KR101429346B1 - Method of pseudoislets culture using artificial extracellular matrix of elastin like multiblock biopolymer - Google Patents

Abstract

The present invention relates to a method for culturing artificial pancreatic islets using an elastin-like artificial extracellular matrix, comprising the steps of: coating an elastin-like artificial extracellular matrix onto a plate; Removing the remaining solution on the plate; And culturing the pancreatic cells in the plate from which the solution has been removed. The elastin-like artificial extracellular matrix is characterized by being arginine-glycine-aspartate (RGD) -elastin like polypeptide (ELP). Further, there is provided an artificial pancreas small intestine cultured by the above culture method.

Description

[0001] The present invention relates to a method for culturing artificial pancreatic islets using an elastin-like artificial extracellular matrix,

The present invention relates to a method for culturing artificial pancreatic islets using an elastin-like artificial extracellular matrix.

The extracellular matrix (ECM) was first discovered about 100 years ago and was initially thought to play a structural role as a simple intercellular junction (Pathol Biol. (2005) 53, 369-371). However, it has been confirmed that it plays an important role as a cell physiological regulator such as cell division, differentiation and death. In particular, the importance of the extracellular matrix has recently been emphasized for cell therapy and regenerative medicine combined with embryonic stem cells or adult stem cells. It is also of great importance for tissue engineering studies that try to create cells by culturing cells in biomaterials that mimic the extracellular matrix.

The extracellular matrix is the product of the cells that make up each tissue. Therefore, it is composed of different components depending on the organization and has special physical properties. In general, the extracellular matrix is mainly composed of structural proteins such as collagen and elastin, and is called polysaccharides called glycosaminoglycan (GAG) Adherent proteins are fixed, and various biochemical factors such as growth factors are transferred and distributed (J Cell Physiol. (2004) 199, 174-80).

Extracellular matrix extracted directly from animal tissue may be the best option for repeated cultivation of live cells on tissue culture plates in a microhabitat environment. For example, Matrigel or Amgel obtained from mouse sarcoma or human amnion cells has been extensively developed for the analysis of a variety of cellular activities and has also contributed in part to understanding the behavior of tumor cells . However, specimens of naturally derived ECMs are complex and have a disadvantage in that they are not only limited in high throughput and mass production but also are expensive. Recently, there has been increasing interest in artificial extracellular matrix (ECMs) that mimic the physical environment of the extracellular matrix. Previously reported artificial pancreatic islets are produced by encapsulating pancreatic beta cells with hydrogels, polysaccharides, or by suspension culture with agitation. However, existing methods have the disadvantage of using cytotoxicity and special devices.

Meanwhile, Korean Patent Application No. 10-2009-7023990 discloses a cell culture system including a 3-dimensional biocompatible skeleton structure and a biocompatible nanoparticle, a cell culture method, and a cell or cell product and tissue , There is no mention of the cultivation of pancreatic islets using the elastin-like artificial extracellular matrix of the present invention.

It is an object of the present invention to provide a method for producing an elastin-like artificial extracellular matrix, in particular an arginine-glycine-aspartate (RGD) -elastin like polypeptide (ELP) And cultivating the artificial pancreas.

It is another object of the present invention to provide an artificial pancreas cannula cultured by the above-mentioned culturing method.

In order to achieve the above object, the inventors of the present invention conducted an easy method of simply coating on a tissue culture plate using an elastin-like artificial extracellular matrix which is safe without cytotoxicity, easily cultured and has good productivity and excellent biocompatibility , Allowing the formation of a three-dimensional cluster of pancreatic beta cells, that is, artificial pancreatic islets. In addition, the artificial pancreas sperm formed by the above method has an insulin releasing ability in response to glucose stimulation, and thus it is possible to provide a novel method of tissue regeneration and tissue transplantation for the treatment of diabetes based on the artificial pancreas sperm.

The present invention provides a method of treating an elastin-like artificial extracellular matrix comprising: coating an elastin- Removing the remaining solution on the plate; And culturing the pancreatic cells in the plate from which the solution has been removed. Specifically, the step of coating the plate is characterized by treating the elastin-like artificial extracellular matrix with a concentration of 0.5 to 10 uM, wherein the pancreatic cell is a beta-TC-6 cell, which is a mouse islet cell do.

On the other hand, the elastin-like artificial extracellular matrix is characterized by being arginine-glycine-aspartate (RGD) -elastin like polypeptide (ELP), which is abbreviated as RGD-ELP Respectively.

In detail, the RGD-ELP is a multi-block biopolymer, wherein one of the 1 to 100 blocks includes peptide I of cell adhesion function for tissue regeneration, and at least one block not selected as peptide I Each of which is a peptide II having a structure of PGX (P is L-proline, G is D-glycine, X is 19 L- or D-amino acid except L- or D-proline), peptide III having a structure of PGXX, A peptide IV having a structure of PGXXX, a peptide V having a structure of PGXGXX, and a peptide VI having a function of promoting mutual binding of a multi-block polymer.

