Abstract
For many bioengineered tissues to have practical clinical application, cryopreservation for use on demand is essential. This study examined different thermal histories on warming and short holding periods at different subzero temperatures on subsequent functional recoveries of alginate encapsulated liver spheroids (ELS) for use in a bioartificial liver device. This mimicked transport at liquid nitrogen (−196°C) or dry ice (∼−80°C) temperatures. Holding at −80°C on warming after −196°C storage resulted in ELS expressing significant (p < 0.001) damage compared with direct thaw from liquid nitrogen, with viable cell number falling from 74.0 ± 8.4 million viable cells/mL without −80°C storage to 1.9 ± 0.6 million viable cells/mL 72 h post-thaw after 8 days storage at −80°C. Even 1 day at −80°C after −196°C storage resulted in lower viability (down 21% 24 h post-thaw), viable cell count (down 29% 24 h post-thaw), glucose, and alpha-1-fetoprotein production (reduced by 59% and 95% 24 h from 1 day post-thaw, respectively). Storage at −80°C was determined to be harmful only during the warming cycle. Chemical measurements of the alginate component of ELS were unchanged by cryogenic exposure in either condition.
Highlights
Successful and reliable transportation and distribution of bioengineered products from point of manufacture to point of care are essential for clinical application
Viability and viable cell number Samples held at À80°C (1, 4, or 8 days), following storage at À196°C, have significantly worse viability on thawing compared with a sample thawed directly from À196°C (Fig. 1)
In samples held for either 1 or 4 days at À80°C, samples held at À80°C for 8 days after liquid nitrogen storage did not recover viability by 72 h post-thaw
Summary
Successful and reliable transportation and distribution of bioengineered products from point of manufacture to point of care are essential for clinical application. Many biological constructs, such as the 2-L bioartificial liver (BAL) device being developed by the authors,[1] require storage at cryogenic temperatures (below À150°C) for effective delivery and treatment of acute liver failure. Samples can be stored indefinitely[2,3] before thawing on demand. Most complicated bioengineered products take several weeks or months to produce and so just in time manufacture is not logistically or economically feasible for patient treatment
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