An iPSC line is not a cell line in the sense you are used to. HEK293 and HeLa will forgive you. They grow, you split them, and if you are two hours late nothing happens. A pluripotent line spends every day deciding whether to stay pluripotent, and the conditions you provide are the vote.
That is why most beginner failures are not contamination. They are drift: a culture that is still alive, still growing, and quietly no longer the thing you thought you were working with. Everything below is organised around preventing that.
Before you touch a cell
Know where the line came from
If you are not reprogramming your own, get the line from a repository that documents it: hPSCreg indexes lines and their provenance, WiCell, Coriell and EBiSC distribute them. A line handed over on ice by a departing postdoc with no paperwork is a line you cannot publish on without awkward questions about consent.
Whatever the source, ask for four things before it arrives:
- Passage number. You are inheriting whatever damage accumulated before you, and you need to know where the clock stands.
- A recent karyotype or CNV result, with a date and a passage number attached.
- Mycoplasma status, tested, not assumed.
- Which culture system it was adapted to. Medium and matrix both. A line that has only ever seen one system will sulk when you move it to another.
Pick one medium and one matrix, then stop changing them
Feeder-free is the default now, which means the cells sit on a protein coating rather than a layer of irradiated mouse fibroblasts. Two medium families dominate:
- Essential 8 and its relatives, from the chemically defined formulation that stripped hPSC medium down to eight components and removed serum albumin, whose batch variability had been causing a lot of unexplained bad months.
- mTeSR1 and mTeSR Plus, the latter stabilised so it tolerates a skipped weekend feed.
For the surface, Matrigel and Geltrex are cheap, effective and undefined, being tumour-derived extracts that vary by lot. Recombinant vitronectin or laminin-521 costs more and behaves the same way every time. For a beginner, either works. What does not work is switching between them halfway through a project and then trying to interpret what changed.
Write your choice down, buy enough of one lot to finish the work, and treat the combination as a fixed part of the experiment rather than a consumable you reorder by whatever is in stock.
Day 0: thawing
Coat your plate first and let it sit while you fetch the vial, following the coating time on the matrix datasheet. Never thaw into an uncoated well.
The thaw itself is fast, and everything after it is slow:
- Warm the vial in a 37 degree water bath until only a small ice crystal remains, usually under two minutes. Speed matters here.
- Transfer the cells into warm medium slowly, adding the medium dropwise at first. Dumping cold DMSO-laden cells into a large volume in one go osmotically shocks them.
- Include a ROCK inhibitor, typically Y-27632 at 10 micromolar, in the first medium the cells see.
- Plate as clumps, not a single-cell suspension, and move the plate in a firm figure of eight rather than swirling, so the clumps land evenly instead of piling in the centre.
The ROCK inhibitor is worth understanding rather than following blindly. Pluripotent cells pulled apart into single cells kill themselves, and the original report showed Y-27632 suppresses that dissociation-induced apoptosis, raising cloning efficiency from roughly 1 percent to roughly 27 percent. It is the difference between a thaw that works and a well of debris.
Take it out after 24 hours. It is a rescue for a specific insult, not a supplement, and cultures left on it chronically look subtly wrong.
Expect day 1 to be ugly. Sparse, lots of floating dead cells, not much attached. Change the medium, which removes the debris, and judge the thaw on day 2 or 3 instead.
The daily routine
Feed every day, at roughly the same time, including weekends unless you are on a medium formulated to skip one. This is the least glamorous instruction here and the one that separates good cultures from bad ones.
Look at the plate under the microscope before you aspirate, every single time. You are checking for four things.
Good colonies are tight and round with sharp, defined borders. The cells inside are small and uniform, with very little cytoplasm around each nucleus and prominent nucleoli you can pick out at 10x. The colony looks like a single flat sheet, not a mound.
Differentiation shows up as loose, flat, spread-out cells, usually starting at colony edges, and as a fuzzy border where a crisp one used to be. Dense or dark centres mean the colony has got too large and the middle is starving.
Small amounts of differentiation are normal. Scrape those regions off with a pipette tip under the microscope, or mark and aspirate them, before you feed. If more than roughly one colony in ten looks wrong, something systematic is happening: stop and work out what, rather than passaging the problem forward.
Density is the fourth, and it is the one beginners misjudge. You are looking for when to passage, which is covered next.
