KR101177273B1 - Method for improving pancreatic islet cell survival or its activity - Google Patents

Abstract

The present invention relates to a method for increasing the survival rate or the function and activity of the islet cells in culture, and more particularly, comprising co-culturing mesenchymal stem cells in the culture of the islet cells. It provides a method or a method for increasing the survival rate or the activity of the culturing islet cells, characterized in that it comprises the step of culturing the islet cells with a culture medium of mesenchymal stem cells in the culture of the islet cells. The present invention also provides a kit for culturing the culture or pancreatic islet cells therefor.

According to the present invention, it is possible to increase the survival rate, activity or function of pancreatic islets during the culturing of pancreatic islets so that the transplantation of pancreatic islets can be performed more successfully.

Islet Transplantation, Mesenchymal Stem Cells, Islet Culture, Umbilical Cord Blood, Bone Marrow, Cytokine

Description

Method for improving pancreatic islet cell survival or its activity

The present invention belongs to the area of cell culture technology and pre-organization pretreatment technology in the field of clinical medicine, and relates to a method for enhancing the survival rate or its function or activity in the culture of islet cells.

Currently, 4.6% of patients worldwide suffer from diabetes, and are expected to continue to grow and exceed 6.0% by 2025. Recently, diabetes treatment through pancreatic transplantation and pancreatic islet cell transplantation has been carried out. However, the current isolation and culture technology for transplanting pancreatic islets is a pancreatic islet cell obtained from 2 to 4 donors, which can be transplanted into one diabetic patient. Insulin-independent maintenance after transplantation is 10% (5 years). The situation is not exceeding. Therefore, as well as studies aiming to optimize the isolation and culture conditions of pancreatic islets and minimize the damage of transplanted pancreatic islets (1-5), various methods of proliferating pancreatic islets, animal tissues other than humans He is also conducting research on methods such as xenotransplantation. In addition, when the pancreatic islet cells are separated from the pancreatic tissue, there is a problem that the function of the pancreatic islets is impaired and apoptosis is caused. Unlike the general cell lines, pancreatic islet cells are known to rapidly increase cell loss depending on the culture period.

On the other hand, various growth factors are now known to improve the function and activity of pancreatic islets (6), and the trend and the mechanisms for their effects on pancreatic islets are being multifaceted. Vascular endothelial growth factor (VEGF (7,8)), interleukin-6 (IL-6 (9,10)), TGF-beta (11), and hepatocyte growth factor (Hepatocyte) growth factor: HGF (12)), and studies have shown that these growth factors are among the growth factors released by mesenchymal stem cells by trophic effect (13) on adjacent cells. . However, the improvement of biological function and activity of pancreatic islet cells by the combination of these growth factors has not been studied until now.

At home and abroad, research is being conducted on various aspects of improving the quality of the pancreatic islet cells or maintaining them in the long term. Many single factors and their mechanisms have also been studied. However, the trophic effects of parenchymal and mesenchymal cells, especially mesenchymal stem cells, on the parenchymal and mesenchymal cells have not been actively studied. There is a situation. Izumida Y et al . Although a group of fibroblasts co-cultured with islet cells to increase their activity and function was reported in 2006 (14), the trophic effect compared to mesenchymal stem cells. Is considered to be weak, and comparative verification in terms of substantially controlling blood sugar at the level of animal experiments has not been conducted, and the mechanism has not been studied. After culturing pancreatic islet cells using a culture medium in which islet cells and co-cultures or mesenchymal stem cells were cultured using bone marrow and cord blood-derived mesenchymal stem cells, the quality and activation process were confirmed in various aspects. Will focus on elucidating the mechanism and the first to demonstrate practical effects at the in vitro as well as animal level. It is believed to be a study.

In the present clinical trial, when pancreatic islet cells of another person are transplanted to the patient through the islet cell transplantation, the 5 year success rate is not high due to the gradual death of the transplanted islet cells (15). This is thought to be due in large part to the deterioration of the activity and function of pancreatic islets in the culture stage prior to islet cell transplantation. Therefore, it is a problem to be solved by the present invention to establish a culture method that can increase the survival rate and prevent activity or reduced function in the culture step of the islet cells before transplantation to the patient.

