I was wondering if anyone can advice on how to store the skin biopsies (bat wing) immediately after collections in the field, before they can be delivered to the lab for establishment of cell culture. For practical reasons, freezing the skin biopsies in the field work location would be the most feasible. The problem is that in the field work location there will be no access to -80C freezer required for use with slow cooling devices like Mr Frosty (only -20C freezer and liquid nitrogen will be available), so it would be hard to achieve the advised slow freezing rate to -80C. I thought that another solution would be to freeze the biopsies in the field work location using the methanol-dry ice slurry and styrofoam box, or floating the samples in the styrofoam box on the surface of liquid nitrogen. However, this way the rate of freezing would be rather poorly controlled. I wonder if anyone has any experience in freezing the mammalian tissue without the use of -80C freezer? I would be very grateful for any suggestions.
Is it impossible to get the bat(s) to the lab? In cost/benefit terms, 5 bats held for 24hr and approriately cared for, before relesase at the point of capture, is better than 50 sampled in the field without successfully recovering a tissue line from any. There is no reason why they wouldn't be released in a better condition than when found (depending on species)
A well organised trip and team can get bats to and from a lab from an awful long way-away
PS I gave up on a bat serum immunology project a few years back because we couldn't reliably stabilise samples in the field and even the 45min back to the lab was long enough to produce unreliable results. In our case we didn't have the funding to cart bats to and from the lab but in retrospect it was the direction we should have gone
james
Dear Joanna Kacprzyk,
For cryopreservation of skin biopsy in field conditions, after washing rapidly with 70% ethanol and cutting in small pieces, we did the freezing in field by two ways:
1- Keep in tube with PBS in box with glass and transported to lab, it take about 5-6 h for air travel and car from airport to our lab, doing cryopreservation to liquid nitrogen after getting lab.
2- Treated tissues pieces with for equilibration in DMSO 10% at room temperature or 4 C for 45-60 minute, transferred to nitrogen vapor, at this high that temperature is about -15-20 C for 30 -45 minute, then transferred directly to LN.
We have good survival more than 85% after thawing and culture in CO2 tube.
Best regards
Bui Xuan NGuyen
Dear Joanna Kacprzyk,
For cryopreservation of skin biopsy in field conditions, after washing rapidly with 70% ethanol and cutting in small pieces, we did the freezing in field by two ways:
1- Keep in tube with PBS in box with glass and transported to lab, it take about 5-6 h for air travel and car from airport to our lab, doing cryopreservation to liquid nitrogen after getting lab.
2- Treated tissues pieces with for equilibration in DMSO 10% at room temperature or 4 C for 45-60 minutes, transferred to nitrogen vapor, at this high that temperature is about -15-20 C for 30 -45 minutes, then transferred directly to LN.
We have good survival more than 85% after thawing and culture in CO2 tube.
Best regards
Bui XN
My suggestion would be to try a vitrification approach using either VS55 or a high glycerol concentration in which samples are permeated with cryoprotectant in a stepwise manner on ice and then cooled as fast as possible by plunging in your case into liquid nitrogen. The method would need to be validated for skin. Generally the smaller the sample the easier to obtain good viability results.
The attachment demonstrates my preferred way to vitrify tissue samples but for very small samples you should be able to put your tissues in straws used for preservation of reproductive tissues and plunge into nitrogen in the field.
Thank for all your answers! Unfortunatelly, taking bats to the lab for tissue collection is not an option for a numver of reasons, although definitely I agree it would be the most convenient, James. I need to read more on vitrification, thank you for the suggestion and recommended paper, Kelvin.
Hi,
I think a version of any of the answers mentioned so far would work. Check out this link/source and perhaps it can be modified to your needs. In my virus isolation experience, typically as long as you can cryoprotect the specimen somehow and get it frozen asap, all will be fine.
http://webcache.googleusercontent.com/search?q=cache:ApNN1Oeyba4J:www.biomedcentral.com/content/supplementary/2047-217X-1-8-S2.doc+&cd=5&hl=en&ct=clnk&gl=us
Bui, thank you very much for the protocol, looks exactly like something I am looking for! (we will have -20 C freezer available)
Cell cultures can be established from skin biopsies even after about 3-4 days without freezing. In this case, collect the biopsies to the cell culture medium and keep on ice or in fridge at 4C until use.
Hi Peter and Mukesh, thank you for your answers. I think that what we're going to do in the end - just kep the samples on ice until they're transported to the lab.
Dear Joanna
My experience is based on vitrification of human oocytes.
The technique for vitrification of oocytes and the subsequent warming process are producing results equal to those using fresh oocytes.
Vitrification is a process by which liquid turns into a non crystalline solid. It might simply be achieved by plunging the sample into LN at .
As described' you might be able to do it in the field.
Technically I would suggest you to divide the cells into small aliquots , suggesting
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