Compositions and methods for biopreservation
View Patent ↗Methods are disclosed for viable preservation of biomaterials including both prokaryotic and eukaryotic cells/materials such as human cells and tissues at subzero and suprazero temperatures. One embodiment provides a method wherein initial desiccation and subsequent cooling of the biological samples is below their glass transition temperature (Tg) to achieve a stable glassy state without exposing the biomaterials to excessive osmotic/chemical stresses for long periods of time. Another embodiment provides a method that includes combining the initial desiccation with subsequent freeze-drying to achieve a glassy state of biomaterials. Another embodiment provides a desiccation medium with low salt, high osmolyte/glass former content and desiccation of biomaterials in a spherical droplet to avoid the edge effect.
1. A method for preserving a biological sample comprising: transferring the biological sample to be preserved comprising one or more cells to a desiccation medium, wherein the desiccation medium comprises trehalose at a concentration of 0.05-0.30, a polymer at a concentration of 5-20%, and a cryoprotective agent at a concentration of 1 to 10%; desiccating the biological sample in the desiccation medium sufficiently close to the glass transition temperature (Tg) until entering into the rubbery region while allowing the biological sample to equilibrate to changing extracellular conditions during desiccation; and crossing the Tg curve of the biological sample by cooling the desiccated biological sample to 10° C. or more below the Tg of the biological sample to achieve a glassy state of the biological sample.
2. The method of claim 1 , wherein desiccation of the biological sample takes place in a spherical droplet of the desiccation medium to achieve a uniform drying.
3. The method of claim 1 , wherein the desiccation medium replaces a portion of salts with osmolytes or glass formers to reduce both osmotic and chemical stresses while raising the Tg of the biological sample.
4. The method of claim 1 , wherein desiccating the biological sample comprises convective or evaporative or vacuum desiccation while raising the Tg.
5. The method of claim 4 , wherein an initial desiccation of the biological sample is stopped at different moisture contents of the biological sample before reaching the rubbery region, and then the initially desiccated biological sample is cooled to different subzero temperatures selected from the group consisting of 20° C., −40° C. and −80° C. to induce extracellular ice nucleation.
6. The method of claim 5 , further comprising freeze-drying biological sample resulting from claim 5 , which results in crossing the Tg curve of the biological sample at subzero or suprazero temperatures.