In order to solve the above problems, the present invention co-cultures the mesenchymal stem cells isolated from human-derived bone marrow and umbilical cord blood with pancreatic islet cells isolated from mice and humans to increase the survival rate of pancreatic islets and maintain the activity or function of the islets. Provide a way to.

The present invention also provides a method of culturing pancreatic islet cells with a culture medium prepared using mesenchymal stem cells to increase the survival rate of pancreatic islet cells and maintaining the activity or function of the islet cells.

According to the present invention, when the islets are cultured, the biological activity and function of the islets are reduced, and the survival rate of the islets is increased, so that the islet cells can be transplanted more successfully.

The present invention relates to a method for increasing the survival rate of pancreatic islet cells and maintaining the activity or function of pancreatic islet cells by co-culturing the mesenchymal stem cells derived from bone marrow or umbilical cord blood and islet cells.

The inventors of the present invention surprisingly found that the co-culture of human-derived mesenchymal stem cells and pancreatic islets increases the survival rate of the islet cells compared to the control group (FIG. 1), and confirmed this by AO / PI staining (FIG. 2). . In addition, the results of GSIS (glucose-stimulated insulin secretion), a test for verifying the ADP / ATP ratio and insulin secretion ability, confirmed that the function and activity of the co-cultured pancreatic islet cells were improved as compared to the control group (FIG. 3A-A). D).

In addition, the inventors of the present invention found that the activity of pancreatic islets is increased even when the islet cells are cultured using the culture supernatant cultured with mesenchymal stem cells (Fig. 3 E-F).

 In order to elucidate the mechanism by which such mesenchymal stem cells or their cultures affect pancreatic islet cells, the inventors of the present invention observed the expression of cytokines and proteins in the culturing stage, and as a result, the function and activity of It has been found that IL-6, HGF, VEGF, FGF, TGF-beta, etc., which are known to help a lot, have been increased (AE of FIGS. 5A and 5B), and it is also noteworthy that the pro-inflammatory cytokine is Interferon-gamma and TNF-alpha have been reduced (EF in FIG. 5B), and these results provide mechanistic clues to prevent the death of pancreatic islets in vitro. have. In addition, a decrease in P27 and an increase in PDX-1 were observed as proteins related to the proliferation of beta cells in pancreatic islets during the coculture. In addition, the expression of PI-3K, which is known as the higher signaling protein of these two proteins, is increased, and it can be inferred that the expression changes of P27 and PDX-1 are derived from the higher signaling level. Suggests that it may come from derived mesenchymal stem cells. Taken together, these results can indirectly confirm that co-culture with human-derived mesenchymal stem cells affects the proliferation of beta cells in pancreatic islets. In addition, the expression of XIAP, which inhibits the expression of CASPASE protein, a mechanism involved in cell death, was observed to be increased by co-culture, confirming the state of inducing the inhibitory effect of islet cell death. In culture, the expression of HSP32 and HSP70 proteins in pancreatic islets increased by inflammatory reaction and oxidative stress, indicating that the activity of pancreatic islets is excellent (FIG. 6).

These pancreatic islets were cultured for 48 hours in mesenchymal stem cell cultures for the purpose of verifying the in vitro test result in co-culture in the type 1 diabetic mouse model using STZ (streptozotocin). The cells were transplanted under the kidney membrane of the mouse, and the change in blood glucose was observed for 2 weeks. As a result, the blood glucose was continuously maintained as compared to the control group using the normal culture medium. It was confirmed in vivo (FIG. 7A), and the weight of the pancreatic islet cells cultured in the culture medium of the mesenchymal stem cells was confirmed to increase normally (FIG. 7B). In addition, in the Intraperitoneal Glucose Tolerance Testing (IPGTT) performed to confirm the function of the transplanted islets, the group transplanted with the pancreatic islet cells cultured in the mesenchymal stem cell culture solution compared to the control group showed even better results. It was confirmed to show (FIG. 7C).

Based on the experimental results as described above, the present invention provides a method for increasing the survival rate of pancreatic islet cells, characterized in that it comprises the step of co-culture with the mesenchymal stem cells in the culture of the islet cells.

In another aspect, the present invention provides a method for maintaining the function or activity of pancreatic islet cells in the culture of pancreatic islets comprising the step of co-culturing with mesenchymal stem cells in the culture of pancreatic islets.

