Rat pancreatic islet cells can be used as (1) experimental materials for in-depth study of islet cell function in the laboratory; (2) cellular screening models for new diabetes drugs.
Operation method
Rat islet cell culture experiment
Principle
Chloral hydrate was used for intraperitoneal anesthesia, and rat pancreatic islets were isolated and purified by collagenase v retrograde perfusion, in situ digestion and Ficoll-400 gradient centrifugation, and islet monocytes were isolated and cultured.
Materials and Instruments
One-week-old Wistar rats Move 1. One-week-old Wistar rats were killed by neck dissection, soaked in 75% ethanol for 15 minutes, the pancreas was aseptically removed, rinsed in ice-cold sterile D-Hanks' solution, and extra-pancreatic tissues, such as fat, peritoneum, and blood vessels, were carefully removed with ophthalmic scissors and transferred to penicillin vials. 2. Add a small amount of sterile D-Hanks' solution, use ophthalmic scissors to cut into pieces of 0.1-1.0 mm3 in size, use a dropper to gently suction out the upper layer of small fat fragments and oil droplets, and then use sterile D-Hanks' solution to repeatedly wash 8-10 times. Add 10 times the volume of sterile digestive enzyme solution [trypsin-collagenase digestive solution: 0.05 g of trypsin, 0.025 g of collagenase V (663 U/mg), 0.05 g of glucose, dissolved in 100 ml of Ca2+ and Mg2+-free 0.01 mol/L PBS (pH 7.4) solution, and filtered through 0.22 µm microporous filter membrane], at 38 ℃ ± 1 ℃. Digestion was carried out at 38°C±1°C with constant shaking for 10 min and the supernatant was discarded. 3. Wash the block 2-3 times with sterile D-Hanks' solution, add fresh enzyme solution and continue digestion, repeating the above steps until the edges of the block are blurred. 4. Immerse the tissue block in digestive enzyme solution, digest at 38℃±1℃ for 10 minutes, separate the digestive enzyme solution from the tissue block, and add fresh digestive enzyme solution to the tissue block for digestion; while the original digestive enzyme solution is centrifuged at 1500 rpm for 10 minutes, and then take the precipitate, which is digested cells, and then re-suspend it with sterile D-Hanks' solution, centrifuge it, and repeat the procedure 1--2 times, and then use culture medium to digest the tissue block. --The cells should be resuspended in sterile D-Hanks', centrifuged, and repeated 1-2 times, then washed 2-3 times with culture medium, and suspended in culture medium to obtain the cell suspension. 5. 5. Repeat the digestion of the digested tissue block 5--6 times until the block is completely digested, and repeat the above operation. 6. 6. Combine the cell suspensions obtained several times, count, adjust the cell concentration to 2×105 cells/mL, inoculate the cell suspension into 24-well plastic culture plates, 1 ml per well, and place them in an incubator at 37℃, 5% CO2, and saturated humidity. 7. Since fibroblasts adhere to the wall more rapidly than islet cells, 15 h after inoculation, gently oscillate the culture plate, and connect the cells that are not attached to the wall to a new culture plate, so as to remove part of the fibroblasts. 8. After culturing the cells in the new culture plate for 48 h, change the freshly configured medium containing 2.5 ng/mL of iodoacetic acid for 5 h, which can remove most of the fibroblasts, while the islet cells are not injured, change the medium without iodoacetic acid for 2 washes, and change the medium without iodoacetic acid for 5 h. Cultivate the islet cells in 37℃, 5% CO2, saturated humidity incubator, and then change the culture medium every 3 days, and then obtain the monolayer of islet cells. Islet cells were obtained. Caveat 1. The islet extraction of rat generally contains more amount of pancreatic tail, generally extracted to pay attention to the thought of the part, filling good or not is very critical for the amount of extraction is crucial. 2. During cell culture, please pay attention to maintain aseptic operation. 3. trypsin digestion time should not be too long during the process of subculture, otherwise it will affect the cell attachment and its growth status. 4. The culture medium can be stored for 3--6 months at 4℃. For more product details, please visit Aladdin Scientific website.
D-Hanks' solution Trypsin Type V collagenase Glucose Iodoacetic acid
Surgical scissors Surgical forceps Ophthalmic scissors Ophthalmic forceps Large head pins Surgical blades Petri dishes