posted on 2023-10-07, 01:03authored byJonian Grosha, Jun-Hung Cho, Shannon Pasley, Peter Kilbride, Claudia Zylberberg, Marsha W. Rolle
Engineered tissues are showing promise as implants to
repair or
replace damaged tissues in vivo or as in vitro tools to discover new
therapies. A major challenge of the tissue engineering field is the
sample preservation and storage until their transport and desired
use. To successfully cryopreserve tissue, its viability, structure,
and function must be retained post-thaw. The outcome of cryopreservation
is impacted by several parameters, including the cryopreserving agent
(CPA) utilized, the cooling rate, and the storage temperature. Although
a number of CPAs are commercially available for cell cryopreservation,
there are few CPAs designed specifically for tissue cryostorage and
recovery. In this study, we present a flexible, relatively high-throughput
method that utilizes engineered tissue rings as test tissues for screening
the commercially available CPAs and cryopreservation parameters. Engineered
test tissues can be fabricated with low batch-to-batch variability
and characteristic morphology due to their endogenous extracellular
matrix, and they have mechanical properties and a ring format suitable
for testing with standard methods. The tissues were grown for 7 days
in standard 48-well plates and cryopreserved in standard cryovials.
The method allowed for the quantification of metabolic recovery, tissue
apoptosis/necrosis, morphology, and mechanical properties. In addition
to establishing the method, we tested different CPA formulations,
freezing rates, and freezing points. Our proposed method enables timely
preliminary screening of CPA formulations and cryopreservation parameters
that may improve the storage of engineered tissues.