In another aspect of the present invention, the present invention provides a method for increasing the survival rate of pancreatic islet cells, comprising culturing the mesenchymal stem cells, taking a supernatant thereof, and culturing the islet cells in the supernatant. to provide.

In another aspect, the present invention provides a method for maintaining the function or activity of pancreatic islet cells, characterized in that it comprises the step of culturing the mesenchymal stem cells and taking the supernatant thereof, and culturing the islet cells in the supernatant. .

The mesenchymal stem cells used in the present invention may be selected from the group consisting of bone marrow derived and cord blood derived mesenchymal stem cells. The mesenchymal stem cells may be derived from mammals including humans and mice.

The present invention also provides a pancreatic islet cell culture medium comprising mesenchymal stem cell culture medium.

The present invention also provides a kit for culturing pancreatic islets comprising mesenchymal stem cell culture.

As the culture medium of pancreatic islets, M199, CMRL1066, Miami medium, RPMI1640 may be used, but are not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example

Example  1: cord blood and Bone marrow  collection ( Cell collection )

Umbilical cord blood was collected from full-term or preterm laborers at Samsung Medical Center according to the institutional guidelines. Blood was collected in a 250 ml standard blood collection bag containing green citric acid-phosphate-dextrose anticoagulant (CPDA).

Bone marrow was collected by aspiration from a brain lion's iliac bone into a 50 mL syringe containing 5 mL of heparin. According to the Institutional Guidelines, Samsung Medical Center was extracted with the donor's prior consent.

Example  2: Intermediate lobe  Stem Cell Isolation and Culture

In order to isolate mesenchymal stem cells, the method presented by Bieback et al (16) group was self-improved. Umbilical cord blood and bone marrow fluid isolated in Example 1 were diluted 1: 1 with phosphate-buffered saline to which 2 mM EDTA (Sigma, St. Louis. MO, USA) was added, and 50 mL for density gradient. Slowly superimpose prediluted bone marrow fluid and umbilical cord blood in a pre- aliquoted Ficoll-Paque Plus (Amersham, Freiburg, Germany) at 20 mL in a tube, then centrifuge at room temperature for 400 g, 30 minutes, without braking, The red blood cell layer, the mononuclear cell layer in the middle, and the serum layer on the top were separated. Using a pipette, the mononuclear cell layer was collected and transferred to a 50 mL tube, filled with 50 mM phosphate-buffered saline with 2 mM EDTA (Sigma, St. Louis. MO, USA), followed by 1800 rpm, 10 After centrifugation at room temperature for minutes, the supernatant was removed, washed with an additional 50 ml of phosphate buffer solution, centrifuged at 1800 rpm for 10 minutes, and resuspended in 25 mL of DMEM-Low glucose culture. After staining using tryphan blue (Gibco, Grand Island, NY, USA), the cell viability and cell number were measured by a hemocytometer. T-75 cm ² 1.5 x 10 ⁶ monocyte cells per square centimeter (cm ² ) were plated on DMEM-low glucose with 10% FBS and 2 mmol / l L-glutamine (Dulbeco's Modified Eagle Media-low glucose: Gibco, Grand Island, NY, USA) medium. Incubate in a 37 ° C. constant temperature 5% CO 2 incubator for one week, and remove cells that have not adhered to the culture dish one week later using phosphate-buffered saline, followed by 10% FBS and 2 mmol / l L. -Passage was cultured using glutamine added DMEM-low glucose (Dulbeco's Modified Eagle Media: Gibco, Grand Island, NY, USA).

Example  3: Intermediate lobe  Stem cell culture Supernatant  Produce

Umbilical cord blood-derived mesenchymal stem cells in culture by the method of Example 2 to 1x10 ⁶ cells T-75 cm ² The culture plate was dispensed using DMEM-low glucose (Gibco, Grand Island, NY, USA) medium with 10% FBS and 2 mmol / l L-glutamine. Cultured in a constant temperature 5% CO2 incubator at 37 ℃ for 24 hours, and observed under a microscope to confirm that the cell number density compared to the cell culture dish was 60 ~ 70%. Cultures were replaced with CMRL1066 with 10% FBS for human pancreatic islet cell culture and M199 with 10% FBS for mouse pancreatic islet cells, and cultured in a 37 ° C. constant temperature 5% CO 2 incubator for 48 hours. After 48 hours, the culture solution on the culture dish was collected by pipette, the supernatant was filtered through a syringe filter (0.22 μm pore size; BD, San Diego, Calif.), And only the pure culture medium from which the suspended solids and dead cells were removed was removed. Collected and stored in 4 ℃ refrigerator.