Further, the present invention provides an artificial pancreas small intestine cultured by the above culture method. Specifically, the artificial pancreatic islets have an insulin secretion function by glucose stimulation.

According to the method of forming a three-dimensional cell cluster of pancreatic beta cells using the elastin-like artificial extracellular matrix according to the present invention and the artificial pancreatic islet prepared on the basis thereof, the present invention is safe without cytotoxicity, Excellent tissue regeneration ability can be provided based on materials having good productivity and excellent biocompatibility. The artificial sole formed by this method maintains insulin secretion ability against glucose response, so that the present invention can provide a method for the development of tissue and cell-forming artificial organs for the treatment of type 1 diabetes.

FIG. 1 shows the appearance of artificial pancreatic islets and the measurement of cell activity of beta-TC-6 cells cultured on RGD-ELP over time.
Figure 2 shows insulin secretion and insulin gene expression results of artificial pancreas sisters formed on RGD-ELP.
Figure 3 shows the expression results of E-cadherin and connexin-36 of the artificial pancreatic islets formed on RGD-ELP.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Artificial pancreas small intestine  Morphology and cellular activity

1. Elastin-like artificial extracellular matrix (RGD-ELP) purification

The elastin-like artificial extracellular matrix used in the present invention was named Arginine-Glycine-Aspartate (RGD) -elastin like polypeptide (ELP), abbreviated RGD-ELP. RGD-ELP is a protein having a ligand having an amino acid sequence of RGD (arginine-glycine-aspartic acid) using ELP as a template.

RGD-ELP was synthesized according to the general peptide synthesis method. A DNA fragment encoding the amino acid of RGD (arginine-glycine-aspartic acid) was successfully cloned by recursive directional ligation (RDL), which was transformed with E. coli BLR DE3). Reverse rotation was carried out three rounds of cycling to increase the purity, and it was confirmed by SDS-PAGE that the purity was 95% or more.

The RGD-ELP used in the present invention is a multi-block biopolymer, wherein one of the 1 to 100 blocks contains peptide I for cell adhesion function for tissue regeneration, and at least one of the blocks (P is L-proline, G is D-glycine, X is 19 L- or D-amino acid except L- or D-proline), pentaide III having the structure of PGXX, Peptide IV having the structure of PGXXX, peptide V having the structure of PGXGXX, and peptide VI having the function of promoting mutual binding of the multi-block polymer.

2. Cell culture

The cells used in the present invention were incubated with beta-TC-6 cells, insulin-releasing mouse islet cells, in a constant temperature and humidity chamber maintained at 37 ° C in 5% carbon dioxide. Beta-TC-6 cells were purchased from the American Type Culture Collection (ATCC). Beta-TC-6 cells were cultured in ATCC-formulated Dulbecco's Modified Eagle's Medium supplemented with 15% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin, and subcultured 30-40 Were used.

3. Formation and observation of artificial pancreatic islets

The concentrations of RGD-ELP were set at 0, 0.5, 1, 5, and 10 μM, and the results showed that 1 μM concentration was suitable for artificial pancreatic islet formation. Therefore, all experiments were carried out with 1 μM of RGD-ELP as final concentration. The plate was treated with 200 μl of 1 μM RGD-ELP in a 24-well plate and kept in a thermostat at 37 ° C for 1 hour. The RGD-ELP is then precipitated and coated onto the plate surface. After removing the remaining solution on the plate, beta-TC-6 cells were added to each well of each well, and cell growth and morphological changes were observed with a microscope for 1, 3, 5 and 7 days.

4. Cell activity measurement

For cell activity measurement, LIVE / DEAD reagent purchased from Molecular Probe was used. The artificial pancreatic islets cultured on the plate were washed three times with phosphate buffered saline (PBS), treated with LIVE / DEAD reagent containing 2 μM calcein AM and 4 μM EthD-1 and incubated at room temperature for 30 And then observed with a fluorescence microscope.

5. Results

In order to examine the effect of RGD-ELP on the growth and morphology of beta-TC-6 cells, 1, 3, 5 and 7 days of incubation were carried out on 1 μM RGD-ELP coated plates and observed under a microscope. Beta-TC-6 cells cultured on normal plates were monolayered and spread on plates. On the other hand, beta-TC-6 cells cultured on RGD-ELP formed small globular aggregates from the first day of culture, and when they were cultured until 3, 5 and 7 days, Forming various sizes (FIG. 1A).

The LIVE / DEAD reagent was used to measure the activity of beta-TC-6 cells forming artificial pancreatic islets. Calcein AM was stained in living cells to show green fluorescence. EthD-1 To give a red fluorescence. Artificial pancreatic islets formed on RGD-ELP were unable to observe dead cells even after 3 days of incubation. On days 5 and 7, however, some dead cells were observed inside the cell cluster (FIG. 1B).

