Transport Mode
High-speed rail transportation
1. Shockproof foam box transportation.
2. Fix the insulated box on the car floor's anti-slip groove to avoid shifting during sudden braking or acceleration.
3. Express delivery after arriving at the high-speed railway station.
Reference Citation
pass over.
Scope of Application
The scientific research uses of islet cells mainly include the following:
Diabetes mechanism and therapeutic research: for analyzing the pathological mechanisms of type 1/type 2 diabetes, such as defective pancreatic β-cell function or insulin resistance, and developing cell replacement therapies (e.g., islet cell transplantation with stem cell differentiation).
Regenerative medicine and cell therapy: transform somatic cells into pluripotent stem cells through chemical reprogramming, and then differentiate them into functional islet cells to achieve a functional cure for diabetes, e.g., RGB-5088 injection of Ripchentron is able to get rid of insulin dependence through autologous cell transplantation.
Drug screening and toxicity assessment: as an in vitro model to test the effect of drugs on insulin secretion, glucose metabolism, or to assess drug nephrotoxicity (e.g. diabetic nephropathy research).
Metabolic regulation and signaling pathway research: to investigate the role of pancreatic islet α/β cells in glucose, fat and protein metabolism, as well as the regulation of cellular function maturation by pathways such as TGF-β, Wnt and ceramide metabolism.
Disease model construction: using primary islet cells or pluripotent stem cell-derived islet cells to construct in vitro models to simulate diabetes progression and complication mechanisms.
These studies not only advance the treatment of diabetes (e.g., clinical transplantation and functional cure), but also expand into the fields of metabolic diseases and drug development.
Due to biannual updates of our product instruction manual, the following procedures are for reference only.
The most recent version included with the product shall prevail.
II Reagents and Materials
- Mouse pancreatic islet cells（Cat# LV-mPICs003）
- Recovery medium（Cat#LV-Rec001）
- Islet cell sustaining medium（Cat# LV-mPICM003）
-D-hank’s solution
- Biosafety cabinet
- Sterile centrifuge tube of 15 ml
-Pipette
- Thermostat water bath
- 37 °C/5% CO2 incubator
- Non-TC-treated 10 cm cell culture dish
- Wide-mouth pipette tip (The tip of a normal one is cut off and sterilized)
-Cell culture flasks (plates)
-FDA/PI (fluorescein double acetates /propidine iodide)
- GSIS kits
-0.25% trypsin-EDTA
III Resurgence and Plating of Cells
1. Preheat islet cell recovery medium and sustaining medium in a 38 °C thermostat water bath for 20 min.
2. In a biosafety cabinet, add 10 mL of islet cell recovery medium to a non-TC-treated 10 cm cell culture dish and set it aside.
3. Quickly transfer the frozen islet cells from the refrigerated position to a thermostat water bath of 38℃. Then，immerse them in as much water as possible at 38°C and rotate clockwise for thawing. Please make sure that the cap of the freezing tube is kept above the water.
4. Thaw the freezing tube for about 90-120s, until only a small amount of crushed ice floats in it.
5. Sterilize the freezing tube with 75% alcohol and transfer it to a biosafety cabinet.
6. Aspirate the cells with a wide-mouth pipette tip and gently add dropwise to a non-TC-treated 10 cm cell culture dish containing pre-heated 10 mL of islet cell sustaining medium (Note: if there are residual cells left on the freezing tube and pipette tip, recovery medium can be used to rinse. Then, mix them into a non-TC-treated 10 cm cell culture dish).
7. After gently shaking the cell culture dish back and forth for 2-3 times, leave it at room temperature for 30 min.
8. Take an appropriate amount of cells and stain them with FDA/PI (fluorescein double acetates /propidine iodide) to observe the survival of islet cells.
9. The cells obtained from step 7 should be treated appropriately according to the experimental requirements. The following processing is for reference only).
a. Islet cells: Treat islet cells with a 20 μL of tip pick up well-integrated islet cells for treatment under the stereo microscope.
a.1 Insulin secretion under glucose stimulation (GSIS): see instruction of the GSIS kit for specific procedures.
a.2 Islet cell culture: According to the experimental requirements, choose different sizes of cell culture flasks or cell culture plates. If adherent culture is required, choose a TC-treated cell culture flask (plate). If suspension culture is required, select a non-TC-treated cell culture flask (plate). The density of islet cell culture is recommended to be 10-20/mL of islet cell maintenance medium, with half a volume of fluid change in 3-4 days, and a full amount of liquid change in 7 days. Different components can be added to islet cell sustaining medium according to experimental requirements.
a.3 Islet cell transplantation: According to the needs of different receptors and transplant sites or specific experimental requirements, select islet cells referred to as islet equivalent quantity (IEQ) and transplant them into specific transplant sites of specified receptors. The recipient's blood glucose changes and physical condition should be monitored and recorded daily.
b. Islet single cell:
b.1 Collect the islet cells in step 7 into a 15 mL centrifuge tube, centrifuge of 500 × g at room temperature for 5 min and discard the supernatant completely. It is then resuspended at 5 mL of pre-heated D-hank's liquid. Repeat the previous steps of centrifugation and discarding supernatant.
b.2 Add ten to twenty times the volume of cell pellet 0.25% trypsin-EDTA digestion solution to resuspend and incubate at 37 °C for 5 min. Then gently pipette in a biosafety cabinet with a 200 μL tip for 30 s. Continue incubation at 37 °C for 3 min. Pipette and scatter for 30 s until the clumps of islet cells are not visible to the naked eye. (Note: The total digestion time should not exceed 20 min, so as not to make the cell activity too low).
b.3 Add five times the volume of digestion fluid to the islet cell maintenance medium to terminate digestion. Centrifuge at 500×g at room temperature for 5 min, completely discard the supernatant, and maintain the medium resuspending in an appropriate amount.
b.4 Cell survival and total cell volume of cell can be measured by trypan blue exclusion.
b.5 According to the specific experimental requirements, the islet single cells are treated accordingly. Islet single cells should be cultured according to the density of 5-10×104/mL