Example  4: Pancreatic islet cells  Isolation and incubation.

4-1) human Islet cells  Isolation and Cultivation

Human pancreatic islet cells used in the present invention used a brain lion pancreas. According to the institutional guidelines, the pancreas was extracted with the donor's prior consent. The pancreatic islets were surgically sterile and immediately placed in an ice-filled University of Wisconsin solution and transferred to the pancreatic islet chamber (17). That is, liberase was slowly injected into the pancreatic duct through a catheter, and then the swelled pancreas was placed in a separate chamber, and the chamber was gently shaken while circulating 37 ° C. HBSS prepared in advance using a peristaltic pump into the chamber. Liberase (Roche; Nutley, NJ, USA) also allowed the circulating fluid to rise 1-2 ° C. per minute in the pancreas, allowing the circulating fluid to flow at a rate of 70-79 mL per minute. At this point, the digested solution was taken at intervals of about 1-2 minutes, stained with 0.2 mM diphenylthiocarbazone, and observed under a microscope to determine that at least one fully isolated islet was digested. 4 ° C. HBSS (Hank's balanced salt solution; Louis, MO, USA) was rapidly injected and the tube drawn into the chamber was immersed in ice to rapidly lower the chamber temperature to inactivate liberase degrading enzyme. Digestive tissue from the chamber was collected one by one in a tube containing HBSS (containing 10% FBS), centrifuged (1500 rpm, 5 minutes, 4-6 times), and purely separated using a cell separator Cobe2992. The obtained pancreatic islets were cultured at 37 ° C. for 2 days, and then the supernatant of each stage was collected and tested for microbial contamination.

4-2) mouse Pancreatic islet  Isolation and Cultivation

Isolation of mouse islet cells was based on the method of injecting collagenase (type P, Sigma) solution directly into the common bile duct of the mouse. Before the experiment, collagenase was dissolved in HBSS solution at a concentration of 0.8 mg / ml, passed through a 0.22 μm syringe filter, and maintained at 4 ° C. to be used for the experiment. Mice were opened under anesthesia by intraperitoneally administering a ketamine mixture prepared by mixing ketamine (45 mg / kg) and xylazine (2.5 mg / kg). The distal portion was ligated and a 30 gauge needle was inserted proximal to the bile duct. Inflate the pancreas by injecting 2.5-3 ml of collagenase solution through an intubated needle, extract the expanded pancreas, place it in a 50 ml tube, incubate for 15-20 minutes in a 37 ° C thermostat, and then vortex for about 10 seconds. ) Crushed digested tissue. 20 ml of HBSS + 10% FBS solution cooled to 4 ° C. was added to the solution to stop the action of collagenase, and the cells were collected by centrifugation (2000 rpm, 1 minute). The turbidity was washed twice with HBSS at 4 ° C., and then passed once through a 1200 μm and 500 μm mesh filter (Bellco, Vineland, NJ, USA), respectively, and the tissue passed through the cooled HBSS solution. After washing twice with centrifugation (1500 rpm, 2 minutes), the supernatant is removed. Add 7 ml of 27% Ficoll solution to the precipitated tissue, mix well, and make a discontinuous gradient with 5 ml of 23%, 20.5%, and 11% Ficoll solution in sequence, after centrifugation (2000 rpm, 20 minutes, no braking). The pancreatic islets were removed using a pipette at the 11% and 20.5% interfaces. The washed pancreatic islets were washed twice with cooled HBSS solution, and then cultured in a culture solution containing 10% fetal bovine serum, 2 mg / ml glucose and antibiotics in a 37 ° C. 5% CO2 incubator.

