Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine.

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity. First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with MeSO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for MeSO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for MeSO. A mixture of MeSO, ethylene glycol and glucose (ratio 4:2:1- termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using MeSO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures. In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale.