Mini-Me: Snippets From Cell Culture

“Once a man has children, for the rest of his life his attitude is – to hell with the world! I can make my own people.”

Jerry Seinfeld

Edited by Navjot Kaur

Ever felt the power of knowing that a hundred thousand lives depended on your mere whim? That you could condemn literal masses to their death by one act of appalling stupidity? Or that you could get to play a pretty helpless version of God day in day out?

If you seem to enjoy the above scenarios, then probably cell culture is your career calling.

One of the most low-key (and high-maintenance) ways to cosplay as God, cell culture is the staple of most biological labs. And since I’ve recently started to find my feet in this literal quagmire, it may be worthwhile to share some of my fledgling experiences.
For something so seemingly puny, cells can be ridiculously fastidious. Keep them at a slightly higher or lower temperature and they’ll die out in hordes. Change the pH of the medium in which they grow and they’ll go all haywire. Forget to add a solution or two, and they’ll just give up their ghosts. For something representing the pinnacle of three billion years of evolution, they can be pretty underwhelming!

I won’t delve into a history of cell culture here, nor too much of the specifics. Just a few tidbits about the kind of straightforward stuff I handle at my amateur level. For those interested in a much more expansive view of this topic, I would very strongly recommend Rebecca Skloot’s “The Immortal History of Henrietta Lacks”. More on Lacks later, if we are still within a reasonable article length.

Cells make up tissues, tissues make up organs, and organs make up an organism. A full three marks in middle school. But real-life cells and tissues are quite a bit more complex to deal with. The term cell culture now tends to cover tissue culturing as well, or both terms are sometimes used interchangeably. For instance, the room my labmates use for their cell culture work is called the “TC” – tissue culture room. A small, isolated square of a sacred space, the TC is a fiercely guarded arena. Two doors separate the inner sanctum from the contamination teeming in the outside world. The room itself is pretty cozy, though occasionally cramped if more than two people should step in. As you enter, you immediately sight two large cabinets – the “hoods”. The hoods are the main workstations – and the most hotly contested property within the lab. Typical cell culture work can eat away anywhere between one to three hours of your time, and understandably, no one wants to keep waiting for an evening slot. Learnt it from hard experience!

A snap of my friend Aditya Dixit working in a laminar hood. While this one is not within the tissue culture room, rather inside the main lab itself, the overall structure is very similar to that of general hood. On that day, we both were learning (rather clumsily) to dissect an unsuspecting, and rather unfortunate mouse.

Two incubators – small white mini-fridge like boxes – chug away in contentment. The incubator is the TC’s most prized asset. It holds the hopes, dreams, and labor of the entire lab locked up within its secure environs. For no one can do what the incubator can – keep those pesky lil’ midgets happy with just the right conditions for their growth. And nothing can cause as much widespread misery as a contamination in the incubator – weeks of effort wasted, and a hell lot of clean-up on top of it. The lab can fall oddly silent on days when a widespread contamination is discovered, as we prepare to dress in black and hunt for deals on Uber Eats to seek solace in comfort food. Two incubators – small white mini-fridge like boxes – chug away in contentment. The incubator is the TC’s most prized asset. It holds the hopes, dreams, and labor of the entire lab locked up within its secure environs. For no one can do what the incubator can – keep those pesky lil’ midgets happy with just the right conditions for their growth. And nothing can cause as much widespread misery as a contamination in the incubator – weeks of effort wasted, and a hell lot of clean-up on top of it. The lab can fall oddly silent on days when a widespread contamination is discovered, as we prepare to dress in black and hunt for deals on Uber Eats to seek solace in comfort food.

The original source of cells can be from almost anywhere. Cell-lines (specially bred lineages of cells) are commercially available at ridiculously high prices – these contain collections of very many single cells of a given type which you can buy and start culturing in your lab. Sometimes graduate students bring in patient samples from a hospital the research lab has a collaboration with. These samples are basically pieces of tissue which can then be converted to cells (we’ll get to this soon). Our lab as a whole cultures a wide variety of different cell lines from breast cancer and ovarian cancer – both human and mouse derived.
Most cells in culture will spend their entire lives either floating around, or dipped, in a fluid called the “culture media”. The media is nothing but a special solution containing all the essential nutrients the cells require for optimum growth and survival. There are a wide variety of media of varying compositions now commercially available, and different cell types show preference for different types of media – not very much unlike the preference we humans have for different types of food!

