KR20180080585A - Preparation method of islets cells clusters with enhanced insulin secretion ability by hanging-drop technique - Google Patents

Abstract

The present invention relates to a preparation method of islet cell clusters (ICCs) with enhanced insulin secretion ability using the hanging-drop technique. The present inventors have optimized the size of ICCs by using the hanging-drop technique. The ICCs exhibited substantially improved viability in a hypoxic culture condition. Based on the result of confocal microscopic analysis, and the spatial configuration of islet singlet cells in the ICCs was identical to an intact islet cell. Expressions of various intracellular proteins related to intercellular interactions in islet cells were not much changed. More interestingly, in a diabetic animal, it was confirmed that the ICCs maintain a normal blood glucose level.

Description

[0002] The present invention relates to a method for producing insulin secretory ability by hanging-drop technique,

The present invention relates to a method for producing islets cells clusters (ICC) by using a hanging-drop technique.

Pancreatic islet transplantation is a promising technology for the treatment of type 1 diabetes. However, cell transfer studies for treatment have several disadvantages, such as unsatisfactory vascularization, immediate blood-mediated inflammatory response, and immune-mediated response, which limits the survival of transplanted islets do. The portal vein is an ideal site for transplantation in clinical practice. However, the immediate blood mediated inflammatory reactions (IBIR) at the site of administration can kill a large number of cancers immediately after transplantation. It is also associated with embolization of the liver in the liver. In addition, strictly invasive techniques such as portal vein grafting expose patients to additional risks such as bleeding, thrombosis, biliary perforation, transient elevation of serum aminotransferase, and arterial-venous fistulae. Subcutaneous sites, on the other hand, can minimize invasion and avoid serious side effects such as IBMIR and thromboembolism associated with portal vein grafting. However, due to unsatisfactory vascularization in the subcutaneous space, transplanted hepatic veins undergo severe hypoxic conditions early in the transplant. This often causes dysfunction of the implanted cancers in the subcutaneous space.

Pancreatic islets are composed of various cell types, collectively known as islets of Langerhans. Each metaplasia consists of α, β and δ cells. The rodent carcinoma has a shell-core structure, the β-cells are located in the core and the non-β-cells are located in the periphery. The volcanic ash exhibits an uneven size distribution in the range of 50 [mu] m to 400 [mu] m.

Previous studies have reported that an islet can be isolated by single treatment with enzyme treatment. In addition, a single isolated hepatic cell can spontaneously form clusters with a structure similar to that of parental cancers. However, the spatial organization of a single tumor cell and the interaction between tumor cells in islet cell clusters (ICCs) have not been clearly elucidated. Despite the normal insulin secretory properties of ICC in glucose-promoted in vitro conditions, the in vivo effect of ICC implanted in diabetic animals was not well known.

Transplant Proc. Mar 2013; 45 (2): 605-10

It is an object of the present invention to provide a method for producing islets cell clusters (ICC) using a hanging-drop technique.

In order to accomplish the above object, the present invention provides a method for isolating ISC, comprising the steps of: isolating islet single cells (ISC) by treating trypsin-EDTA with a separate pancreatic islet; Hanging-drop culturing the isolated ISC; And recovering islets cell clusters (ICC) from the current culture. The present invention also provides a method for producing a canola cell having improved insulin releasing ability.

Also, the present invention provides a cane clone improved in insulin releasing ability, comprising 250 to 4000 ISCs and having a diameter of 70 to 150 탆.

In addition, the present invention provides a composition for preventing or treating diabetes mellitus comprising the above-described cancellous clusters as an active ingredient.

The present invention relates to a method of producing islets cell clusters (ICC) by using a hanging-drop technique, and the present inventors have developed a method for producing ICCs by using a hanging-drop technique. The size was optimized. The viability of ICC was significantly improved under hypoxic culture conditions. Confocal microscopy analysis showed that the spatial organization of single tumor cells in ICC was identical to that of original tumor cells. Expression of various intracellular proteins involved in cellular interactions in tumor cells has not changed significantly. More interestingly, ICC in diabetic animals was found to maintain normal levels of blood glucose.

