I want to work with iPSCs, what do I need to get started

Good morning and happy Monday, no, not feeling it. Yes, I agree, I hate Mondays too. I never fully understood why the day existed except to be worse then Tuesday, and don’t even get me started on Wednesdays. Anyway, I digress, I trust everyone had a lovely weekend, here in Montreal the sun was shining, 25-30s, and I got my pink Irish hue from 5 minutes of sun exposure. So, all good. And yes, we do get sun and summer in Canada. For anyone thinking we live in a northern Tundra with snow filled caps all year long. The snow is only 6 months of the year, the remaining 6 months we get rewarded with glorious sun and thunderstorms.

So, on Friday I did a double-header of a blog and article, and the response has been overwhelming, so thank you everyone for all the support. And to hold up my end of the bargain of 10 blogs/notebook entries before the end of August, I now switch from fun Tom to serious-work Tom and start with Blog 1 on “I want to work with iPSCs, what do I need to get started”. This is what I needed to get everything off the ground, and for new trainees, if you want to work with stem cells, here are some friendly tips and tricks to help get you set up. As a disclaimer, this is what I used, I am not saying this is the only way, but this can be used as a starting point for any discussion as I hope to also learn from the community at large. So here is my easy five step process (why five steps, well every blog has 4-15 steps, so I figured five was a solid number).

Step 1: Ensure you have dedicated equipment to work with these cells. What do I mean by this. Firstly, find a room that is to be reserved for tissue culture use only, and which also has negative pressure. Ask your local facilities, they will know. Then make sure you have a biosafety laminar (BSL) flow hood, which should be class II, type A2. There are many models and any conventional BSL hood used for working with mammalian cells or other cell-lines pretty much fits the bill. We try to ensure hoods for iPSCs are used only for iPSCs, so not to cross-contaminate with other cell types.

But your work doesn’t stop there. You will also need a dedicated incubator to house your cells, set to 37oC and 5% CO2. There are new models coming online with varying oxygen levels, which can assist in reprogramming and other procedures, but I am not going to get into these details at this time. You will also need a brightfield microscope in your TC room for checking your cells on a daily basis, ideally one with capabilities to snap images for your own records (we have the EVOS core) and some form of cell counter, either manual or automated, whatever your budget can stretch to. Outside of the TC room, you will need a -20 and -80 freezer for storing reagents, a four-degree fridge for storing your media, and space for your cells in a liquid nitrogen tank. Based on the size of your group, you can have a small tank like a locator, or if you are in larger group with many lines, you might need a much larger dry phase freezer. Great, so you have everything you need, time to get started, WRONG. Now the paperwork begins.

Step 2: Safety training. All universities have requirements for safety training before you can even lift a pipette in the lab. This goes especially for working with stem cells. At McGill, we need to complete four training courses to work with cells. These include Workplace Hazardous Materials Information System (WHMIS) training, handling hazardous materials, biosafety training, and safe use of a biosafety cabinet. This varies from place to place, but you should expect to take a panel of these four courses. Once completed and passed, you will receive certification to be renewed every 3 years (this is for McGill and it might vary elsewhere). Super, I have my certificates, ready to start. Well no, you have no cells. How are you going to get cells?

Step 3: Ethical approval for working with human stem cells. Before you can even work with stem cells, your lab requires ethical approval to work with stem cells. This applies to all human cells, so what you need to do is contact your local Research Ethics Board (REB) and tell them you plan to work with iPSCs, and they can provide you all the paperwork needed. At the MNI, we have to complete a full human study approval form to to work with these cells. This is submitted to the ethics board, and after several rounds you get approval once you meet all the requirements needed. This needs to be renewed on an annual basis. One tip though, knowing what cells you will need will be a big plus here, and this is what I address in Step 4.

Step 4: Finding iPSCs to work with. You are now ready to work with iPSCs, so the big question is what cell-line you want, and where do you get them from. There are many reputable repositories worldwide, including HipSci, Coriell, Wicell, EBISC, ATCC, to name just a few we got cells from. I really like HipSci as they have so much associated information with their cells, but it really depends on your experimental needed, you might need a specific cell for a specific disease, so only one source will have them. You can also email other scientists to ask them for their cells. Obtaining cells will require an MTA of some kind, and a large pile of paperwork, they also all charge, so be prepared to pay a fee per line, plus shipping to be added on top. They don’t come cheap. We will have our first batch of iPSCs to be made available very shortly through the MNI’s C-BIG repository, the first panel will be ten healthy control lines, and I will fill you in more on these lines in the next few weeks as the blogs progress. The more people who work with them the better. I will also be making our QC pipeline overview available so more to follow on this.

Step 5: Decontaminate room and create a sterile environment for working with iPSCs. The day has come, your cells have arrived, you store them in the liquid nitrogen, and you are just so happy. You call your parents, friends, neighbors and tell them you are ready to work with stem cells. They reply with a that’s nice, or a who is this, why are you calling me, we can just WhatsApp. But it doesn’t matter, you are ready to conquer the world. Just a tiny, small insignificant detail, well actually very significant. You have to ensure that where you work with these cells is of the highest sterility, as most of your work will be done sans antibiotics, so you are walking a tightrope. Any bacteria, yeast, or other contaminating agent even looks at your cultures, kaput.

This means, before you start, decontaminate the entire room and hood. Use mycoplasma removing sprays to ensure you don’t have mycoplasma and use in both hoods and incubators. They smell, so use during a quiet time for the room. Also, 70% ethanol spray for cleaning all surfaces. And lest we forget, the fans in both hood and incubator. If not already serviced, get them serviced. The HEPA fans are your barrier from contamination so making sure they all work and are in good shape is essential. Ideally, technicians should come and service your incubators and hoods on an annual basis.

Once everything is clean, you want it to stay that way, that means a lab coat for use only in the cell culture room, a wearable facemask, a hairnet, latex gloves, and booties. All this apparatus is to minimize contamination, and to ensure you work in the cleanest environment possible. Also, safety goggles and mycoplasma testing on a regular basis. And the room is only for people working with stem cells, no-one is allowed in otherwise unless they are coming to work with their cells. This prevents people bringing in dust, dirt and all kinds of everything. But at the end of the day, how you set things up is up to you, this is what we do.

Great, now I am ready to go, I sit down, and am ready to start. Oh no, what reagents do I use. Well fear not, here is everything we work with, I did say I was giving away all my protocols, so this is the big one, all the reagents we use. I don’t argue for or against a reagent from a particular company, these are the ones we use, and we try to stick to them so as to avoid changing SOPs. Feel free to use it in your group. It’s all clickable, so you can view any reagent and all the details are associated. If you want to make your own reagent list in this format, happy to help, we were fortunate to have the expertise of Trisha Rao in setting up this clickable pdf, along with the hard work of members in the team who helped putting it together. We consider this our recipe book, as its everything across nearly all our iPSC culture SOPs. Any feedback is very much welcome.

Well that’s it for now, next blog, thawing and seeding my iPSCs. I promised you this two blogs ago, so finally we will get to it. I know, I know. The excitement is too much to bear. Just wait a few more days and I will have the next installment up online with how we do it, with our next SOP to follow as we head on the road to the EDDU.

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