KR20200089054A - Hydrogel composition comprising retinal pigment epithelium cells and use of the same - Google Patents

Abstract

The present invention relates to: a hydrogel composition comprising a scaffold including polyethylene glycol and gellan gum, and retinal pigment epithelial cells; and a composition for retinal pigment epithelial cell regeneration, the treatment and prevention of retinitis pigmentosa, or the treatment and prevention of macular degeneration using the hydrogel composition.

Description

Hydrogel composition comprising retinal pigment epithelium cells and use of the same}

The present invention relates to a hydrogel composition comprising retinal pigment epithelial cells and uses thereof, and more specifically, a hydrogel composition containing a support comprising gellan gum and polyethylene glycol and retinal pigment epithelial cells, and the hydrogel composition The present invention relates to a composition for the treatment and prevention of retinal pigment epithelial cell regeneration or retinal pigment epithelial degeneration, or the treatment and prevention of macular degeneration.

Retinal Pigment Epithelium plays an important role in maintaining the normal visual function of the retina, and degeneration of the retinal pigment epithelium causes various retinal degenerative diseases. The macula, the nerve tissue in the central region of the retina, is densely packed with visual cells and plays an important role in visual acuity.

However, degeneration of the retina occurs due to various causes such as aging, genetic factors, inflammation, stress, excessive ultraviolet exposure, and thus damages eyesight. This macular degeneration (Macula Degeneration, MD) is, in particular, mainly affects elderly patients due to aging, and this is called Age-related Macular Degeneration (AMD).

Although technologies for treating the age-related macular degeneration have been studied worldwide (non-patent documents 1 to 3), it is difficult to expect a therapeutic effect with current technology.

Patent documents related to retinal pigment epithelial cells include a method of inducing differentiation of retinal pigment epithelial cells from cystic structures (patent document 1), cell model for angiogenic disease using EV-infected human retinal pigment epithelial cells (patent document 2), A composition for inducing retinal pigment epithelial differentiation (Patent Document 3) and the like are disclosed, but there are no known cases where retinal regeneration is attempted using gellan gum and polyethylene glycol as biomaterials.

Accordingly, in the present invention, a hydrogel was prepared using gellan gum and polyethylene glycol as biomaterials for effectively transplanting RPE cells, and retinal regeneration was confirmed using the same.

Patent Publication No. 10-2013-0048468 Patent Publication No. 10-2014-0079067 Patent Publication No. 10-2017-0049775

Ophthalmology 122(4) (2015) 809-816. Biomaterials 31(9) (2010) 2555-2564. Acta Biomaterialia 49 (2017) 329-343.

The present invention has been devised to solve the problems of the prior art as described above, the object of the present invention is to effectively transplant the retinal pigment epithelial cells, including retinal pigment epithelial cells using gellan gum and polyethylene glycol as biomaterials It is to provide a hydrogel composition.

Another object of the present invention is to provide a method for preparing a hydrogel composition using gellan gum and polyethylene glycol as biomaterials to effectively transplant retinal pigment epithelial cells.

Another object of the present invention is to provide a use of the hydrogel composition.

In order to achieve the above object, the present invention provides a hydrogel composition comprising retinal pigment epithelial cells, comprising a support comprising gellan gum and polyethylene glycol and retinal pigment epithelial cells.

In addition, the present invention (a) preparing a retinal pigment epithelial cell by culturing a retinal pigment epithelial cell line obtained from a human, (b) dissolving gellan gum in distilled water to obtain an aqueous gellan gum solution, (c) the (b) As a separate step from the step, obtaining polyethylene glycol aqueous solution by dissolving polyethylene glycol in distilled water, (d) mixing the gellan gum aqueous solution and polyethylene glycol aqueous solution, and adding a crosslinking agent to include gellan gum and polyethylene glycol Obtaining a support, and (e) mixing the retinal pigment epithelial cells prepared in step (a) with a support comprising gellan gum and polyethylene glycol, of a hydrogel composition comprising membrane pigment epithelial cells. Provide a manufacturing method.

In addition, the present invention provides a composition for retinal pigment epithelial cell regeneration including the hydrogel composition.

In addition, the present invention provides a composition for the treatment and prevention of retinal pigment epithelial degeneration comprising the hydrogel composition.

