Experience at Cell culture

My first day at 'training' (very informal) was shadowing the Senior lab manager, Wasim at the brady Research Lab, JHH. The first thing he said tme before he started was, "Cell culture is more unrelenting/unforgiving than a surgery (with respect to aseptic techniques to be followed). A surgeon who screws up can cover up by antibiotics but  someone performing cell culture does not hav that option. One goof up and cells die or get contaminated."  The next one week was spent getting acquainted to terms, techniques of cell culture -- passaging cells, changing media, splitting cells. Under the guidance of General Tao (alias Dr. tao ping shi), a rigorous, military training was undertaken! Every hand movement, every finger touch was scrutinised. But every advice was logical, rational and had some significance. While working in the Bio safety cabinets with laminar air flow, many things had to be kept in mind. The liberal use of 70% ethanol was the foremost! Many bottles used to be finished in a day! not thinking about wastage of lab reagents was one things to overlook. No Stingy measures! No watches, rings and miscellaneous items. Every inch of probable contaminated equipment was to be excluded. Many more such things that i can not really explain.




Peering through inversion microcope into my
T150(150 refers to area of flask on which cells
grow; 150cm2) flask containing LNCaP cells.



Once my mentors thought that i was ready to 'make the plunge', I was given LNCaP M. Luc (prostate cancer cell lines isolated from the Lymph node metastasis, therefore the name). M.Luc refers to refers enzyme which helped in counting the cells acording to their luminiscence which was counted in the presence of an appropriate reagent. These are one of the oldest and are also very sturdy. Good for a novice I thought. After passaging and maintaining cells for about 2 weeks, the cells had stabilised (in growth, population,characterstics) and ready for an experiment. I can not disclose the experiment but it was related to chemosensitivity of LNCaP cells to Valproic acid and other m-TOR inhibitors.

After adding to required concentrations of cells and its serial dilutions in the 6 well plates, the plates were incubated. After 12 hours the first sample was pipetted out, 100microliters into SEL tubes for 3 consecutive days. the thin about M.Luc is that the enzyme accumulates in the soulution over time. So by pippetting out small quatities we can predict the amount of cells by the luminiscence values we receive and extrapolate it to the entire solution. Thi helps in predicting the no. of cells in the solution. We had 3 time points --12 hours, 36 and 60 hours. We were analysing 4 drugs-- VPA and 3 m-TOR inhibitors. And then the day of the assay, using an elecronic repaeat pipetter i used to pipette out solution and then plate it into 3 wells of the 96 well plates. So, basically i triplicated the sample.

During all this process, the lab manager used to look over me sometimes. Sometimes when i faulted, he used to say, "Its Ok. Cell culture is more an art than a science. Small errors would not make such an impact."  Art..wow! A person with experience can only say such things! And yes, he did.

The first experiment was a big mistake as we did not plate the appropriate control to really comment on the data we had received. There is this thing about basic research, one wrong step and it can really screw up days and weeks of hardwork. So, we had to redo the entire 3 day process again to record the data. I was just relieved that we were able to complete before i left the place.

Encouraging results, said the lab manager. I was joyous. :)