< Example  2> Artificial pancreas  insulin Secretory function  Expression of insulin gene

1. Insulin secretion measurement

To determine the amount of insulin secreted by glucose stimulation in the artificial pancreatic islets formed by RGD-ELP, the Rat / Mouse Insulin ELISA kit purchased from Millipore was used. Briefly, beta-TC-6 cells were cultured on a 24-well plate coated with RGD-ELP for 1, 3, 5 and 7 days. Krebs ringer bicarbonate containing 5 mM glucose and 2% FBS And maintained in a buffer solution (Krebs Ringer bicarbonate buffer; KRBB) for 5 hours using a constant-temperature humidifier at 37 ° C. After 5 mM glucose or 25 mM glucose was treated for 1 hour, the supernatant was recovered and 10 μl of the supernatant was used for the analysis.

2. Quantitative real time PCR (qRT-PCR)

Quantitative real-time PCR (qRT-PCR) was used to confirm the expression of the altered gene during artificial pancreatic islet formation. Trizol reagent from Invitrogen was used to isolate RNA from artificial pancreatic islets formed by RGD-ELP, and cDNA was synthesized using High-Capacity cDNA Reverse Transcrition Kit from Applied Biosystem. QRT-PCR was performed using the SYBR Green PCR master mix kit. The conditions of the PCR cycle were 2 minutes at 50 ° C, 10 minutes at 95 ° C, 15 seconds at 95 ° C, 1 minute at 60 ° C for 40 times It was to repeat. The expression of insulin-1, insulin-2, E-cadherin and connexin-36 gene expression was analyzed by qRT-PCR.

3. Results

An important factor to evaluate whether the beta-TC-6 cell aggregates formed on RGD-ELPs function in artificial pancreatic islets is insulin secretion by insulin synthesis and glucose stimulation. Therefore, insulin secretion was confirmed by insulin ELISA assay and qRT-PCR was performed to confirm insulin gene expression. Insulin secretion of artificial pancreatic islets formed on RGD-ELP was increased to 1.3-1.6 times as compared to cells cultured in the plane (Fig. 2A). The artificial pancreas shed formed on the RGD-ELP showed that the insulin secretion ability by glucose stimulation was maintained until the fifth day (Fig. 2B). The expression of insulin-1 and insulin-2 gene in artificial pancreatic islets formed on RGD-ELP was markedly increased at 3 days, and at 5 days and 7 days, (Fig. 2C, D).

< Example  3> In artificial pancreas  E- E-cadherin and Connexin ( connexin ) -36 expression

1. Western blot analysis

The Western blot method was used to confirm protein expression involved in artificial pancreatic islet formation. Cell lysates were extracted with RIPA buffer in artificial pancreatic islets formed on RGD-ELP. Changes in these protein expression were confirmed using anti-E-cadherin and anti-connexin-36 antibodies.

2. Statistical processing

Results were expressed as mean ± standard error, and Duncna's test was used for analysis of variables. P values less than 0.05 were considered to be statistically significant, and all experiments were performed three times or more independently and statistically.

3. Results

To confirm the expression of E-cadherin and connexin -36, important factors involved in maintaining the structural form and function of pancreatic islets, qRT-PCR and western blot were performed . E-cadherin gene and protein expression were significantly increased from day 1 in artificial pancreatic islets formed on RGD-ELP compared to cells cultured on planar surfaces. E-cadherin gene and protein expression in artificial pancreatic islets was slightly reduced by 3, 5, and 7 days, but the expression level was consistently higher than that of the flat culture 3A, B). Also, the expression of connexin-36 gene and protein was higher in artificial pancreatic islets than in planar cultures (Fig. 3C, D).

Claims (7)

Coating an arginine-glycine-aspartate (RGD) -elastin-like polypeptide (ELP) as an elastin-like artificial extracellular matrix onto a plate;
Removing the remaining solution on the plate; And
Culturing the pancreatic cells in a plate from which the solution has been removed. The method of claim 1, wherein the step of coating the plate comprises treating the RGD-ELP at a concentration of 0.5 to 10 uM. delete 2. The method of claim 1, wherein the RGD-ELP is a multi-block biopolymer, wherein one of the 1 to 100 blocks comprises a peptide I of cell adhesion function for tissue regeneration, wherein at least one Each of the blocks is composed of a peptide II having a structure of PGX (P is L-proline, G is D-glycine, X is 19 L- or D-amino acid except L- or D-proline) III, a peptide IV having a structure of PGXXX, a peptide V having a structure of PGXGXX, and a peptide VI having a function of promoting mutual binding of a multi-block polymer. The method of claim 1, wherein the pancreatic cell is a beta-TC-6 cell, a mouse islet cell. delete [Claim 2] The method according to claim 1, wherein the artificial pancreas islet has an insulin releasing ability by glucose stimulation.

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