In addition, the present invention provides a composition for the treatment and prevention of macular degeneration, including the hydrogel composition.

The composition for retinal pigment epithelial cell regeneration according to the present invention is a polymer polysaccharide extracted from aquatic plants, gellan gum having excellent physical properties and biocompatibility, and RPE cells in a hydrogel containing polyethylene glycol, which is harmless to the human body and widely used for improving physical properties Increases the adhesion and proliferation rate of, and the effect that the function of cells can be normally expressed occurs.

In addition, the market for biomaterials containing stem cells that are currently commercially available is insufficient, and according to the present invention, there is an advantage that the retinal therapeutic agent can be used as a formulation for commercializing stem cell agents and hydrogels.

Figure 1a is a photograph showing a naked eye image of the prepared PEG / GG hydrogel, Figure 1b is a SEM image confirming the shape of the pores, Figure 1c is a graph showing the size of the pores.
2 is a graph showing PEG and PEG/GG hydrogels through FT-IR.
Figure 3 is a result showing the porosity of the PEG / GG hydrogels.
4 is a result showing the swelling degree of PEG / GG hydrogels.
5 is a result showing the decomposition of PEG / GG hydrogels.
6 is a result showing the cell proliferation rate of PEG / GG hydrogels.
7A to 7C are results obtained by quantifying gene expression of PEG/GG hydrogels.
8 is a result showing the cell viability evaluation in PEG / GG hydrogels.
9 is an SEM photograph showing the adhesion and proliferation of cells in PEG/GG hydrogels.
10 is a H&E staining photograph showing the distribution and morphology of cells in PEG/GG hydrogels.

Hereinafter, the present invention will be described in more detail as one embodiment as follows.

The present invention relates to a hydrogel composition comprising retinal pigment epithelial cells containing retinal pigment epithelial cells and a support comprising gellan gum and polyethylene glycol.

The gellan gum (GG) contained in the hydrogel composition containing retinal pigment epithelial cells is one of biomaterials having excellent biocompatibility, and has excellent physical properties such as heat resistance, acid resistance, and enzyme resistance. In addition, since the manufacturing process is not complicated, and gelation occurs at a temperature close to body temperature, it is suitable for use in an injectable form.

The polyethylene glycol (PEG) contained in the hydrogel composition containing the retinal pigment epithelial cells is a substance harmless to the human body because it does not cause any special interaction with biological chemicals, and has a function of giving viscosity.

In particular, since it is a hydrophilic polymer, it is widely used in medical materials, and is widely used for improving physical properties through compounding with other materials and surface modification of materials.

Accordingly, in the present invention, a hydrogel obtained by mixing the gellan gum and polyethylene glycol was prepared to increase retinal pigment epithelial cell proliferation and regeneration efficiency.

In the hydrogel composition comprising the retinal pigment epithelial cells, the support comprising the gellan gum and polyethylene glycol may contain 1 to 10% by weight, preferably 1 to 5% by weight, based on the total weight of the composition.

In the hydrogel composition containing the retinal pigment epithelial cells, when the support comprising the gellan gum and polyethylene glycol exceeds 10% by weight based on the total weight of the composition, the viscosity increases, thereby inhibiting cell growth and growth. There is, when the support comprising the gellan gum and polyethylene glycol is less than 1% by weight relative to the total weight of the composition, there is a problem that the crosslinked product of the gellan gum and polyethylene glycol can not serve as a support.

In the hydrogel composition containing the retinal pigment epithelial cells, the crosslinking agent of the support comprising the gellan gum and polyethylene glycol is calcium chloride (CaCl ₂ ), lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), cesium chloride (CsCl), endogen polyamine spermidine, and the like, but is not limited thereto.

In the hydrogel composition comprising the retinal pigment epithelial cells, the gellan gum and polyethylene glycol may be contained in a weight ratio of 90 to 99: 1 to 10, preferably 95 to 99: 1 to 5.

In the hydrogel composition comprising the retinal pigment epithelial cells, the retinal pigment epithelial cells may be of human origin, but are not limited thereto.

In the hydrogel composition comprising the retinal pigment epithelial cells, the composition may be for retinal pigment epithelial cell regeneration, but is not limited thereto.