Passaging
Passage at roughly 70 to 85 percent confluence, before colonies touch each other and before centres compact. In practice that is every four to six days for most lines. The single most common beginner mistake is leaving them one more day, and one more day is how a clean culture becomes a differentiating one.
Clump passaging is the default. The cells survive better in small groups than as singles, so unless you specifically need a cell count, do not dissociate fully. A typical EDTA passage:
- Aspirate the medium and rinse the well with DPBS.
- Add 0.5 millimolar EDTA in DPBS, enough to cover, and leave it at room temperature for about three to five minutes.
- Watch the plate rather than the clock. You want colony edges to lift and the cells to look bright and separated with gaps opening between them, but the colonies still attached.
- Aspirate the EDTA completely. This is the step people rush; residual EDTA carried into the new well is a common cause of poor attachment.
- Add fresh medium and detach the colonies by pipetting across the surface a few times, breaking them into clumps rather than a smooth suspension.
- Distribute into fresh coated wells at a split of roughly 1:6 to 1:12, and adjust next time based on how fast you reached confluence.
ReLeSR and similar reagents do the same job with a selection step that favours undifferentiated cells, which is genuinely useful when a culture has started to drift.
Single-cell dissociation, with Accutase or TrypLE, is for when you need an accurate count: seeding a differentiation, sorting, cloning. Always add ROCK inhibitor when you do it, and accept that it is harder on the cells than a clump passage. It is a tool for specific jobs, not a routine.
Do not passage too sparsely either. These cells signal to each other and a well with a handful of isolated clumps often recovers badly.
Bank early, and bank properly
The passage you are on now is the healthiest the line will ever be. Freeze vials at the earliest passage you can, and once the culture is clearly happy, freeze a proper bank of ten or more.
Freeze as clumps, in a purpose-made cryopreservation medium or 10 percent DMSO, cooling at about one degree per minute in an insulated container, then move to liquid nitrogen for long-term storage. Label with line, passage number and date, and keep a register somewhere that is not a sticky note on the dewar.
Then thaw one vial and confirm it recovers. A bank you have never tested is a hypothesis, and the moment you discover it is a bad one will be the moment you actually need it.
The three checks that save projects
These are not optional extras. Each one catches a failure that otherwise surfaces months later, in an experiment you have already paid for.
- Mycoplasma, monthly. It does not make the medium cloudy and it does not kill the cells. It changes their behaviour, quietly, and it spreads through an incubator.
- Karyotype or CNV, roughly every ten passages and before any experiment you care about. Pluripotent cultures accumulate genetic changes over time, and abnormal cells are abnormal precisely because they grow better, so they take over. The International Stem Cell Initiative analysed 125 human ES lines and 11 iPSC lines from 38 laboratories and found a progressive tendency to acquire changes on prolonged culture, most commonly on chromosomes 1, 12, 17 and 20.
- Identity, at bank creation. An STR profile takes one submission and settles forever the question of whether the vial contains what the label says.
Pluripotency markers such as OCT4, NANOG, SSEA4 and TRA-1-60 are a reasonable periodic sanity check, by flow cytometry or staining. Keep in mind what they do and do not tell you: they confirm the cells still look pluripotent, not that the line can still differentiate into the lineage you need. Only a differentiation tells you that.
How this usually goes wrong
In rough order of how often it happens:
- Cultures left to overgrow because passaging was inconvenient that day.
- Feeding that slips: a missed day, or wildly different intervals across a week.
- Single-cell dissociation without ROCK inhibitor, or rough pipetting that effectively achieves the same thing.
- Passage number creeping up unnoticed, with no karyotype since the line arrived.
- Small amounts of differentiation tolerated week after week until the culture is majority something else.
- A new Matrigel lot, or a switch of medium, in the middle of a time-course that then cannot be interpreted.
Notice that none of these are technically difficult. They are all consistency problems, which is the honest summary of iPSC culture: the skill is not in any single manipulation, it is in doing the same unremarkable thing every day and looking at the plate properly before you do.
A realistic first month
Week one, thaw a vial and do nothing ambitious. Week two, passage twice and start recognising what a good colony looks like in your hands, on your microscope. Week three, freeze a few vials back down and confirm one thaws. Week four, run mycoplasma and send for a karyotype if the line arrived without a recent one.
Only then start the experiment you actually wanted to do. A differentiation protocol that fails because the starting culture was drifting looks exactly like a differentiation protocol that fails because you got a step wrong, and you will burn weeks telling those two apart.