When it rains, it pours

It's surprising how things can change so quickly. A week ago, I wrote about how we tried to rescue our 293T stock with my cells...

On Monday, all our 293Ts croaked (if you can call it that. Maybe individual cells actually do make tiny gasps when they die) Talk about cockiness. The medium changed colour completely, and all the cells detached themselves from the bottom of the plates. And we have absolutely no idea why that happened. The cells may have grown too fast, used up all the medium, run out of nutrition and starved to death. But that's just my theory.

To make things worse, on Thursday all the HeLa cells died. Every last one. They were in trouble right from the beginning, so it wasn't entirely unexpected.

Now that there aren't any cells around, most of my work over the next few days will be reading about checkpoint proteins. When DNA in cells is damaged (by UV or some chemical agent) and if the cell's DNA replicating machinery detects the damage, the cell is prevented from multiplying till the damage is repaired. Checkpoint proteins bring about this arrest. And I will be studying one of these proteins over the course of my project.

Of course, to study these proteins, we need to kill the cells and extract them. And we need to prepare 30 plates of cells each time. So far, we haven't even been able to sustain cultures of 3 plates. Apparently, this kind of trouble is always there while starting a new lab in India. So wish me luck. I'm going to need lots of it to get through.

Errare humannum est..

So, as it turned out, my guide's cells were contaminated. There was zero growth in one of the plates, and very patchy growth in the other one. One of the other professors said it looked like there was a lot of debris in her plates. My cells, on the other hand, grew quite nicely. Almost 90% confluency (almost the whole plate was covered with a lawn of cells) and they looked pretty healthy.

So, on Friday, we split my 293Ts into two plates, in case we aren't able to revive the one other plate. And when I checked on Saturday, cells in both plates seem to have settled down quite nicely. We still haven't figured out what went wrong on Monday, though. Just hope it doesn't happen again.

A second batch of HeLa cells came in from Hyderabad on Thursday. The first lot came in two weeks ago, but they were in pretty bad shape when we received them. They were contaminated and we couldn't salvage anything. We tried splitting the second lot of HeLa cells as well on Friday. Come Saturday, and it looked like the same contaminant which hit the first lot had hit these cells too. There were clumps of white cell-like structures floating in the medium ( I couldn't see a distinct nucleus in any of 'em). Funnily though, HeLa were growing just fine at the bottom of the plate. I really hope we can fix this soon.

Proudest moment this week: The prof who was supervising my work while my guide was away told me I had good hands, and that I'd taken to cell culture like a duck to water. Granted he didn't watch me for more than 20 mins, but it still felt good to hear stuff like that. Makes me feel much more at ease with what I'm doing.

The spy

My guide’s gone out of town. So I had to split not just my cells, but her cells as well today. I followed the protocol correctly for the most part, but I may just have messed up splitting her cells. I think I added too many cells to her plate while splitting them. As usual, I’ll know only two days later when I change the medium.

Lately, I’ve been thinking about what it means to be a biologist. Back when I was picking a college to join, a lot of people expected me to take up medicine. That’s how it works in India. If you’re good in bio, you’re expected to become a doctor. If you’re good in math or physics, you’re expected to become an engineer. If you’re good with people and talk well, you’re expected to become a lawyer. And if you do none of these, you’re expected to do an MBA and get into management. Thankfully, attitudes are changing now.

I don’t think I would’ve been happy as a doctor. I’m using a very crude analogy here, and I don’t mean to offend anyone, but a doctor is a little like a car mechanic. He doesn’t have to be involved in the design side of things. He needs to know how to patch things up. As a doctor, most of your day is spent looking at things that aren’t working right.

I’m not saying I’m on the design side. No sir! We’re nowhere near design, as far as life is concerned. No, I’m kinda like the industrial spies car companies employ to find out how a rival’s new thingamajig works. And when your rival car company is natural selection (which produced you in the first place), boy, do you have a lot of spying and analysis ahead of you!

Oh, and in case you’re wondering about the car analogies, I’m a car nut. I have a soft spot for Italian and German cars. And I think most American cars are just a waste of money and metal.

Every small step...

The cells are okay! They’re not growing nearly as quickly as my guide’s. But it’ll do. Somehow, I already feel an attachment of sorts to them. That I’m responsible for them. Yes, I know it sounds lame, and very unprofessional. I guess I’m just completely caught up in the rush of it all. New lab. New equipment. The gloves and extra sterility. Plus, it’s been a full year since I worked with cells of any kind. And those were bacterial cells [S.aureus, to be precise. They’re the bacteria you find in pimples :D]

After checking them, we changed the medium in the plates. The cells should have fresh nutrition available now. Remember, TLC.