In addition, the present invention (a) preparing a retinal pigment epithelial cell by culturing a retinal pigment epithelial cell line obtained from a human, (b) dissolving gellan gum in distilled water to obtain an aqueous gellan gum solution, (c) the (b) As a separate step from the step, obtaining polyethylene glycol aqueous solution by dissolving polyethylene glycol in distilled water, (d) mixing the gellan gum aqueous solution and polyethylene glycol aqueous solution, and adding a crosslinking agent to include gellan gum and polyethylene glycol Obtaining a support, and (e) mixing the retinal pigment epithelial cells prepared in step (a) with a support comprising the gellan gum and polyethylene glycol, of a hydrogel composition comprising retinal pigment epithelial cells. It relates to a manufacturing method.

In the method of preparing a hydrogel composition containing the retinal pigment epithelial cells, step (a) is a step of preparing the retinal pigment epithelial cells by culturing the retinal pigment epithelial cell line ARPE-19 cells in a DMEM/F-12 culture medium. Can.

In the method of preparing a hydrogel composition containing the retinal pigment epithelial cells, step (b) is a gellan gum solution by heating the gellan gum powder in distilled water heated to a temperature of 90 to 110°C and heating for 10 to 40 minutes under stirring. It may be a step of manufacturing.

In the method of preparing a hydrogel composition containing the retinal pigment epithelial cells, the step (c) is put in polyethylene glycol powder in distilled water heated to a temperature of 90 ~ 110 ℃ and heated for 10-40 minutes under stirring to a gellan gum aqueous solution It may be a step of manufacturing.

In the method of preparing a hydrogel composition comprising the retinal pigment epithelial cells, the step (d) is 90 to 99: 1 to 9 by weight of the gellan gum aqueous solution and the polyethylene glycol aqueous solution, preferably 95 to 99: 1 to After mixing at a weight ratio of 5, a crosslinking agent may be added and crosslinked for 1 to 30 minutes to obtain a support comprising gellan gum and polyethylene glycol.

In the method of preparing a hydrogel composition comprising the retinal pigment epithelial cells, the step (e) is mixing the retinal pigment epithelial cells prepared in the step (a) on a support comprising the gellan gum and polyethylene glycol, the gellan It may be a step of adding and mixing such that the weight ratio of the support comprising gum and polyethylene glycol and the retinal pigment epithelial cells is 1 to 10:90 to 99.

In addition, the present invention relates to a composition for retinal pigment epithelial cell regeneration including the hydrogel composition as a use of the hydrogel composition.

In addition, the present invention relates to a composition for the treatment and prevention of retinal pigment epithelial degeneration comprising the hydrogel composition as a use of the hydrogel composition.

In addition, the present invention relates to a composition for the treatment and prevention of macular degeneration comprising the hydrogel composition as a use of the hydrogel composition.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are intended to illustrate the present invention, and the contents of the present invention are not limited by the following examples.

< Example 1> cell culture

ARPE-19 cells, an RPE cell line obtained from a human (American Type Culture Collection ^® ), were cultured in DMEM/F-12 culture.

At this time, the culture medium was DMEM/F-12 (Dulbeco's Modified Eagle's Media, Gibco, USA), 10% fetal calf serum (FBS, Gibco) and 1% penicillin (PS, 100 U/mL) and 100 It was used by adding μg/mL streptomycin (Gibco).

The ARPE-19 cells were cultured in an incubator at 37°C and 5% CO ₂ conditions. ARPE-19 cells were cultured until an appropriate cell number was obtained, and the medium was changed every 2 days.

< Example 2> PEG / GG Preparation of crosslinked products

After dissolving PEG (concentrations of 0, 1, 3 and 5% by weight) in distilled water (dH ₂ O) at a temperature of 100° C., and continuously stirring for 20 minutes, completely dissolve, and then GG (1% (w/v) ) Were added to each solution to dissolve all, then 0.03% CaCl ₂ was added and the mixture was crosslinked for 10 minutes.

< Example 3> ARPE -19 encapsulation

The PEG/GG crosslinked product prepared in Example 2 was sterilized at 120° C. for 30 minutes, and then 5×10 ⁵ cells/ml of ARPE-19 cells were mixed. After pouring into the Petri dish, gelation occurred by decreasing the temperature at room temperature for 15 minutes.

