b) 1016.2 ± 72.0 pg / ml, (c) 972.4 ± 102.9 pg / ml] (P <0.05, fig. 4A), compared to the control group (p <0.05). In addition, mature dendritic cells induced by PolyHEMA hydrogel coated culture conditions [Electroporation (-); (a) 1001.6 ± 163.5 pg / ml, (b) 1316.4 ± 159.9 pg / ml, (c) 1179.8 ± 107.9 pg / ml, Electroporation (+); (a) 728 ± 130.7 pg (a) of mature dendritic cells induced in culture conditions without coating of (a) 1282.2 ± 49.5 pg / ml, (b) 1480.4 ± 59.7 pg / (p <0.05, Fig. 4B), compared to the control (b) (864.6 ± 66.6 pg / ml and 912.8 ± 56.7 pg / ml, respectively). In particular, polyHEMA coated culture conditions further increased the production of IL-12 and IFN-r in dendritic cells loaded with the antigen using electroporation [(a) electroporation (-). electroporation (+), P &lt;0.05; (b) electroporation (-) vs. electroporation (+), P &lt;0.05; (c) electroporation (-) vs. electroporation (+), P &lt; 0.05]. Thus, this result indicates that the polyHEMA hydrogel coated culture condition improves the function of mature dendritic cells.

Claims (14)

A method for producing dendritic cells which are antigen-presenting cells comprising the steps of:
a) separating peripheral blood mononuclear cells (PBMC) from white blood cells of the blood;
b) isolating monocytes from said peripheral blood mononuclear cells;
c) culturing said single cells to differentiate into immature dendritic cells;
d) loading the immature dendritic cells with the antigen by electroporation; And
e) immature dendritic cells loaded with the antigen are placed in a culture container coated with hydrogel and cultured to differentiate into mature dendritic cells.
The method for producing mature dendritic cells according to claim 1, wherein the separation of peripheral blood mononuclear cells in step a) is performed using centrifugation.
The method according to claim 1, wherein the separation of the single cells in step b) is performed using a culture-container adherent method, a MACS MicroBeads method, or a density gradient method. Lt; RTI ID = 0.0 &gt; dendritic &lt; / RTI &gt;
The method according to claim 1, wherein the culture in step c) is cultured in an animal serum medium supplemented with rhIL-4 and rhGM-CSF.
[6] The method of claim 1, wherein the electroporation of step d) comprises mixing an immature dendritic cell with an antigen and then using a square wave pulse.
The method according to claim 1, wherein the hydrogel coating in step e) uses PolyHEMA (poly-2-hydroxyethyl methacrylate) hydrogel coating material.
The method according to claim 1, wherein the culture of step (e) is cultured by adding a maturation inducing factor.
8. The method of claim 7, wherein the maturation-inducing factors are TNF-alpha, IL-1 beta, IL-6 and PGE2.
The method of claim 1, wherein the hydrogel coating improves the yield of mature dendritic cells compared to no hydrogel coating.
The method of claim 1, wherein the hydrogel coating enhances the expression of the CD83 surface antigen, which is a maturation marker of dendritic cells, as compared to the hydrogel coating.
The method according to claim 1, wherein the hydrogel coating is a hydrogel-coated mature dendritic cell, wherein the mature dendritic cell has a longer and strong dendritic cell shape. How to.
The method of claim 1, wherein the hydrogel coating improves the production of cytokines (IL-12 and IFN-r) that exhibit dendritic cell function as opposed to hydrogel-free coatings. A method for producing dendritic cells.
The method according to claim 1, wherein the electroporation enhances the expression of the CD83 surface antigen, which is a maturation marker for dendritic cells, and inhibits cytokines (IL-12 and IFN-r ) Of the antigen-presenting cells.
A cancer immune cell therapeutic composition comprising mature dendritic cells differentiated by the production method according to claim 1 as an active ingredient.

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