Example  5: human origin Intermediate lobe  Stem cells Pancreatic islet cells  Coculture

Bone marrow and umbilical cord blood-derived mesenchymal stem cells isolated in Example 2 were prepared in a 24-well culture dish with 3 x 10 ⁴ cells per well. The cells were pre-dispensed and cultured in DMEM-low glucose (Gibco, Grand Island, NY, USA) culture medium with 10% FBS and 2 mmol / l L-glutamine in a 37 ° C. constant temperature 5% CO 2 incubator for 24 hours. After 24 hours, it was observed under a microscope to confirm that the cell number density became 60-70% compared to the cell culture dish, and after removing the supernatant, washing with phosphate buffer solution (PBS) three times, and having a pore size of 12 μm. Cell culture inserts (Millicell; Millipore. Mississauga, Ontario) were inserted into the wells of the culture dish, respectively, and cultures for pancreatic islet cell culture [for human islets: CMRL1066 medium (Gibco, Grand Island, NY, USA), mouse islets In the case of: M199 medium (Gibco, Grand Island, NY, USA)] was dispensed with islet cells 700 μl per well. Incubate for about 48 hours in a 37 ° C. constant temperature 5% CO2 incubator to create a condition in which cytokines secreted from mesenchymal stem cells are mixed on the basal medium, and by using a cell culture insert, the culture medium is shared while the mesenchymal stem cells are shared. And pancreatic islet cells were not mixed.

Example  6: Intermediate lobe  Stem Cell Culture Supernatant  Culture of Pancreatic Islet Cells

Using the supernatant obtained by culturing the mesenchymal stem cells by the method of Example 3, the isolated islet cells were cultured by the method of Example 4, and the function and survival rate of the islets were observed. An appropriate number of Petri dishes of 100 mm size was prepared for culturing isolated islet cells. The pancreatic islets immediately after the separation process were washed with HBSS and centrifuged (1500 rpm, 30 seconds) to remove supernatant. The supernatant obtained by culturing the mesenchymal stem cells was resuspended, and about 700-1000 islets were divided and divided into each Petri dish. The supernatant obtained by culturing the mesenchymal stem cells in a petri dish containing each divided pancreatic islets is added so that the total amount is about 12 ml. Pancreatic islet cells dispensed in 12 mL of culture were incubated for approximately 48 hours in a 37 ° C. 5% CO ₂ incubator.

Example  7: cultured Pancreatic islet cells Survival rate

Human pancreatic islet cells were cultured for 24 and 72 hours using the human-derived mesenchymal stem cell supernatant by the method of Example 6, and then counted by counting the live islet cells without being killed. Figure 1 shows the culture of pancreatic islets in cultured Miami media containing M199 + 10% FBS, CMRL1066 + 10% FBS, and cytokines currently used in pancreatic islet transplantation clinical trials, which are commonly used for the study of mouse and human islets. When the mesenchymal stem cells were cultured in a conditioned media in CMRL1066 obtained by culturing in CMRL1066, the number of viable islets is expressed in EIN unit, which is a unit of the number of islets. When the same number of pancreatic islets were dispensed, it was observed that the number of remaining pancreatic islets is relatively high in the experimental group co-cultured with the mesenchymal stem cells as compared with the conventional culture medium (FIG. 1). .

Example  8: AO Of PI  By dyeing Islet activity  Measure

Mouse pancreatic islet cells were co-cultured with human-derived mesenchymal stem cells and cultured without mesenchymal stem cells (control), and AO (Acridine Orange) / PI (Propidium iodide), a staining method for measuring activity of pancreatic islet cells The results of staining were compared. In Vitro Cell Dev Biol. Based on the method of 1988 Apr; 24 (4): 266-73, mouse pancreatic islet cells from each of the co-cultured experimental groups were collected and centrifuged for 15 seconds at 8 ° C and 1500 rpm, and the pancreatic islet cells were placed on a 15 mL tube. Down. Resuspended in 1 mL of HBSS and transferred to 1.5 mL of E-Tube (Eppendorf tube). 100 μl of each Acridine orange (0.67 μmolL) and PI (75 μmolL) and 100 μl of the islet cell suspension were mixed, dispensed into a slide glass, and observed under a fluorescence microscope. In FIG. 2A, the upper part is a control group, and the lower part is a case of co-culture with mesenchymal stem cells. When co-cultured with mesenchymal stem cells, it was confirmed that PI is stained with red color in dead cells on the islets of mice.

