© Split one of the T25 flasks 1:2, resulting in two T25 flasks. This set of two T25 flasks will be your “working” set of cells.
© Split the other T25 flask 1:3, and place the contents into one T75 flask (protocol follows). After the cells in this T75 flask are confluent, they should be split into two T75 flasks. When the cells in these two flasks reach confluency, they can either be frozen (protocol follows under “Freezing”), or further split 1:2, resulting in 4 T75 flasks to be frozen. We recommend freezing away four flasks.
© Thus, within a week of receiving the HL-1 cells, you should have four cryovials of these cells frozen away for future use.
© It is recommended that cultures be split only after full confluence.
© Rinse each T25 flask briefly with 3 ml of 0.05% trypsin/EDTA warmed to 37oC (use 6 ml for T75) by pipetting the trypsin/EDTA onto the bottom of the flask (side opposite the cap), trying not to hit cells directly with the enzyme. Rinse gently and remove by aspiration.
© Add another 1.3 ml trypsin/EDTA per T25 flask (3 ml per T75). Incubate at 37oC for 2 minutes.
© Remove and add fresh trypsin/EDTA. Incubate for an additional 2-3 minutes. Look at the cells microscopically, as this may only require 2 minutes.
© Examine microscopically and, if cells are still adhered, rap the flask to dislodge remaining cells.
© To inactivate the enzyme, add an equal amount (1.3 ml) of soybean trypsin inhibitor directly onto cells.
© Transfer cells from the flask into a 15 ml centrifuge tube.
© Rinse the empty flask with 5 ml wash medium (Claycomb Medium containing only 5% FBS and penicillin/streptomycin), and add to the cells already in the 15 ml centrifuge tube.
© Centrifuge at 500×g for 5 minutes.
© Meanwhile, remove the gelatin/fibronectin solution from each T25 flask, and add 4 ml supplemented Claycomb Medium/flask. Set aside.
© Remove the tube containing the HL-cardiomyocytes from the centrifuge. Remove the supernatant by aspiration, and gently resuspend the pellet in 3 ml of supplemented Claycomb Medium.
© Transfer 1 ml into each of three labeled, gelatin/fibronectin-coated T25 flask. Each flask now contains 5 ml.
© If the cells are passaged on a Friday, use 2´ the volume of supplemented Claycomb Medium per flask.
Freezing:
IT IS RECOMMENDED THAT YOU FREEZE 4 OR MORE VIALS AS SOON AS POSSIBLE AFTER RECEIPT OF THE CELLS (please see note under “Passaging”).
This allows you to return to this passage, and also protects you in case of contamination.
© We generally freeze the contents of one confluent T75 flask into one cryovial. (When cells are needed, this cryovial is thawed into one T75 flask.)
© Briefly rinse the T75 flask containing the HL-1 culture with 5 ml of 0.05% trypsin/EDTA warmed to 37oC. Remove by aspiration.
© Transfer 3 ml of trypsin/EDTA into the flask.
© Incubate the flask at 37oC for 2 minutes.
© Remove the trypsin/EDTA, and replace with 3 ml of fresh trypsin/EDTA.
© Incubate at 37oC for 2-3 minutes.
© Check under a microscope that cells are dislodged. If not, rap to dislodge any adherent cells.
© Add 3 ml of soybean trypsin inhibitor to the flask, and transfer the 6 ml into a 15 ml centrifuge tube.
© Rinse each empty flask with 8 ml wash medium, and add to the cells already in the 15 ml centrifuge tube. Total volume is now 14 ml.
© Centrifuge tube for 5 minutes at 500×g.
© Remove wash medium by aspiration.
© Gently resuspend each pellet in 1.5 ml of freezing medium (95% FBS/5% DMSO).
© Pipette resuspended cells into a cryovial. Place the cryovial containing the cells into a Nalgene freezing jar containing room temperature isopropanol.
© Immediately place the freezing jar into a –80° C freezer, and freeze cells at a rate of -1°C/minute.
© Six to twelve hours later, transfer the vial to a liquid nitrogen dewar.
Thawing:
© Gelatin/fibronectin-coat a tissue culture flask overnight in a 37°C incubator.
© Next morning, remove the gelatin/fibronectin from the culture flask, and replace with 10 ml of supplemented Claycomb Medium. Place this flask back into incubator.
© Transfer 10 ml wash medium into an empty 15 ml centrifuge tube. Incubate tube in a 37°C water bath.
© Quickly thaw the cells in a 37°C water bath (about 2 min), and transfer into the 15 ml centrifuge tube containing the wash medium.
© Centrifuge for 5 minutes at 500×g.
© Remove the tube from the centrifuge and remove the wash medium by aspiration.
© Gently resuspend the pellet in 5 ml supplemented Claycomb Medium, and add to the 10 ml of medium already in the T75 flask.
© Replace the medium with 15 ml of fresh supplemented Claycomb Medium 4 hours later (after cells have attached).
Vendor Catalog #
Claycomb Medium JRH Biosciences 51800
*Fetal Bovine Serum (Lot # 5J0994) JRH Biosciences 12103-500M
Penicillin-Streptomycin (104 U/ml P and 104 mg/ml S) Life Technologies 15140-122
Norepinephrine [(±)-Arterenol] Sigma A-0937
L-Ascorbic Acid, Sodium Salt Sigma A-7631
L-Glutamine, 200 mM Life Technologies 25030-081
Trypsin-EDTA, 1´ Life Technologies 25300-054
Trypsin inhibitor, soybean Life Technologies 17075-029
Fibronectin Sigma F-1141
Bacto© Gelatin Fisher Scientific DF0143-17-9
Cryovials, Corning VWR 66021-920
Sterile Acrodisc syringe filters, 0.2 mm Gelman Sciences 4192
Distilled Water, cell culture grade Life Technologies 15230-147
*If you have any problems obtaining this lot of serum, please contact:
Ms. Joan Carlson
Research Scientist, JRH
Phone: 913-469-5580 ´ 287
E-mail: joan.carlson@jrhbio.com
This FBS has been pre-tested by the Claycomb lab for use with HL cells AND IT IS AN ABSOLUTE REQUIREMNENT THAT THIS PARTICULAR LOT OF SERUM BE USED WHEN CULTURING THESE CELLS. THE CELLS WILL NOT MAINTAIN THE CONTRACTING PHENOTYPE IF THIS FBS IS NOT USED. IT IS ONE OF THE MOST IMPORTANT REAGENTS!!