1 is a schematic diagram showing a process of isolating pancreatic islets and producing islets cell clusters (ICC).
Figure 2 shows the size distribution of intact cancellous and functional pseudo-islets.
Figure 3 shows a live / dead image of pseudo-iso-cep under normal oxygen conditions (magnification: 100).
Figure 4 shows a Live / Dead image of Pseudo-Dog Sappho under hypoxic conditions (24 hours) (magnification: 100).
Figure 5 shows the results of Glucose stimulated insulin secretion (GSIS) of pseudo-ischemia. (a) and (b) normal oxygen conditions, (c) and (d) hypoxic conditions.
Fig. 6 shows immunofluorescence analysis results of pseudo-toxoplasma.
Fig. 7 shows the result of immuno-fluorescence analysis of collagen secretion of pseudo-isoepsin. (a) green-collagen, (b) fluorescence intensity, and (c) confocal image.
FIG. 8 shows the results of confirming the role of various proteins necessary for maintaining the mechanism of metaplasia interaction and the function of metaplasia.
FIG. 9 shows the result of measuring the fasting blood glucose level of the ICC-transplanted mouse.
FIG. 10 shows the results of performing intraperitoneal glucose tolerance test in normal mouse, diabetic mouse, and all transplanted groups.

Thus, the present inventors optimized the size of ICC using a hanging-drop technique. The viability of ICC was significantly improved under hypoxic culture conditions. Confocal microscopy analysis showed that the spatial organization of single tumor cells in ICC was identical to that of original tumor cells. Expression of various intracellular proteins involved in cellular interactions in tumor cells has not changed significantly. More interestingly, we confirmed that ICC maintains normal levels of blood glucose in diabetic animals and completed the present invention.

The present invention relates to a method of isolating islet single cells (ISC) by treating trypsin-EDTA in isolated pancreatic islets; Hanging-drop culturing the isolated ISC; And recovering islets cell clusters (ICC) from the current culture. The present invention also provides a method for producing a canola cell having improved insulin releasing ability.

Preferably, the step of culturing the isolated ISCs is cultured to include 250 to 4000 ISCs, but the present invention is not limited thereto.

Preferably, the sacrificial clusters may have a diameter of 70-150 탆, but are not limited thereto.

Also, the present invention provides a saccharide cluster having an improved insulin releasing ability, comprising 250 to 4000 ISCs and having a diameter of 70 to 150 탆.

In addition, the present invention provides a composition for preventing or treating diabetes mellitus comprising the above-described cancellous clusters as an active ingredient.

Preferably, the composition can be administered subcutaneously, but is not limited thereto.

The composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, and examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A solubilizing agent such as a flavoring agent can be used. The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.

The effective amount of the active ingredient of the composition of the present invention means the amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Islet (pancreatic islet)

250-300 g of healthy male Sprague-Dawley (SD) rats were used as donor cells. Experimental rats were housed at Yeungnam University Animal Care Center (Gyeongsan, Korea) under specific pathogen-free conditions. The rats were first anesthetized with ketamine (90 mg / kg) and xylazine (10 mg / kg) and sacrificed via cervical dislocations. All experimental protocols were carried out in accordance with the guidelines of Yeungnam University Animal Ethics Committee. Islet isolation and purification were performed as previously reported (Biochem Biophys Res Commun, 2013. 433 (4): 513-8).

< Example  2> Soap  Manufacture of islets cell clusters (ICC)

After 24 hours of additional incubation, the tissues were carefully removed from the culture plate and centrifuged to remove the medium. Thereafter, the tissue suspension was suspended in 0.25% (w / v) trypsin-EDTA (Hyclone) at 37 ° C for 10 minutes. Enzyme activity was terminated with RPMI-1640 supplemented with FBS. Thereafter, the isolated single cells (ISC) were washed away by centrifugation. Spoloid production was performed in RPMI-1640 medium containing 20% FBS (Hyclone). Briefly, to produce various sizes of ICCs, ISCs were counted using a hemocytometer and suspended in RPMI supplemented with 20% FBS and 1% antibiotic / antifungal agent. To prepare an ICC containing 250 ISCs, 5x10 ⁵ ISCs were suspended in 50 mL medium. Thus, 25 μL of cell suspension contained 250 ISC. Similarly, to produce ICCs containing 500, 1000, 2000, and 4000 ISCs, respectively, the same number of cells were suspended in 25, 12.5, 6.25, and 3.125 mL media. ICC was produced using the current culture method. To manufacture the clusters, droplets containing 25 [mu] L of each cell suspension were applied into a Petri dish lid containing distilled water to prevent cells from drying. Five days after incubation, ICCs were recovered using capillaries to determine the shape and size of ICC (Fig. 1).

