Tissue Testing Technologies LLC, 2231 Technical Parkway, Suite A, North Charleston, SC 29406, USA.
Cells. 2022 Mar 25;11(7):1115. doi: 10.3390/cells11071115.
The demand for human bioengineered tissue constructs is growing in response to the worldwide movement away from the use of animals for testing of new chemicals, drug screening and household products. Presently, constructs are manufactured and delivered just in time, resulting in delays and high costs of manufacturing. Cryopreservation and banking would speed up delivery times and permit cost reduction due to larger scale manufacturing. Our objective in these studies was development of ice-free vitrification formulations and protocols using human bioengineered epithelial constructs that could be scaled up from individual constructs to 24-well plates. Initial experiments using single EpiDerm constructs in vials demonstrated viability >80% of untreated control, significantly higher than our best freezing strategy. Further studies focused on optimization and evaluation of ice-free vitrification strategies. Vitrification experiments with 55% (VS55) and 70% (VS70) cryoprotectant (CPA) formulations produced constructs with good viability shortly after rewarming, but viability decreased in the next days, post-rewarming in vitro. Protocol changes contributed to improved outcomes over time in vitro. We then transitioned from using glass vials with 1 construct to deep-well plates holding up to 24 individual constructs. Construct viability was maintained at >80% post-warming viability and >70% viability on days 1−3 in vitro. Similar viability was demonstrated for other related tissue constructs. Furthermore, we demonstrated maintenance of viability after 2−7 months of storage below −135 °C.
由于全球范围内正在逐步停止使用动物进行新化学物质测试、药物筛选和家用产品测试，因此对人体生物工程组织构建体的需求正在增长。目前，构建体是及时制造和交付的，这导致了制造过程的延迟和高成本。冷冻保存和存储可以加快交付时间，并由于大规模制造而降低成本。在这些研究中，我们的目标是开发无冰玻璃化配方和协议，使用人体生物工程上皮构建体，可以从单个构建体扩展到 24 孔板。在小瓶中使用单个 EpiDerm 构建体进行的初步实验表明，未经处理的对照物的存活率>80％，明显高于我们最好的冷冻策略。进一步的研究集中在无冰玻璃化策略的优化和评估上。使用 55％（VS55）和 70％（VS70）的冷冻保护剂（CPA）配方进行玻璃化实验，在复温后不久即可获得具有良好活力的构建体，但在复温后的体外几天内，活力下降。方案的改变有助于在体外随时间的推移改善结果。然后，我们从使用装有 1 个构建体的玻璃小瓶过渡到可容纳多达 24 个单个构建体的深孔板。在复温后，构建体的活力保持在>80％，在体外第 1-3 天的活力保持在>70％。其他相关组织构建体也表现出相似的活力。此外，我们还证明了在低于-135°C 的温度下储存 2-7 个月后仍能保持活力。
