This protocol is really to culture the isolated and obtained pancreatic vesicle epithelial cells in collagen-coated petri dishes. In turn, two-dimensional aggregated clones are created for use in research. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Scheme 23.7 Pancreatic epithelial cell isolation and culture experiments
Principle
Cells are isolated from guinea pig pancreatic tissue and the cell suspension is filtered through gauze or nylon mesh, and the filtered cell suspension is gently added on top of the BSA solution. The clumped cells were dispersed by 3 successive centrifugations and suspension of the cells. The cells were then inoculated into collagen-coated culture vessels.
Materials and Instruments
F12K Tissue Culture Solution Containing 20% Calf Serum HBSS HBSS-DVC Glucose Trypsin Solution Trypsin Solution Citrate Buffer Where Citrate Trisodium Salt NaCl Glucose Phenol Red Collagenase Solution Collagenase Dissolution Siliconized Conical Flasks Siphons Move makings (2) Centrifuge For more product details, please visit Aladdin Scientific website.
Blichner funnels Dialysis tubing Sidearm bottles Polypropylene centrifuge tubes Gauze Petri dishes
1. Aseptic
(1) F12K tissue culture medium: containing 20% calf serum (F12K-CS20)
(2) HBSS
(3) HBSS-DVC: Ca2+ and Mg2+ free HBSS
(4) Glucose (Difco, 0155-174, BDBiosciences)
(5) Trypsin solution: 0.25% trypsin solution (1:250, Difco, BDBi-osciences) was prepared with citrate buffer. The citrate buffer (pH 7.6) contained 3 g of van citrate trisodium salt, 6 g/L NaCl, 5 g/L glucose and 0.02 g/L phenol red.
(6) Collagenase solution: 1800 U/ml with 1XHBSS-DVC (pH 7.2) to dissolve collagenase (GIBCO). Adjust the pH of collagenase solution to 7.2 and dialyze with 12Kda dialysis membrane at 4℃ for 4 h. The dialysate is HBSS-DVC containing 0.2% glucose. After removing HBSS, repeat the procedure with HBSS-DVC for 16-18 h. The solution is filtered and sterilized. The dialysate is filtered for sterilization, dispensed into 10-20 ml, and stored at 70°C or below.
(7) 25 ml siliconized conical flask (Bellco)
(8) Pipette: 5 or 10 ml, large bore (Bellco)
(9) Blichner funnel
(10) Dialysis tubing (SpectroPor)
(11) Sidearm bottle: 250 ml
(12) Polypropylene centrifuge tube: 50 ml
(13) Gauze
(14) Petri dishes coated with collagen (Biocoat, BD Biosciences)
2. Non-sterile
(1) Water bath oscillator (Model M5B, 11-22-1, Backer)
Procedure
1. Prepare a mixture of collagenase and trypsin (1:2) and heat to 37°C. 2. Prepare the digestive solution.
2. Remove the whole pancreas (0.5-1 g) and place it in F12K-CS20 under aseptic conditions.
3. Cut off the mesentery and other tissues, and cut the pancreas into 1-3 mm pieces.
4. Transfer the pieces of tissue to a 25 ml siliconized conical flask containing 5 ml of preheated digestive solution and digest by shaking in a water bath (120 r/min for 15 min) at 37°C. Repeat this step 2-3 times with fresh digestive solution until most of the tissue is dispersed. Repeat this step 2-3 times until most of the tissue has been digested and dispersed.
5. After each oscillation, allow large pieces of tissue to settle and transfer the supernatant into a 50 ml polypropylene centrifuge tube. The centrifuge tube contains approximately 12 ml of cold F12K-CS20 to neutralize the digestion solution.
6. After centrifugation (600 r/min for 5 min), suspend the cells with 5-10 ml of cold F12K-CS20 and place the tube on ice.
7. Collect the cell suspension and filter through several layers of sterilized gauze (in a Blichner funnel). During filtration, insert the funnel handle into the vacuum flask and gently aspirate the cell suspension. A small amount of cell suspension was taken for quantitative analysis.
8. Typically, 1X105 to 2X105 cells are obtained per milligram of tissue, and 90% to 95% of the cells are viable.
9. 5X107 to 5X108 cells are added to the surface of a column of 2 to 4 50 ml polypropylene centrifuge tubes, each containing 35 ml of HBSS-DVC (pH 7.2) with 4% cold BSA. Then, centrifugation (600 r/min, 5 min ) was performed. This step is essential to efficiently separate the vesicular cells of the pancreas from the islet cells, ductal cells and stromal cells. The supernatant was aspirated and this separation step was repeated 2 times, and after each separation, the cells were collected and suspended in cold F12K-CS20.
10. Collect the cells with 5-10 ml of cold F12K-CS20. Take a small amount of cell suspension and count the cells. 2X 104-5X104 cells per mg of tissue were obtained, and 80%-95% of the cells were vesicular cells. Inoculate at a density of 3X105 cells/cm2. Within 72 h, the cells adhered to the wall and formed clones.