I will be freeze-drying small skeletal muscle samples (~60mg) per sample using a Virtis SP Scientific Benchtop Pro Freeze Dryer. I've had difficulty findings a reference protocol to determine the optimal length and would love input. There is seemingly plenty amongst various animal models using larger samples (200-300mg) running them for ~24h, but I'd like to save time if possible.
Has anyone seen any such methodology and with the above device if possible?
Hello Steven,
Key in this kind of drying it a path out of the tisssue for the water. Muscle or other animal tissues can be a complex mix of substances, and it is therefore challenging to model the drying process well. Typically, dry until you reach a constant mass is the method, however, how you weigh the sample in the dryer is a challenge. This leads to long, perhaps over long, methods for drying tissues.
Challenge here is to keep the water all frozen. The tissues is complex, but also the places that the water exists are complex too, full of peptides, sugars proteins and salts, all of which change the freezing behaviour of water. The low pressure, 200mTorr = -40°C freezing / sublimation point, is a funtion of making sure that the whole sample remains frozen so that there is minimal tissue damage and creation of artefact in your sample. Remember, the vacuum pressure in the system will control the sublimation temperature of the water.
The thing on your side is that your samples are low mass. I would, if you can, ensure they are thin, and prefreeze them. However, the challenge is that most tissues processors use a shelf based freeze dryer which enables the tissue to be frozen on the shelf, this gives far superior control to a benchtop basic lab dryer. It also allows the addition of heat, which when in steady contition speeds up drying to a certain extent. To speed things up in a Benchtop, the best we can suggest is put the dryer in a sunny window such that IR from the sun will help out.
If you can, I suggest a couple of experiments with samples that you don't need for your research, e.g. use some meat from the store, and see how that goes. If you prep this like your samples, load up 3 or 4 flasks and then you can remove them sequentially say at 6 or 8 hour intervals to evaluate the process of drying. With such small samples, counterintuitive though this may sound, it may help the process to pre-freeze them a small block of buffer to overcome the antifreeze effects of plasma.
Kind regards
Rob
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