After culture, the cells were stained with AO (Acridine Orange) / PI (Propidium iodide) on days 1, 3, and 5, respectively, and when the islets were observed, bone marrow (D in FIG. 2) and cord blood (C in FIG. 2) were derived. When co-cultured with mesenchymal stem cells, it was confirmed that the ratio of viable islets is higher than that of the control group (B of FIG. 2) .

Example  9: cultured Pancreatic islet cells  Comparison of active or functional

Pancreatic islet cells isolated from mice and humans were co-cultured with human bone marrow and cord blood-derived mesenchymal stem cells, and an ADP / ATP assay, which is an experimental method for measuring the quality of pancreatic islet cells, was performed. The experimental method provided on the Aspoglow BioAssay Kit (Lonza, Rockland, ME, USA) was changed to fit the islet experiments and hand-picked the pancreatic cells cultured under the respective experimental conditions according to the number of samples (N value). Collected by hand-picking and transferred to 1.5 mL E-Tube (Eppendorf tube). After centrifugation at 4000 RPM for 2 minutes, washing with phosphate buffer again, centrifugation was performed once again under the same conditions, and the supernatant was removed. 100 μl of NRR buffer provided on the assay kit was added and allowed to stand at 4 ° C. for 15 minutes, 20 μl of NMR buffer was added and measured by a luminometer (A value), and then at room temperature for 10 minutes. After standing still and measuring again with a luminometer (B value), putting ADP-CR buffer in each sample again, and after 5 minutes at room temperature, measuring again with a luminometer (C value), the ADP / ATP ratio value is (C value-B value) / A value. Coculture with mesenchymal stem cells in both mouse islets (FIG. 3A) and human islets (FIG. 3B) resulted in reduced ADP / ATP results, demonstrating that the quality of pancreatic islets is relatively good. It is reported that pancreatic islets with a low ATP / ADP ratio have better activity or function (18).

In addition, in the case of conducting GSIS (glucose-stimulated insulin secretion), a method for verifying insulin secretion, co-culture with mesenchymal stem cells in both mouse islets (FIG. 3C) and human islets (FIG. 3D) was compared with the control group. It was confirmed that insulin secretion is enhanced, and even more excellent results were obtained when cultured in cord blood-derived mesenchymal stem cells.

In the case where pancreatic islet cell cultures were prepared using mesenchymal stem cells, the results of confirming that the culture medium in which the mesenchymal stem cells were cultured were effective for improving the quality of the islet cells through the ADP / ATP assay (FIG. 3E). 2 days, the culture medium of the mesenchymal stem cells increased the quality of mouse islet cells the most.

In addition, the mice were cultured for 1, 2, 3, and 5 days using mesenchymal stem cell cultures for 2 days after screening for 2 days, followed by ADP / ATP assay. It was confirmed that the results showing the quality of the islet cells in the group using mesenchymal stem cell culture medium (FIG. 3F).

Example  10: Intermediate lobe  Cytokines in Stem Cell Cultures

Cytokines detected on human-derived mesenchymal stem cell cultures for culturing pancreatic islets were screened by ELISA method (FIG. 4). Significant concentrations of TGF-beta, HGF, VGEF, IL-6, and FGF, which are known to help a lot of the function and activity of islets, were detected. A) Culture medium using bone marrow-derived mesenchymal stem cells B) Culture medium using cord blood-derived mesenchymal stem cells

Example  11: Islet cells Intermediate lobe  Changes in Cytokines in Stem Cell Co-Culture

When co-culturing human-derived pancreatic islets and mesenchymal stem cells, supernatants were collected from the culture medium in coculture to observe changes in cytokines in the culture medium, and changes in cytokines were observed by ELISA (FIG. 5). ). The control group is a group cultured with islets alone, and the experimental group is a group in which the islets are co-cultured with mesenchymal stem cells. The results showed that IL-6, HGF, VEGF, TGF-BETA, and FGF-2, which are known to have positive effects on pancreatic islets, were increased during co-culture. Also, the pro-inflammatory cytokine Interferon-gamma and TNF-alpha have been reduced, and this result provides a mechanism for preventing the death of pancreatic islets in vitro.