Oh, and in case you’re interested, here’s the protocol to dilute (or split) a culture of 293T. Note that I’m using 10ml of medium and 10cm dia. plates.

1) Aspirate (remove using a vacuum pump) the media using vacuum
2) Rinse with 5ml PBS (Phosphate Buffer Saline) gently
3)Aspirate PBS. Add 2ml trypsin. Leave for max. 2 mins. (This is to loosen the cells from the plate. Trypsin is an enzyme that digests protein)
4) Add 5ml of medium to stop the reaction.
5) Loosen the cells by tapping the side of the plate
6) Transfer 7ml of medium + trypsin to a 50 ml Falcon tube
7) Centrifuge for 10 mins at 1000 rpm, 25°C . Use balance.
8) Aspirate supernatant.
9) Add 1 ml medium to tube. Re-suspend cells.
10) Add 10ml of medium to fresh petri dishes. Add required dilution of re-suspended cells.
11) Move cells around in plate to spread evenly. Keep in incubator.

And that’s how it’s done :)

In splits

I have a small confession to make. When I said worked with, in the previous post, it’s not entirely true. I just stood by and watched while my guide separated them out into culture plates. I actually handled the cells on my own today. I diluted the cells and plated them all by myself. My guide was there to make sure I didn’t mess up, though.

Remember what I said about 293T needing TLC? It’s like this. As the cells grow, they multiply. And when they multiply it leads to crowding. See, 293T grows best when it has some surface to cling onto. But there’s only so much area on a single culture plate for them to grow on. Secondly, when crowding happens, there’s more waste products being released into the culture medium, and less nutrition available to each individual cells. That’s why we need to dilute the cells, to make sure there’s enough room for all of them.

Dilution isn’t a simple matter of just pouring more medium into the plate. It’s a cell culture, not orange squash. First, you have to remove the old medium, peel the cells off the plate (not literally) and then put a fraction of the cells you’ve recovered into a fresh plate with fresh medium. And all of this has to be carried out under sterile conditions. 293T is particularly sensitive to bacterial infection. (FYI, most culture media are banquets to bacteria and fungi. The media are so chock-full of nutrients it’s almost as if they beg to be contaminated)

The whole procedure took me around an hour and half, from start to finish. And I think I’ve done a reasonably good job. I won’t know till the 16th, though. That’s when the cells should have settled on the plate and started multiplying. Hope they’re OK. Fingers crossed!

TLC for 293T

I started cell culturing today. Okay, I know that that isn’t the correct term, it’s tissue culture. I’m really excited about it though. This is the first time I’m going to handle mammalian cells, barring that silly experiment in the 10th grade where they make you scrape out cheek cells and look at them under a microscope.

No, this time it’s proper human cells from a proper cell line. A cell line is a strain of cells that are unique in their genetic composition. And they’re stable, as in they don’t mutate or change properties in any way easily. Stable bacterial cells are called strains. Stable plant and animal cells are called cell lines.

The cells I worked with today are from a line called 293T. These cells are mutants from embryonic kidney cells. They’re not aggressively invasive, like a lot of other lines. The HeLa line, which I hope to work with some time down the line, is aggressive. Also, 293T is kinda sensitive to temperature and nutrients. It needs a lot of TLC (37°C, perfect osmotic balance, the works), which is probably why it isn’t very aggressive either. Can’t really survive outside the body.

And since they need so much TLC, we’re gonna focus on just keeping them alive and getting them to proliferate, for the next two weeks. After that, I’m not quite sure where we go. I’ll keep you posted, though.

Number one

When I started this blog, I meant it to be a site that explains biology, in its broadest sense. But then I realized that, for one, I’m just an undergraduate student and that there are far, FAR more qualified people besides me out there in the internet. And a lot have done an admirably good job of explaining things. For another, the field is just so goddamn vast and varied that I don’t know where to begin.

So, Not Quite Alchemy will be the diary of a biology grad student. Follow me as I go about another semester of my course.

Just to get you up to speed, I’m doing a newfangled five year course, from which I will graduate with an M.Sc. And I’m currently in the third year of the course. Now that that’s settled, wish me luck. A new semester, new challenges. And new insights, hopefully. Keep your rubber gloves handy :D