[Open_electroporator] culture shock tests on live cells

[John Griessen]

On 07/11/2017 08:49 PM, Mitchell Altschuler wrote:
 > It also seems that the old EP process was changed from what was done in the past I assume because of the cost and safety issues 
... some of those capacitors seemed extremely dangerous if the charge was not discharged ....
 >
 > Since I am new to this project it would be useful to know why we think these new parameters will still work?  Is that 
documented anywhere?

I found plenty googling "electroporation cells" "electroporation bacteria" "electroporation cell wall" "electroporation voltage"

Here is some of the early theory and experiment by Dr. James C. Weaver* on electroporation:

http://onlinelibrary.wiley.com/wol1/doi/10.1002/jcb.2400510407/abstract
http://cicbs.com/rife/Electroporation97.html

His long list of references is good for finding some more papers also.    http://www.freefullpdf.com  helped.

Reading Weaver et al, you get a conclusion that if the cells are not close and touching by a good bit, they will
get pores in the sides closest to either end along the lines of electron flow, and if they are moved around some
you can have pores all around.  This seems natural to me from my studies of fields.  Pores all around gives good transfection -- 
it lets molecules go in and out of good sized holes that appear in a couple of microseconds, then go away, (zip up), after seconds 
or minutes pass.

Another conclusion from this and other researchers' papers is that there is a threshold for making pores, after which
they stay open and any higher volts is destructive, but repeated lower volts tends to move charged molecules and push and pull 
them through the pores.

Beyond that it's all details.  Many of the current bragging "new tech" instrument sellers say they have
made choosing the volts to transfect at automatically.  That amounts to zapping, then measuring the effective impedance of the 
whole cuvette and comparing it to the before condition.  When the impedance drops some, you infer that pores have opened, and stop
increasing the volts then.  the culture shock will probably be able to do this function also and without running into any patents, 
since this phenomenon is mentioned in JCW's early papers from 1992 - 1995, which is in the patent expired range now.

So, to give you the short answer of "why we think these new parameters will still work?", it's that  volts matter, and they have
quick effect in the microseconds range, then the cells stay  "porated" for a while, (minutes), and that means volt level matters, 
and nothing in particular about the waveform matters.  An effective waveform from the theory is a peak at X volts with a top that 
lasts at least 10 microseconds, then it can fall and be jaggy or smooth, and come back up to 0.9 * X volts for another twenty 
times over 10 milliseconds falling to .5 * X volts in between or just stay between 0.9 * X and 0.89 * X for up to 10 milliseconds
and be good.

Either way -- smooth or jagged volts will work fine.  Square pulses or exponential rises and falls will work just as well.

That's my reading of JCW's work.  Next to prove it with the convenient culture shock gear.  Much easier than experiments in 1992.

-- 
John Griessen
cibolo.com  Austin TX  building lab gear for biologists