The prepared hydrogel was prepared by using a punch to form a hydrogel having a diameter of 8 mm and a height of 5 mm, and was put in a DMEM/F-12 culture solution and cultured in an incubator of 5% CO _{2 and} 37°C.

< Experimental Example 1> Hydrogel Characterization of the support

1.PEG/ GG Hydrogel Visual observation and internal shape observation

Visual observation of the prepared PEF/GG hydrogel was shown in FIG. 1A, and there was no external change according to the PEG content. At this time, the surface properties and morphology of the hydrogel were confirmed by an electron scanning microscope (Bio-LV-SEM, SN-3000 Hitachi, Japan).

It was fixed with a carbon tape of a freeze-dried PEG/GG hydrogel, and after coating with platinum, porosity was observed. The gel containing PEG had pores smaller than that of gellan gum, and the size of porosity decreased as the content of PEG increased. At this time, the size of the pores was approximately between 200 and 350 μm.

2. PEG/ GG Hydrogel FT-IR analysis

To confirm the structural changes of the prepared PEG, GG and PEG/GG hydrogels, a qualitative analysis was performed in the range of 4,000-500 cm ^-1 using FT-IR, and the results are shown in FIG. 2.

As for the peak of PEG, -CH group peak was generated at 841 cm ^-1 and 2882 cm ^-1 , and -COC- group peak was generated at 1093 cm ^-1 .

The main peak of GG was a broad peak at 3369 cm ^-1 due to the presence of the -OH group of the glucopyranose ring, a carboxylate group at 1425 cm ^-1 , 1630 cm ^-1 , and a 2924 cm ^-1 Peak -CH ₂ appeared.

As the content of PEG increased, it was confirmed that the peak near 1150 cm ^-1 deepened, which confirmed that the characteristics of the hydrogel were modified through the overlapping peaks of PEG and GG, resulting in a strong band.

3. Porosity measurement

In order to measure the pores in the support, the hydrogel was freeze-dried, soaked in distilled water for a period of time, and measured through volume change of distilled water. The measured formula is as follows.

Porosity (%) =

In the above formula, V ₁ is the volume of the first distilled water, V ₂ is the volume of distilled water increased after adding the support, and V ₃ means the volume of distilled water reduced after removing the support.

Porosity is an important property of hydrogels because it can induce cell adhesion, penetration, and nutrient exchange. FIG. 3 shows the porosity of the produced hydrogel, and the porosity of the GG hydrogel and the PEG/GG hydrogel is 60%. It showed abnormality.

According to these results, it was confirmed that as the PEG concentration of the hydrogel increased, the viscosity increased, and the porosity decreased and the pore size decreased.

4. Measurement of swelling degree

To measure the swelling degree of the support, the hydrogel was freeze-dried, soaked in PBS to measure the weight change of the support over time, and the measurement formula is as follows.

Swelling degree (%)

In the above formula, W _s is the weight soaked in PBS, and W _d is the lyophilized weight.

From the measurement results shown in Fig. 4, it was confirmed that the moisture absorption of all the supports was very high.

5. Decomposition rate measurement

To measure the decomposition rate of the support, the hydrogel was freeze-dried, soaked in distilled water, the weight change of the support was specified over time, and the measurement formula is as follows.

Decomposition rate (%)

In the above formula, W _f denotes a basis weight and W _d denotes a lyophilized weight.

Since hydrogels are cell carriers and replace ECM, they must provide adequate biodegradability. In particular, the hydrogel for retinal transplantation should be evenly distributed in the cell layer and the binding of the RPE cell layer.

The degradability of the PEG/GG hydrogel was measured for 21 days, and the degradation rate of the GG hydrogel was faster than that of the PEG-containing hydrogel. The initial decomposition rate was less as the PEG content increased, but after 10 days, there was no significant change in the decomposition rate, and after 21 days, GG and 3% by weight of PEG hydrogel showed a decomposition rate of more than 65%.

< Experimental Example 2> Hydrogel Evaluation of encapsulated cell properties

1. Evaluation of cell proliferation rate

To confirm the biocompatibility and proliferation of the hydrogel, the cell proliferation rate was evaluated using MTT assay (3-[4,-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; thiazolyl blue, Amresco, USA).