Housing options for cells are regrettably limited. Almost all the standard offerings are some variant of a transparent, covered glass/plastic dish – circular or rectangular in shape, and maintained sterile. To get into this, we need to first cover a few basic terms, as listed:
• Adherent Cultures (settle down for life)
• Suspension Cultures (the rolling stones)

Stock photo of the usual culture dishes used to grow cells (I forgot to take some before I left!)

Adherent cultures are those wherein the cells literally stick to the bottom of the dish, divide and grow further. Usually, the culture plates/dishes we buy are pre-treated with chemicals/coated with electric charges such that the cells will easily adhere to the glass/plastic surface.

Adhesion to a surface is a particularly important feature of very many cell types. Unless they have a base to anchor to, it is difficult, if not impossible, for the cells to develop properly and carry out all their regular functions. This is a consequence of the fact that even within our body – where these cells have originally been derived from – they have evolved such that they grow on solid surfaces. Suspension cultures are a special subtype which involve cells that do not stick to a surface, but can remain suspended in a liquid medium and still grow well. Though there are specialized devices for suspension culturing (the most common being a simple rotating instrument which keeps the liquid media with cells in constant motion, thereby not allowing them to settle down), even the regular plastic dishes can be customized to allow for a suspension culture.

Recollect that the basis of adherent cultures was cells being able to stick to the bottom of the dishes. Herein, we introduce a small modification. Even if you had a table, you couldn’t really sit unless you bought chairs, right? Along similar lines, we remove the metaphorical “chairs” by simply coating the bottom of these dishes with a polymer which does not allow the cells to “sit” still anymore – thereby forcing them to remain suspended in the liquid media. And voila! with minimal fuss, you have a suspension culture too.

Image taken from a culture dish which had ovarian cancer cells growing in it. The small circular blobs are single cells, whereas the larger clumps are aggregations of cells.

Once the cells have been added to the dishes (called as “seeding”), it’s now time to let them grow and mature. Usually, only a miniscule amount of cells is added to a dish – think of it like making curd at home. Cells will grow, divide, and increase in number over time to eventually fill up the dish completely. The term “confluence” is used to describe the situation wherein the cells have completely filled up all the available space within a given dish. At this stage, we first take them out (the actual process isn’t that straightforward), and seed back a small amount of them into the dish. The majority are used for whatever experiments we would like to run, while the dish goes back into this loop.

This is basically the core use of cell culture – providing cells for experiments. The excess cells which are not needed for seeding a fresh culture are the ones which are used for these studies. And usually, since we need so little for seeding, we almost always get cells in large excess.

All this is honestly just the tip of the iceberg. Actual cell culture is a vastly complex field, with hundreds of specializations, optimized protocols, and demanding techniques. There’s infact a thousand-page book on animal cell culture by Ian Freshney (which I’m supposed to be reading :P).

Actual cell culture work demands high levels of patience and good practices with a keen attention to detail. Since the entire process is so fundamentally radical – attempting to grow a body’s cells in a plastic dish (think about it for a second, its indeed a testament to humanity’s never ending faith in its ingenuity), even the slightest of errors can be catastrophic.

Forgot to wear your gloves? That’s one of the biggest red flags you can have. You’ll end up contaminating not just your cultures, but the entire workstation. Forgot to keep the cells stabilized while changing their media, even for mere minutes? Might as well as throw the culture away – they’ll be writhing under stress and mess up all future experiments. Left them outside for just a bit too long because you stepped out for a quick tea? Congrats, you won’t have any cells left to revive.  And the worst of all is the contaminations you bring in from outside – god alone knows what kinds of fungal spores and bacterial strains we carry around on a daily basis.

But by all accounts, despite its grueling demands, it’s an oddly satisfying endeavor. The very fact that YOU get to nurture and propagate a few hundred thousand living wonders is an incredibly powerful feeling, and simultaneously scary – every inadvertent mistake is another cohort of your people condemned. For the more active cell culture users in the lab, it may even seem like bringing up a kid – feed them, change their dishes, protect them from infections and contamination, make sure they’re happy enough, …. but most of all, for God’s sake keep them alive. As Seinfeld quipped, once you can maintain your own cells, to Hell with the rest of the world!

I know I promised earlier to talk about Henrietta Lacks, but that’ll have to wait. We’re already past our redline of 1500 words, and stop now we must. I’ll be making a couple more posts related to cell culture soon, so stay tuned!

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