Hello! :) I am looking for a protocol for lyophilization/freeze-drying (primary drying only) of organs/tissues to obtain at least 1 g of drying sample at the end of the process, temperature and time needed.
The process could be very simple, it all depends on what you want to do with the tissue afterwards. If you don't care about protein activity or structure, then full vacuum, lots of heat and make biltong all day!
"organs/tissues" covers a wide range of material.
To freeze dry, you need good freezing - water, sugar, dissolved proteins and lipid content, plus any buffer or preservatives you have - will matter. Freezing also "sets" the structure you will see after drying, if you want to do some kind of microscopy where structure is important, then you will need to ensure that things don't collapse, so will need to freeze in a block of ice. The buffer / reagents in which you freeze also can affect protein activity when constituted (if that's what you are doing) - particularly buffer choice as you can see pH shift in buffers when freezing.
When drying - all of the above affect the time and conditions, more water takes more time. More complex organ or tissue, with different surface area to volume ratio - different drying times / conditions need.
Finally - the nature of your freeze dryer is important. If you have a basic lab unit, then you don't have much control. If you have access to a unit with shelves and good controls, then you can hope to preserve more structure and activity.
Final note - dry tissue is almost impossible to macerate - do this before freeze drying if that is in the future processing stages. It will also make drying easier.
Dear Dr Rob Darrington,
Thank you very much for your answer. I only need to do primary drying of the tissues (kidney, liver...). The idea is to perform trace element determination afterwards so it is not important to preserve proteins or anything, they will be digested afterwards. The freeze-dryer is a simple common laboratory one. I am afraid I will need 48 hours at least for the process but I was concerned about time and pressure.
Regards,
Catarina Baptista
Catarina Jota Baptista then suggest grinding / digesting first, and drying that (or an extract of that) as it is less to process in the freeze dryer. Suggest making the tissues as small as possible and freezing each in some water BEFORE placing in the freeze dryer. 1 small vial or microtube, your tissue sample and some water - the water will help things to freeze well.
Hard to say what the melting point of the water in the tissues will be - to play safe, then target -40°C processing temperarture, which needs a vacuum level of ~275uBar or 200mTorr. Pressure = temperature of the ice. Time, best guess, 48 hours seems right. If you can, run over the weekend. If you want it to go faster, put the freeze dryer in a sunny window or shine a light on the samples - IR light will speed things up. More energy input = faster drying.
Kind regards,
Rob
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