After the ARPE cells were encapsulated in a hydrogel, 1 ml of culture solution and 100 µl of MTT reagent were added on the 1st, 3rd, 5th, 7th, 14th day, and cultured in an incubator of 5% CO ₂ , 37°C for 4 hours.

After that, when purple crystals were formed, 1 mL of dimethyl sulfoxide (DMSO, Sigma, USA) was added to completely dissolve the purple crystals, and 0.1 mL of each was dispensed into a 96-well plate to measure absorbance at 570 nm.

The measurement results are shown in FIG. 5, and the cells proliferated as the incubation period increased, and the highest cell proliferation rate in 3 wt% PEG/GG hydrogel was confirmed.

2. mRNA Expression analysis

The expression of mRNA extracted from ARPE cells cultured in a hydrogel was analyzed using RT-PCR (Reverse transcription-polymerase chain reaction) and is shown in FIG. 6.

Samples on the 3rd, 7th, and 14th days were synthesized and amplified by cDNA, placed in a 1% agarose gel, and electrophoresed under 100V conditions, irradiated with ultraviolet rays having a wavelength of 360 nm, and RPE65, CRALBP, and NPR-A bands. Was observed.

The expression level of Cellular Retinaldehyde-Binding Protein (CRALBP), Retinal Pigment Epithelium-specific 65-kDa (RPE65), and Natriuretic Peptide-A Receptor (NPR-A) is expressed using the anti-zone gene beta-lactin. As a result of normalization and digitization, it was confirmed that mRNA expression of cells cultured in 3% by weight PEF/GG hydrogel was highest (see FIG. 6 ).

3. Evaluation of cell viability

To evaluate the viability of the cells, a Live/Dead cell imaging kit (Invitrogen, USA) was used, each PEG/GG hydrogel was thinly cut, and the reagent was pre-soaked in a confocal dish, and then the hydrogel was placed on top again. Covered and incubated under conditions of 5% CO ₂ and 37° C. for 3 hours to allow the reagent to enter the interior.

After blocking the light, live cells and dead cells were observed inside the hydrogel with a high-resolution confocal laser scanning microscope (LSM 880 with Airycsan, Carl Zeiss, Germany).

As a result of confirming the samples on the 1st and 14th days, it was confirmed that the green color representing living cells increased over time, and appeared most in the 3 wt% PEG/GG hydrogel.

4. PEG/ GG Hydrogel Analysis of cell adhesion and proliferation inside ( Electron scanning microscope observe)

In order to confirm the adhesion and migration patterns of ARPE cells in the PEG/GG hydrogel, the results were observed using an electron scanning microscope (Bio-LV-SEM, SN-3000 Hitachi, Japan) and shown in FIG. 8.

5×10 ⁵ cells/ml Hydrogels with seeded cells were cultured for 1 and 14 days. After removing the culture solution and washing with PBS, it was fixed with 2.5% glutaraldehyde (Sigma-aldrich) for 24 hours, frozen in a freezer for 4 hours after freezing for 4 hours, and freeze-dried for 24 hours after freezing for 4 hours.

After observing the cross section after cutting the dried support, a carbon tape was attached to the metal plate to fix the support and coated with platinum. This was observed using a scanning electron microscope to observe the cell attachment and migration patterns on the support.

It was confirmed that attachment and migration of ARPE cells occurred better in the PEG/GG hydrogel containing 3 w% PEG than the control GG hydrogel (see FIG. 9). 10 is a H&E staining photograph showing the distribution and morphology of cells in PEG/GG hydrogels.

Although described with reference to preferred embodiments and experimental examples of the present invention as described above, those skilled in the art in the technical field of the present invention will not depart from the spirit and scope of the invention described in the claims below It will be understood that the present invention can be variously modified and changed within.

The composition for retinal pigment epithelial cell regeneration according to the present invention is a polymer polysaccharide extracted from aquatic plants. It has excellent physical properties and biocompatibility. Since it increases the attachment and proliferation rate of cells and has the effect that cell functions can be normally expressed, it is applicable to tissue regeneration of the retina, and it can be applied to the tissue regeneration medicine field of other organs.

