I have also used 0.5mM EDTA for passaging cells as clumps from Geltrex to Geltrex, as Mark James Davies has suggested.  One shouldn't need to use ROCKi all the time other wise cells can get 'addicted' to it, that is they require it all the time. Generally I use ROCKi only for freezing and thawing of cells or if I need to seed them at single cell density.

Despite this, I have found that with the use of Geltrex, like Matrigel, cultures can  suddenly stop behaving. They can eitherr die or differentiate, be they human ES or IPS. I have since switched to using Laminin 521 and my maintenance cultures are much more reliable and I very rarely see any spontaneous differentiation.

One final thought concerning your line - if it was growing fine and has now stopped doing so, there could be a genetic change. Have you checked the karyotype recently, or even better, the molecular karyotype (i.e. using SNP arrays)?

Hope this helps,

What type of cells are you passaging?

For example: we use an enzymatic process on Geltrex/Matrigel matrix such as Releaser. Then gentle fragment the colonies into clumps with tapping the plate or pipette up and down only once. Then when splitting the cells we add ROCK inhibitor for the first 24-48 hr before the next media change. This aids cell viability in our hands.

IPSCs derived from peripheral blood mononuclear cells. I'm adding ROCK inhibitor still colonies don't attach to the surface. 

Thats very surprising, as if the colonies are established on geltrex matrix to begin with, one would hope they would reattach to the same matrix on passaging.

iPS cells are tricky to handle. All i can suggest is ReLeSR (available from StemCell Tech). We think its essentially EDTA but it works very well and is reproducible on passaging hES/iPS cells. It has the added bonus of not having to be spun out so reducing the time cells spend off matrix support.

Allow colonies to grow a little bigger before replacing might help a little. Just before they start to develop a denser differentiating centre.

Best of luck

Thank you Mark! 

I have also used 0.5mM EDTA for passaging cells as clumps from Geltrex to Geltrex, as Mark James Davies has suggested.  One shouldn't need to use ROCKi all the time other wise cells can get 'addicted' to it, that is they require it all the time. Generally I use ROCKi only for freezing and thawing of cells or if I need to seed them at single cell density.

Despite this, I have found that with the use of Geltrex, like Matrigel, cultures can  suddenly stop behaving. They can eitherr die or differentiate, be they human ES or IPS. I have since switched to using Laminin 521 and my maintenance cultures are much more reliable and I very rarely see any spontaneous differentiation.

One final thought concerning your line - if it was growing fine and has now stopped doing so, there could be a genetic change. Have you checked the karyotype recently, or even better, the molecular karyotype (i.e. using SNP arrays)?

Hope this helps,

Hi, I had the same problem with picking an IPSC colony from Matrigel.  I found that the cells need to be briefly washed with EDTA solution first:

1.       Aspirate media, and then rinse well with EDTA passaging solution

2.       Aspirate and then add 1 ml fresh EDTA passaging solution

3.       Incubate for no longer than 5 min, just until the colonies loosen up a bit but the centers of the larger colonies are still tight

4.       Remove EDTA solution and very gently add PBS

5.       Under a microscope use a p200 to scrape a colony off the plate. (expel air first).  When the colony is loose, suck it into the tip

6.       Deposit colony in a single well of a matrigel-coated 6 or 12 well plate containing E8 and Rock inhibitor.  Pipette up and down 2-3 times max and move tip around to free colony.

7.       24 hours later replace media with E8 alone.

EDTA solution recipie:

500 ml PBS with 0.5 ml of 0.5 M EDTA added, along with 0.9 g NaCl

 Note- the EDTA step greatly helps with subsequent cell attachment efficiency

I think what happens is that without the EDTA treatment, the IPS cells remain attached to the ECM even though you picked the colony.  This prevents the cells from reattaching to the new ECM coated plate.