Microplate-in-a-Box: thermophysical exploration of cold storage high-throughput microplate designs for enhanced rate control in cryopreservation

High-throughput experimental screening is desirable to minimize data acquisition time from vast workloads. Cell cryopreservation experiments are routinely performed in single-sample cryovials despite cell seeding being performed in 96-well microplates because these substrates are known to induce microliter supercooling, are prone to thermal compressibility and their lengthy preparation period extends cell exposure time to potentially cytotoxic cryoprotectants. Rather than improving the methodological preciseness of cooling, latest efforts have focused on refining cryoprotectant formulations and supplement precautionary ice nucleators. Here, we built 16 microplate-in-a-box cold storage apparatus by iterative design which allow multi-sample slow freezing cryopreservation in 96-well microplates while ensuring the biologically optimal and reproducible cooling rate (1-3 °C/min) required to minimize the deleterious effects of intracellular and extracellular ice formation. The optimal recipient, a 31.9 × 25.8 × 20.5 cm, 4.3 cm thick Styrofoam recipient pre-equilibrated at -80 °C with internal Styrofoam insulation, yielded a linear -1.2 °C/min cooling rate with minimal variability (±0.2), absent of non-linear thermal lag. Throughput increased by 5.3-fold for single and 10.7-fold for double microplate setups. The most impactful features were recipient thermal pre-equilibration before microplate insertion (IR = 7.0 ± 0.5), Styrofoam insulation of the microplate (IR = 6.9 ± 0.2), increased recipient wall thickness (IR = 5.8 ± 0.5), microplate elevation inside the apparatus (IR = 3.5 ± 0.1) and microplate pre-equilibration (IR = 2.7 ± 0.2), all of which contributed to an attenuation of heat exchange mechanisms. The development of easy-to-use, easy-to-build apparatus with common laboratory materials that significantly enables an enhancement of cooling rate control, improving throughput, reproducibility and procedural uniformity is essential to minimizing the impact of non-biological factors in post-thaw cell viability.