Example  12: Islet cells Intermediate lobe  Protein Expression in Stem Cell Co-Culture

In order to confirm the mechanism by which pancreatic islet function and activity are increased through co-culture with human-derived mesenchymal stem cells, protein expression in pancreatic islet cells was compared using western-blotting (FIG. 6). . As a result, as previously reported as a protein involved in the proliferation of beta cells, it is known that the decrease in P27 (19,20) and the increase in PDX-1 (21,22) are related to the proliferation of beta cells. In this experiment, decrease and increase of P27 and PDX-1 were observed. In addition, the expression of PI-3K, known as the higher signaling protein of these two proteins, increased (23, 24, 25), suggesting that the expression changes of P27 and PDX-1 are derived from higher signaling levels. And suggest that the onset of this signal may come from human derived mesenchymal stem cells. Taken together, these results can indirectly confirm that co-culture with human-derived mesenchymal stem cells affects the proliferation of beta cells in pancreatic islets.

In addition, the expression of XIAP (26, 27), which inhibits the expression of the CASPASE protein, a mechanism involved in cell death, was increased, indicating that the inhibitory effect of apoptosis is also induced. In the case of HSP32 and HSP70 islets (28-31) induced by inflammatory reactions and oxidative stress, the activity of pancreatic islet cells increased according to the results of the well-known islets.

Example  13: Intermediate lobe  Cultured using the stem cell culture solution Pancreatic islet cells  Effect validation in animal models of diabetes

In vitro testing confirmed the tendency of the quality of pancreatic islet cells cultured with stem cell cultures to be enhanced, and whether mice with Streptozotocin (Sigma, St. Louis. MO, USA) induced better experimental results in vivo. Was verified in the diabetic model. The experimental group and the control group were compared to verify the effect in the animal experiment. Animal experiments were carried out under pancreatic islet cells cultured with pancreatic islet cells cultured using control pancreatic and mesenchymal stem cells under the kidney membrane of mice prepared with each diabetic model. After comparing the increase and decrease of glucose metabolism capacity and body weight, the activity of the settled islet cells was compared.

Diabetes Effect Verification: The in vitro test confirmed the tendency of improving the quality of pancreatic islet cells cultured with stem cell culture, and whether Streptozotocin (Sigma, St. Louis. MO, USA) can show better experimental results in vivo. Were validated in the diabetic model of the mouse. The experimental group and the control group were compared to verify the effect in the animal experiment. Animal experiments were carried out in the renal capsule of the mouse prepared with each diabetic model and transplanted into the renal capsule, which is expected to increase the biological activity of the pancreatic islet cells cultured using control islet and mesenchymal stem cells. After comparing the increase and decrease of glucose metabolism ability and body weight, the activity of the settled islet cells was compared.

① Diabetes model induction

Preparation of a Type 1 DM mouse model to verify the effect of pancreatic islet cell transplantation consisted of administration of Streptozocine (STZ). STZ solution for administration is prepared fresh by dissolving in Citric buffer on each day of administration. Mice were fasted the day before dosing, and blood glucose was measured immediately prior to the administration of STZ to provide a basis for verifying the effect after dosing. STZ is administered intraperitoneally at a dose of 180 mg / kg. After administration, the food was fed normally, and the blood glucose was measured daily to confirm the induction of the diabetes model (blood glucose above 300 mg / dL).

② Islet Transplantation

Transplantation of renal subcapillary islet cells in mice proceeded after maintenance anesthesia by intraperitoneal injection of a mixture of ketamine (45 mg / kg) and xylazine (2.5 mg / kg). The anesthetized mouse is incised and the kidney is taken out with a sterile cotton swab to remove the kidney membrane using a glass swab. Then, the micro-injector (self-made) islet is transplanted and the kidney is After it is put in place, the incised skin is closed. Thereafter, the recovery was sufficiently on a hot plate with sterilized air bubbles.

③ Measure of metabolism

After transplantation, the difference in glucose metabolism ability was confirmed using the blood glucose measurement and IPGTT (Intraperitoneal Glucose Tolerance Testing) in experimental and control mice.

  ④ weight measurement

By observing weight gain in experimental and control mice after transplantation, the activity of transplanted islet cells was indirectly confirmed.