Claims (16)

A hydrogel composition comprising retinal pigment epithelial cells, comprising a support comprising gellan gum and polyethylene glycol and retinal pigment epithelial cells. According to claim 1, The gellan gum and the support of polyethylene glycol is characterized in that it contains 1 to 10% by weight relative to the total weight of the composition, a hydrogel composition comprising retinal pigment epithelial cells. According to claim 1, The gellan gum and the support of polyethylene glycol is characterized in that it contains 1 to 5% by weight relative to the total weight of the composition, a hydrogel composition comprising retinal pigment epithelial cells. The method according to claim 1, wherein the crosslinking agent of the support is calcium chloride (CaCl ₂ ), lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), cesium chloride (CsCl) and endogen polyamine spermidine Hydrogel composition comprising a retinal pigment epithelial cell, characterized in that selected from. According to claim 1, The gellan gum and polyethylene glycol is characterized in that contained in a weight ratio of 90 to 99: 1 to 10, a hydrogel composition comprising retinal pigment epithelial cells. The method of claim 1, wherein the retinal pigment epithelial cells, characterized in that derived from human, hydrogel composition comprising retinal pigment epithelial cells. According to claim 1, The composition is characterized in that for retinal pigment epithelial cell regeneration, a hydrogel composition comprising retinal pigment epithelial cells. (a) preparing a retinal pigment epithelial cell by culturing a retinal pigment epithelial cell line obtained from a human;
(b) dissolving gellan gum in distilled water to obtain an aqueous gellan gum solution;
(c) a step separate from step (b), wherein polyethylene glycol is dissolved in distilled water to obtain an aqueous solution of polyethylene glycol;
(d) mixing the aqueous gellan gum solution and the polyethylene glycol aqueous solution, and then adding a crosslinking agent to obtain a support comprising gellan gum and polyethylene glycol; And
(e) a method of manufacturing a hydrogel composition comprising retinal pigment epithelial cells, comprising mixing the retinal pigment epithelial cells prepared in step (a) on a support comprising gellan gum and polyethylene glycol. The method of claim 8, wherein the step (a) is a retinal pigment epithelial cell comprising a retinal pigment epithelial cell, characterized in that the retinal pigment epithelial cells are prepared by culturing ARPE-19 cells, which are retinal pigment epithelial cell lines, in a DMEM/F-12 culture medium. Method of preparing a gel composition. The method of claim 8, wherein the step (b) is characterized in that the gellan gum powder is placed in distilled water heated to a temperature of 90 to 110° C. and heated for 10 to 40 minutes under stirring to prepare a gellan gum aqueous solution. Method of manufacturing a gellan gum hydrogel composition comprising epithelial cells. The method of claim 8, wherein the step (c) is characterized in that to prepare a gellan gum aqueous solution by placing polyethylene glycol powder in distilled water heated to a temperature of 90 to 110°C and heating for 10 to 40 minutes under stirring. Method of manufacturing a gellan gum hydrogel composition comprising epithelial cells. The method of claim 8, wherein the step (d) is a gellan gum aqueous solution and a polyethylene glycol aqueous solution are mixed in a weight ratio of 90 to 99: 1 to 9, and then a crosslinking agent is added and crosslinked for 1 to 30 minutes to form a gellan gum. Method of producing a gellan gum hydrogel composition comprising retinal pigment epithelial cells, characterized in that to obtain a support comprising polyethylene glycol. The method of claim 8, wherein step (e) comprises mixing the retinal pigment epithelial cells prepared in step (a) with the crosslinked product of the gellan gum and polyethylene glycol, wherein the support comprising the gellan gum and polyethylene glycol and the Method for producing a gellan gum hydrogel composition comprising retinal pigment epithelial cells, characterized in that the weight ratio of retinal pigment epithelial cells is added and mixed so that it is 1 to 10: 90 to 99. A composition for retinal pigment epithelial cell regeneration, comprising the hydrogel composition according to any one of claims 1 to 7. A composition for the treatment and prevention of retinal pigment epithelial degeneration comprising the hydrogel composition according to any one of claims 1 to 7. A composition for the treatment and prevention of macular degeneration, comprising the hydrogel composition according to claim 1.

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