Five days after the incubation, a light microscope image was observed to observe the morphology of the pseudo-islets. Unlike the perfect canopy, a constant size distribution was observed in each group of Pseudo - Doséo. The mean diameters of macroparticles and intact capsules were 201.5 ± 27.9 and 154.6 ± 47.5 ㎛, respectively. Similarly, the mean diameters of ICCs including 250, 500, 1000, 2000, and 4000 ISCs were 69.9 ± 5.3, 95.7 ± 5.1, 118.1 ± 5.8, 148.4 ± 9.6, and 206 ± 15.2 μm, respectively (FIG. The diameter of the ICC with 4000 ISCs was similar to the diameter of the giant metaplasia. Therefore, ICC with 4000 ISC was excluded in the subsequent experiments. Previous studies have shown that in vivo ICCs with diameters approaching 100 μm could survive in extreme hypoxic conditions. In addition, ICCs with extremely small diameters also had very low islet equivalence (IEQ). For animal model studies or clinical treatments, transplantation requires a large number of ICCs. Therefore, ICCs with extremely small diameters were excluded from further studies.

< Example  3> ICC's Survival

Cell viability was measured using Live / Dead Assay kit (Molecular Probes, Eugene, OR, USA). Briefly, in order to remove the media from hanging drops, the dog tissue clusters were harvested and centrifuged. The cells were then resuspended in 1 mL HBSS containing 2 μL of 50 μM calcein AM and 2 μL of 2 mM ethidium homodimer-1. The suspension was incubated in the dark for 15 minutes at room temperature. To remove the residual dye, the spoloids were washed twice with HBSS and resuspended in RPMI-1640 containing 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin, Under a microscope (Nikon Eclipse Ti). To determine the effect of hypoxia in in vitro culture conditions, control dogs and ICC were cultured at 37 ° C for 24 hours under hypoxic conditions (1% O ₂ , 5% CO ₂ and 94% N ₂ ). Viability was measured similarly to the previously reported method.

Under normal oxygen conditions, there was almost no difference in viability between the ICC groups and the whole tumor (Fig. 3). Conversely, when measuring the survival rate of the tumor cells after 24 hours of hypoxic culture, the morphology of intact metacercariae and giant ICCs collapsed severely. In addition, the live / dead assay results showed a significant reduction in viability in intact cancers compared to ICC (FIG. 4). The rate of apoptotic cells in ICC, including 4000 ISCs, was significantly higher than in the other groups. This clearly demonstrates that the harmful effect of hypoxia on cell survival increases with increasing size of metaplasia.

< Example  4> Glucose  Glucose stimulated insulin secretion; GSIS )

GSIS was performed to determine the functionality of pseudo-islets at elevated glucose concentrations. Islet cell is KRBB (pH 7.4; 115 mM NaCl , 5 mM KCl, 24 mM NaHCO 3, 2.5 mM CaCl 2, 1 mM MgCl 2, 10 mM HEPES and 1% bovine serum albumin (BSA) containing) (affymetrix, Cleveland, Ohio, USA). Spheroids were then pretreated with low glucose (2.8 mM) dissolved in KRBB for 1 hour. Cells were centrifuged to remove KRBB, and the spoloids were resuspended in low glucose KRBB solution. Cells were incubated for 2 hours and supernatant was collected to determine insulin secretion. KRBB solution containing high glucose (28 mM) was added to a tube containing HSs and cultured for 2 hours. To measure insulin secretion under high concentration conditions, the supernatant was collected via centrifugation. The amount of insulin secreted from each sample was measured using a rat / mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (EMD Millipore Corporation, Billerica, MA, USA). The stimulation index (SI) value was calculated by dividing the amount of insulin secreted under high glucose conditions by the amount of insulin secreted under low glucose conditions.

Insulin concentrations at low and elevated glucose concentrations were not significantly different from those at complete glucose tolerance. As a result, significant changes in the facilitation index could not be observed. In contrast, insulin levels secreted by intact capsaicin were significantly lower than ICC (Fig. 5) when GSCs were cultured for 24 hours in hypoxic conditions prior to GSIS. That is, the above results demonstrate the superiority of ICC under hypoxic conditions.