Mouse pancreatic islet cells cultured with mesenchymal stem cell culture for 48 hours were transplanted under the kidney membrane of the mouse, and the blood glucose change was observed for 2 weeks. As a result, the blood glucose was continuously maintained as compared to the control group using the normal culture medium. , Compared with the control group, it was confirmed that the blood glucose control ability in vivo (Fig. 7A). In addition, as a result of observing the change in body weight, it was confirmed that the body weight was normally increased in the group transplanted with the islet cells cultured in the culture medium of the mesenchymal stem cells (FIG. 7B). In addition, in the Intraperitoneal Glucose Tolerance Testing (IPGTT) performed to confirm the function of the transplanted islets, the group transplanted with the pancreatic islet cells cultured in the mesenchymal stem cell culture solution compared to the control group showed even better results. It was confirmed to show (FIG. 7C).

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Figure 1 shows a comparison of the number of live islet cells without killing after culturing pancreatic islet cells in the case of co-culture with human-derived mesenchymal stem cells and pancreatic islets.

Figure 2 compares the results of AO / PI staining when mouse islets are co-cultured with human-derived mesenchymal stem cells and when they are cultured without mesenchymal stem cells.

FIG. 3 compares the results of ADP / ATP assay and insulin index in co-culture of human or mouse islet cells with human-derived mesenchymal stem cells and in the absence of mesenchymal stem cells (control). The results of the daily ADP / ATP assay were confirmed for the establishment of pancreatic islet cells cultured using mesenchymal stem cells and for the culture of pancreatic islets. BM-MSC: bone marrow-derived mesenchymal stem cells, CB-MSC: umbilical cord blood-derived mesenchymal stem cells

Figure 4 is the result of screening the cytokine detected in human-derived mesenchymal stem cell culture medium for culturing pancreatic islet cells by ELISA method. A) Culture medium using bone marrow-derived mesenchymal stem cells B) Culture medium using cord blood-derived mesenchymal stem cells

Figure 5 shows the cytokine changes in the culture medium when the co-culture of human-derived pancreatic islets and mesenchymal stem cells by ELISA method.

Figure 6 compares the expression of proteins in pancreatic islet cells when pancreatic islets are co-cultured with human-derived mesenchymal stem cells and when cultured without mesenchymal stem cells.

Figure 7 is a result of verifying the effect in the animal model of pancreatic islet cells cultured using the cultured mesenchymal stem cells.

Claims (7)

A method of increasing the islet cell survival rate in the culture of pancreatic islets, characterized in that it comprises the step of co-culture with the mesenchymal stem cells in the culture of the islets, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells and cord blood-derived mesenchyme The pancreatic islets are not cryopreserved and the co-culture is done in vitro at 37 ° C. A method for maintaining the function or activity of pancreatic islet cells in islet cell culture, characterized in that it comprises the step of co-culturing with the mesenchymal stem cells in the culture of the islet cells, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells and Cord blood is selected from the group consisting of mesenchymal stem cells and the islet cells are not cryopreserved and the co-culture is in vitro at 37 ℃. A method for increasing pancreatic islet cell viability in culturing pancreatic islet cells, comprising culturing the mesenchymal stem cells and taking their supernatants and culturing islet cells in the supernatant, wherein the mesenchymal stem cells are bone marrow-derived intermediate The islet is selected from the group consisting of mesenchymal stem cells and cord blood-derived mesenchymal stem cells and the islets are not cryopreserved and the islets are cultured in vitro at 37 ° C. A method for maintaining the function or activity of pancreatic islet cells in the culture of pancreatic islets comprising culturing the mesenchymal stem cells and taking their supernatants and culturing islet cells in the supernatant, the mesenchymal stem cells Is selected from the group consisting of bone marrow-derived mesenchymal stem cells and umbilical cord blood-derived mesenchymal stem cells, wherein the islets are not cryopreserved and the islets are cultured in vitro at 37 ° C. delete A medium for islet cell culture comprising mesenchymal stem cell culture, wherein the mesenchymal stem cell is selected from the group consisting of bone marrow-derived mesenchymal stem cells and umbilical cord blood-derived mesenchymal stem cells and the pancreatic islet cells are frozen Medium which is not conserved and said islet cell culture is in vitro at 37 ° C. A pancreatic islet culture kit comprising mesenchymal stem cell culture, wherein the mesenchymal stem cells are selected from the group consisting of bone marrow-derived mesenchymal stem cells and umbilical cord blood-derived mesenchymal stem cells and the pancreatic islet cells are frozen And not conserved and said islet cell culture is in vitro at 37 ° C.

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