Recently, while working with a cell line that I had obtained from our cell culture facility, I noticed a peculiar change in the cells. After a couple of passages, their growth rate slowed markedly, they increased in size, and were infiltrated with numerous large vacuoles. I explained the symptoms to the cell culture mavin from whom I had obtained the cells and she immediately asked if I routinely left trypsin in the culture medium after passaging. I replied that I did since that was the method of passaging these cells recommended by ATCC. She stated that ATCC now recommended removing the trypsin by centrifugation prior to reseeding the cells but their website had not been updated.
According to what she had heard, it takes 25 ml of medium containing 10% fetal bovine serum to neutralize 1 ml of 0.05% trypsin and 125 ml to neutralize 1 ml of 0.25% trypsin. While some cell lines don’t seem to mind having active trypsin in their growth medium, others are quite sensitive to it and may suffer dramatic changes in growth characteristics and morphology that are not readily reversed. Apparently, the cells I was working with were the sensitive type. My choices were to start removing the trypsin after detaching the cells or do what she recommended, which was to switch to HyQTase from HyClone.
HyQTase is a trypsin-free cell detachment solution composed of a proprietary blend of proteases and collagenases. It does not have to be removed following cell detachment since the enzymes are inactivated after about 1 h at 37°C. It is very gentle on the cells and can be left on the cells at full-strength for up to 45 min without a noticeable loss of cell viability.
I’ve used HyQTase to passage 3 different lung cancer cell lines on several occasions and it has worked perfectly on all of them. The protocol is essentially identical to that used for trypsin detachment: Aspirate the medium, rinse the cells once with Ca++/Mg++-free PBS, add 2 mls HyQTase (for a 10 cm diameter Petri dish), and put the dish back in the incubator for 5-10 min. Two rinses with PBS may help for cells that are difficult to detach. Once the cells are detached, I add 6-8 mls growth medium and pipet the suspension up and down a few times to break up any clumps of cells. I then dispense 3-4 ml into fresh dishes containing enough medium to give a final volume of 10 ml. I’ve looked at the cells under the microscope after dilution into Trypan blue and they are mostly single cells with very high viability.
HyQ®Tase™ Cell Detachment Solution From HyClone
A very effective, gentle, and inexpensive alternative to trypsin for cell detachment and disaggregation.
The only potential negative is the fact that since HyQTase contains proteases, membrane proteins may be damaged during detachment. For this reason, I always use PBS containing 0.5 mM EDTA to detach cells that I am going to use for procedures requiring intact cell surface proteins.
The Bottom Line
A great product. Passaging cells is made simpler by the fact that the detachment reagent doesn’t have to be removed or neutralized prior to reseeding cells.