< Example  5> Within ICC ISC  Distribution

Five days after the current culture, ICC was recovered using a capillary tube. About 50-80 spoloids were collected in 1 mL e-tubes. The medium was completely removed and intact capecitabine or ICC was fixed with 4% paraformaldehyde (PFA) solution at room temperature for 15 minutes. PFA was removed by washing twice with phosphate-buffered saline (PBS). To avoid nonspecific binding of the antibody, a blocking reaction was performed with 1% BSA solution (affymetrix) and 0.1% tween 20 at room temperature for 1 hour. Then, 500 μL of the primary antibody cocktail, which was dissolved in PBS containing 1% BSA and 0.1% tween 20, was prepared. Briefly, primary antibody mixtures diluted 1/500 were prepared. Metabolism and ICC were incubated for 2 hours at room temperature in an antibody solution containing anti-insulin (Abcam, Cambridge, UK), anti-glucagon (Abcam) and anti-somatostatin (Abcam). To completely remove unbound antibody, the cells were rinsed 5 times with PBS. Secondary antibody solutions containing Alexa Fluor 647 anti-chicken (Abcam), Alexa Fluor 405 anti-mouse (Abcam) and IgG-FITC anti-rabbit (Santa Cruz) were diluted 1/500. After that, the cells were reacted with the secondary antibody solution mixed in PBS containing 1% BSA for 1 hour at room temperature. Unbound secondary antibody was removed by washing with PBS 5 times. Immunohistochemical staining and ICC were observed in a confocal microscope (Nikon Eclipse Ti, USA).

Confocal microscopy analysis shows that the 3-dimensional alignment of ISCs in ICC is identical to that of intact cells. Various molecules secreted by ICC were found to be involved in aggregation in a manner similar to intact cancellation (Fig. 6).

< Example  6> Collagen release by ICC

In order to confirm collagen secretion ability, ICC and intact capsaicin were prepared according to previously reported methods. Cells were harvested, fixed and permeabilized in a similar manner. Cells were then incubated with anti-collagen antibody (Abcam) diluted 1/500 at room temperature for 2 hours. After the unbound antibody was removed, the cells were reacted with IgG-FITC anti-rabbit antibody (Santa Cruz) for 1 hour at room temperature. After the unbound secondary antibody was completely removed, the cells were observed under a fluorescence microscope with a green channel (Nikon Eclipse Ti). The fluorescence intensity of collagen staining was quantified using software (Nikon Eclipse Ti). High resolution images were also obtained using a confocal microscope (Nikon Eclipse Ti).

Collagen is an extracellular component important in pancreatic islet cells. Collagen plays an essential role in protecting the islet viability and functionality. ICC exhibited green fluorescence intensity similar to intact cancellous cells under fluorescent staining (Figure 7). This strongly supports the preservation of the viability and functionality of ICCs.

< Example  7> Within ICC ISC  Interaction

Western blot analysis was performed to quantitatively evaluate various proteins involved in ISC interaction. Expression of connexin-36 (Cx36), pannexin-1 (pnx-1) and integrin-α (int-α) was measured in intact cancellous cells, ISCs, free cultured cancellous clusters and ICC. Freely cultured dog tissue clusters were prepared by culturing ISCs. Briefly, it was inoculated with 6 × 10 ⁵ ISC in 12-well plates. Thereafter, the ISC spontaneously refloated for 6 days. ISC was carefully pipetted daily in the wells to induce re-aggregation. To prepare samples for western blot analysis, dissociate ISC, ISC, free culture ISC and ICC in ice cold RIPA buffer (Thermo Scientific) containing dissolved Pierce protease and phosphatase inhibitor mini tablets (Thermo Scientific) . Protein concentration was measured using Pierce 660 nm protein assay reagent (Thermo Scientific). Equal amounts (30 μg) of protein from each sample were mixed in sample buffer, loaded, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins isolated by SDS-PAGE were transferred to Immobilin-P membrane (Millipore Corporation, Billerica, MA, USA) and incubated overnight at 4 ° C with Cx36 (Abcam), pnx-1 (Abcam), int- And the housekeeping protein β-actin (Abcam). The membrane was reacted with mustard peroperoxidase-conjugated secondary antibody at room temperature for 1 hour. Blots were identified using an enhanced chemiluminescence detection system (Thermo Scientific), fluorescence was recorded and bands were imaged. Quantitative determination of protein expression was performed three independent experiments using GelQuantNET software.

