8 replies to this topic

[viobio]

I am trying to differentiate mouse ES cells (E14-TG2A or D3 line). Any lineage would be OK. The Wobus et al 1997 protocol for cardiomyocytes did not give me more than one hollow sphere and no beating cells after: 2 days hanging drop, 8 days in bacteriological plate -LIF, then 9 days on gelatin -LIF +10-8M all trans retinoic acid. Any ideas? Should I have gotten rid of the MEFs for several passages before the hanging drop expt? Or avoid trypsin (they got very confluent in the center of the 24 well plates towards the end)?

[Sandy_8]

I work with mouse embryonic stem cell (D3) and I differentiate them by hanging drops (500 cells in 20ul) for two days, then for 5 days in a bacterial dish, and then I transfer each embryoid body on dish coated with 0.1% of gelatin.
My ES grow in suspension, so I don't need a feeder layer, but you have to get rid of fibroblast before doing the hanging drops by several preplating or by subcolturing them for few passages on a nude dish.

[viobio]

Sandy_8, on Sep 22 2009, 02:20 AM, said:

I work with mouse embryonic stem cell (D3) and I differentiate them by hanging drops (500 cells in 20ul) for two days, then for 5 days in a bacterial dish, and then I transfer each embryoid body on dish coated with 0.1% of gelatin.
My ES grow in suspension, so I don't need a feeder layer, but you have to get rid of fibroblast before doing the hanging drops by several preplating or by subcolturing them for few passages on a nude dish.

Thanks, Sandy! Do you get some clumps sticking to the bacterial dish? How do you get them off?
How do you recognize an embryoid body?
Do you withdraw LIF for the hanging drops?

[viobio]

P.S. Do you treat the cells with trypsin or collagen during any of this process?

[Sandy_8]

yes, sometimes it happens that some EBs stick on the bacterial dish...I tried to detach them with the tip, trying not to damage them..otherwise, I let them pn the dish...
You have to find the best bacterial dish for you...we've changed several brands before finding the right one, but it's an Italian brand only....

Now I'm using Tryple (form Invitrogen) to split the cells; we've used trypsin 0.25%/EDTA 0.53mM until the last year...the cells seem to look better with tryple now....

In the differentiating medium I don't add LIF.....

Edited by Sandy_8, 23 September 2009 - 07:46 AM.

[Radish]

Sandy_8, on Sep 23 2009, 08:45 AM, said:

yes, sometimes it happens that some EBs stick on the bacterial dish...I tried to detach them with the tip, trying not to damage them..otherwise, I let them pn the dish...
You have to find the best bacterial dish for you...we've changed several brands before finding the right one, but it's an Italian brand only....

Now I'm using Tryple (form Invitrogen) to split the cells; we've used trypsin 0.25%/EDTA 0.53mM until the last year...the cells seem to look better with tryple now....

In the differentiating medium I don't add LIF.....

Hello to all of you

I work with mouse E14 cells and I use a simple protocol to produce EBs (if the starting ES cells were of good quality you will have cardiomyocytes-you will see something similar to a heart beat in the EBS-at day 5):
I trypsinize a 6 well from a 6 well plate, after this I centrifuge the cells for 3 minutes in growth media, ressuspend the cells, plate in a low attachment 6 well plate (about the same price of regular plates), feed every two days and wait

note:the EB media is the same of regular ES cell media, just lacks esgro/LIF.

I hope I could help

[swatisatb1]

Radish, on Nov 22 2009, 05:38 PM, said:

Sandy_8, on Sep 23 2009, 08:45 AM, said:

yes, sometimes it happens that some EBs stick on the bacterial dish...I tried to detach them with the tip, trying not to damage them..otherwise, I let them pn the dish...
You have to find the best bacterial dish for you...we've changed several brands before finding the right one, but it's an Italian brand only....

Now I'm using Tryple (form Invitrogen) to split the cells; we've used trypsin 0.25%/EDTA 0.53mM until the last year...the cells seem to look better with tryple now....

In the differentiating medium I don't add LIF.....

Hello to all of you

I work with mouse E14 cells and I use a simple protocol to produce EBs (if the starting ES cells were of good quality you will have cardiomyocytes-you will see something similar to a heart beat in the EBS-at day 5):
I trypsinize a 6 well from a 6 well plate, after this I centrifuge the cells for 3 minutes in growth media, ressuspend the cells, plate in a low attachment 6 well plate (about the same price of regular plates), feed every two days and wait

note:the EB media is the same of regular ES cell media, just lacks esgro/LIF.

I hope I could help

hello everyone..
i am very new in field of stem cell, and working with D3 cell line. when i transffered th EBs to gelatin coated plates, its not getting adhered to the plates? is this problem is occuring with anyone else?? wat shud i do????
Plz help me

[vitalgene]

swatisatb1, on 31 March 2010 - 03:42 AM, said:

Radish, on Nov 22 2009, 05:38 PM, said:

Sandy_8, on Sep 23 2009, 08:45 AM, said:

yes, sometimes it happens that some EBs stick on the bacterial dish...I tried to detach them with the tip, trying not to damage them..otherwise, I let them pn the dish...
You have to find the best bacterial dish for you...we've changed several brands before finding the right one, but it's an Italian brand only....

Now I'm using Tryple (form Invitrogen) to split the cells; we've used trypsin 0.25%/EDTA 0.53mM until the last year...the cells seem to look better with tryple now....

In the differentiating medium I don't add LIF.....

Hello to all of you

I work with mouse E14 cells and I use a simple protocol to produce EBs (if the starting ES cells were of good quality you will have cardiomyocytes-you will see something similar to a heart beat in the EBS-at day 5):
I trypsinize a 6 well from a 6 well plate, after this I centrifuge the cells for 3 minutes in growth media, ressuspend the cells, plate in a low attachment 6 well plate (about the same price of regular plates), feed every two days and wait

note:the EB media is the same of regular ES cell media, just lacks esgro/LIF.

I hope I could help

hello everyone..
i am very new in field of stem cell, and working with D3 cell line. when i transffered th EBs to gelatin coated plates, its not getting adhered to the plates? is this problem is occuring with anyone else?? wat shud i do????
Plz help me

try fibronecting coating, or collagen

[vitalgene]

in general, make hd of 500 +/-100 cells per 20ul drop, , grow untill 2 days in co2 incubator, wash them with 20% FBS containg IMDM medium, and take them in bacterial-petridish, and grow them on shaker kept in incubator. 8-10 day you sld be getting beating focis on EB
one can use RA also to cuase cellular diffection instantly