Environmental support

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Environmental support

Post  mcs on Fri Jun 13, 2008 2:25 am

We work on cultured cardiac myocytes using MEA plates.

Recently we have noticed that although a plate may continue to “beat” on the bench for several hours, after a plate has been removed from the incubator we rapidly (~10 mins) see subtle changes to the properties of the signal which we think may be early precursors of cell death.

Ideally we would like to get rid of these changes or at least extend the window of recording before these changes emerge, so we are looking to provide better environmental support for cells while recording from MEA plates.

We were thinking to better control and monitor Air/CO2, pH, humidity and light levels i.e to best mimic the inside of a CO2 incubator. The simplest solution would be to record directly inside a CO2 incubator however our gut feeling is that this may damage the amplifier particularly with the issue of humidity not to mention the practical problems of setting the system up. Despite which we still have to ask, is the MEA1060 capable of being used inside a CO2 incubator?

Another possibility which came to us would be to place the whole amplifier inside an airtight box which we could prime with Air/CO2 and keep humid and dark, the pH could be maintained by perfusing with media and the temperature maintained like usual. Has anyone attempted anything similar or can recommend a commercial product which can do this?

Secondly, are there any other variables we’ve missed? We are aware that osmolality may also be a factor and are considering the use of CMOS arrays to allow monitoring of the bath pH, osmolality and temperature.

Thanking you in advance.

s.broadbent@zi-medical.com

mcs

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Re: Environmental support

Post  mcs on Fri Jun 13, 2008 2:26 am

Dear SBroadbent,

you are right, that we would not recommend to place the amplifier in a humid incubator.

However, you could use the Teflon Membranes provided by ALA (permeable for CO2, but not for Water) as a lid on the MEA dishes. These closed dishes could be placed into an icubator with 5% CO2 in the amplifier.

Our cells seem to be very sensitive to osmolarity changes, pH and temperature. We use a HEPES buffered medium from about 1h prior to recording to the end of the recording. A continous perfusion of prewarmed medium will reduce osmotic effect due to evaporation from the heated MEA stage.

The change from bicarbonate buffered Medium to HEPES buffer is tolerated quite well by our cells.

Thomas

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Re: Environmental support

Post  mcs on Fri Jun 13, 2008 2:26 am

Thanks, we'll look into it.

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Re: Environmental support

Post  mcs on Fri Jun 13, 2008 2:26 am

I routinely record from probes sitting in an Asahi 4020 CO2 Incubator.
These are small incubators well tested for electrophysiologic recordings by a number of labs. They are practically noise-free (electrically).
There is now actually a version of the Asahi incubator with a built-in Panasonic MED64 amplifier cable. But even with the standard version, one can slide the amplifier cable in the door and still keep it closed and airtight.
I record without any moisture in the incubator because I rely on probe caps with Steve Potter's patented teflon membrane seal, which allows just the passage of CO2 and O2 and nothing else. With that, you also avoid microbial contamination.

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Re: Environmental support

Post  mcs on Fri Jun 13, 2008 2:27 am

Here is an alternative for a home-built mini-incubator for recording from cell cultures outside the incubator while maintaining pH, CO2, oxygen, and humidity. This solution however does not allow you to still see your sample, unless you use a glass top cap.

You will need either one of the culture chamber caps, or a cut 50ml tube (it fits around the regular MEA well).

-Use a culture chamber cap or Cut a 50 ml tube in half (then flame the end a bit to round it and make it fit nicely around MEA ring)
-Drill two small holes near the top of the cap/tube
-Pass a small plastic tubing through the holes, holes should be small enough to hold tubing tightly (hermetic seal not needed as you'll be flowing air out anyway, you need that as pressure relief!)
-poke many pin-size holes through the tubing in the section inside the cap/tube
-rotate tubing so that holes face up toward top of cap
-cover the "out" end of the tubing with parafilm, and poke a hole through that (use this to help regulate air pressure and flow)
- connect a 5%CO2/95%O2 tank to a regulator, then to an air filter, then through a water flask (tube goes into to water to bubble, bubbles go out and take moist gas to next step), then pass the purified and humidified 5%CO2 to the "in" end of the mini-incubator you just built.

Set your pressure to make sure you have gas coming out of the small holes in the chamber and not too much that you are causing waves (adjust flow rate and pressure, and you can close/open the other end of the tube to help with that)

I hope this helps,
Walid

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Re: Environmental support

Post  mcs on Fri Jun 13, 2008 2:27 am

Thank you for the suggestions.

We've started using the Potter membrane and ring system within a Plas Lab 850-LCS incubator (Jencons). We have seen a marked improvement in cell health and longevity (up to around 48 hours) and stability of pH with relatively low noise. We do however notice that an evapouration/condensation/drip-return cycle forms within the bath off the membrane. This is mildly annoying if it should happen to "drip" during a recording however we did notice afterwards that a film of plastic fibres appears to form in the bath solution. We suspect this is something which the membrane has either shed or has been washed off by the condensation, has anyone else seen this and does it constitute a problem?

Secondly has anyone worked out a reasonable way to set-up a system for drug application inside the incubator? We are currently using a manual syringe system which is less than perfect.

Thanking you in advance.

Steven Broadbent

s.broadbent@zi-medical.com

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