As shown in FIG. 8, when trypsinized monocytes with tumor cells, the expression of the protein in ISC was significantly reduced, indicating that the signal molecule was lost. Expression of In-α3 and Cx-36 in ISC free culture was similar. Interestingly, the expression of Pnx-1 in free-cultured cancellosa was identical to that of intact cancellation, which was shown to protect the α-β interaction in free-cultured ISCs. More importantly, the expression of these cell-interacting molecules in ICC is nearly identical to intact cells. Expression of Cx-36 in ICCs accounted for approximately 80% of intact cancers, which is noteworthy.

< Example  8> in vivo in  ICC evaluation

To confirm in vivo efficacy, ICCs were transplanted into streptozocin-induced diabetic mice. For the preparation of transgenic animals, 200 mg / kg streptozocin was injected into the abdominal cavity of BALB / C nude mice. Briefly, before injection, STZ was dissolved in cold citric acid buffer (pH 4.5). Mice with blood glucose levels above 300 mg / dL for two consecutive days were selected as diabetic donors. For subcutaneous delivery of the canine, the following six group mice were prepared. (i) 600 IEQs in Matrigel, (ii) 300 IEQs in Matrigel, (iii) 150 IEQs in Matrigel, (iv) 1000 ICC in Matrigel, (v) 500 ICC in Matrigel and (vi) 250 ICC in Matrigel. Prior to injection, Matrigel was dissolved overnight at 4 ° C. On the next day, doxepsin and ICC were collected, suspended in 20 μL of medium, and mixed with ice-cold Matrigel to the subcutaneous space of anesthetized mice. Before the mice woke up, the injections solidified. Non-fasting blood glucose levels were measured from the tail vein using a mobile blood glucose meter. If the blood glucose level was more than 250 mg / dL for more than two consecutive days, the immune rejection was judged. The body weight of the transplanted mice was observed at regular intervals.

In addition, intraperitoneal glucose tolerance test (IPGTT) was performed at 30 days after transplantation to confirm the reactivity of grafted canine to immediate rise in blood glucose level. The transplanted mice were starved overnight and administered 2 mg / kg of 20% glucose intraperitoneally. Blood glucose levels were measured after 0, 5, 10, 15, 20, 30, 45, 90 and 120 min.

As shown in Figure 9, diabetic BALB / c nude mice transplanted with 600 IEQ and the same amount of ICC (1000 ICC) had blood glucose levels regulated for more than a month. Unstable blood glucose profiles were observed in the 300 and 150 IEQs transplanted groups. Conversely, blood glucose levels were well controlled in mice transplanted with 500 ICC (equivalent to 300 IEQ), suggesting that the problem of ICC transplantation into the subcutaneous space has improved.

Intraperitoneal glucose tolerance test was performed in normal mouse, diabetic mouse, and all transplanted groups. Within hours of fasting, blood glucose levels dropped below 200 mg / dL in all transplanted mice. However, the mean blood glucose level in 150 IEQ transplanted mice did not decrease, indicating that 150 IEQ was insufficient to restore diabetic conditions. Significant differences can be identified by comparing the blood glucose levels of 250 ICC and 150 IEQ transplanted mice with significantly reduced blood glucose levels in the fasting state (Figure 10). When 20% glucose was administered intraperitoneally, all groups adjusted blood glucose levels in a manner similar to that of normal mice. In contrast, 150 IEQ transplanted mice did not recover normal blood glucose until 1 hour after administration.

While the present invention has been described with reference to exemplary embodiments and drawings, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be apparent to those skilled in the art that various changes and modifications It is to be understood that various changes and modifications may be made without departing from the scope of the appended claims.

Claims (6)

Isolating islet single cells (ISC) by treating the isolated pancreatic islets with trypsin-EDTA;
Hanging-drop culturing the isolated ISC; And
And recovering islets cell clusters (ICC) from the current culture. 2. The method of claim 1, wherein the step of culturing the isolated ISCs is performed so as to include 250 to 4000 ISCs. 2. The method of claim 1, wherein the wet tissue clusters have a diameter of 70 to 150 mu m. And 250 to 4000 ISC, and having a diameter of 70 to 150 占 퐉. A composition for preventing or treating diabetes comprising the metapoker cluster according to claim 4 as an active ingredient. The composition for preventing or treating diabetes according to claim 5, wherein the composition is administered subcutaneously.

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