StemCell Freezing Medium contains DMSO and is optimized for the cryopreservation of hepatic and biliary tree stem cells.
Required Supplies:1. Kubota’s StemCell Growth medium2. StemPro Accutase3. StemCell Freezing medium for hepatic and biliary tree stem cells
Preparing Hepatic and Biliary Tree StemCells for Cryopreservation:1. For all volumes refer to Table 1 below
2. Working with multiple plates or flasksa. Only work with a stack of 4 x 100mm plates or 4 flasks at a timeb. Minimize time out of the incubator during the processc. Work swiftly but carefully to minimize cell loss3. Remove the media from the plate or flask and add cold Accutase (see table 1 for volumes) (Do not warm Accutase in the waterbath)4. Take a plate or flask of cells immediately to the microscope so that you can observe the dissociation
Note: Do not place the plates or flasks in an incubator during the dissociation process. Accutase will lose activity if allowed to warm up to 37°C
5. Tap the plates or flasks against the palm of your hand to dislodge the cells and when the cells are free floating, return the plates to the safety cabinet 6. Gently pipette up and down to ensure single cell suspension, then transfer cell suspension into appropriate size conical tube
7. Wash the plate or flask with Kubota’s StemCell Growth medium and add to the tube, pipetting up and down to ensure a homogenous cell suspension8. Take an aliquot for cell countinga. Take 20ul cell suspension and add it to 20ul Trypan Blueb. Pipette up and downc. Load 10ul of cell suspension to both counting chambers of a hemacytometerd. Count the cells within the center gride. Calculate the total cell count8. Take an aliquot for cell countinga. Take 20ul cell suspension and add it to 20ul Trypan Blueb. Pipette up and downc. Load 10ul of cell suspension to both counting chambers of a hemacytometerd. Count the cells within the center gride. Calculate the total cell count9. Divide the total cell count by 1.5 x 10e6 to determine the number of cryovials needed for freezing down the cells for long-term storage
10. Pellet the cells in all the tubes by centrifugation at 500 x g for 5 minutes
11. While the cells are in the centrifuge print the labels needed for each cryovial12. Place one label on each cryovial
13. After the centrifuge stops, resuspend the cell pellet in enough StemCell Freezing Medium to freeze 1.5 x 10e6/ml as calculated in step 9 above
14. Transfer 1ml StemCell Freezing cell suspension into each cryovial15. Transfer the cryovials into a controlled rate freezer or a styrofoam container then place the container into a -80°C freezer over night16. Next day transfer the vials of cells into the LN2 freezer for long-term storage
