Kilbride Peter, Lamb Stephen, Gibbons Stephanie, Bundy James, Erro Eloy, Selden Clare, Fuller Barry, Morris John
Asymptote, General Electric Healthcare, Cambridge, United Kingdom.
Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom.
PLoS One. 2017 Aug 25;12(8):e0183385. doi: 10.1371/journal.pone.0183385. eCollection 2017.
For large and complex tissue engineered constructs to be available on demand, long term storage using methods, such as cryopreservation, are essential. This study optimised parameters such as excess media concentration and warming rates and used the findings to enable the successful cryopreservation of 2.3 litres of alginate encapsulated liver cell spheroids. This volume of biomass is typical of those required for successful treatment of Acute Liver Failure using our Bioartificial Liver Device. Adding a buffer of medium above the biomass, as well as slow (0.6°C/min) warming rates was found to give the best results, so long as the warming through the equilibrium melting temperature was rapid. After 72 h post thaw-culture, viable cell number, glucose consumption, lactate production, and alpha-fetoprotein production had recovered to pre-freeze values in the 2.3 litre biomass (1.00 ± 0.05, 1.19 ± 0.10, 1.23 ± 0.18, 2.03 ± 0.04 per ml biomass of the pre-cryopreservation values respectively). It was also shown that further improvements in warming rates of the biomass could reduce recovery time to < 48 h. This is the first example of a biomass of this volume being successfully cryopreserved in a single cassette and re-cultured. It demonstrates that a bioartificial liver device can be cryopreserved, and has wider applications to scale-up large volume cryopreservation.
为了能够按需提供大型复杂的组织工程构建体，采用诸如冷冻保存等方法进行长期储存至关重要。本研究优化了诸如过量培养基浓度和升温速率等参数，并利用这些研究结果成功冷冻保存了2.3升藻酸盐包封的肝细胞球体。这个生物量体积是使用我们的生物人工肝装置成功治疗急性肝衰竭所需的典型体积。研究发现，在生物量上方添加培养基缓冲液以及缓慢（0.6°C/分钟）的升温速率能产生最佳效果，前提是在平衡融化温度时的升温要迅速。解冻培养72小时后，2.3升生物量中的活细胞数量、葡萄糖消耗、乳酸产生和甲胎蛋白产生已恢复到冷冻前的值（分别为冷冻前每毫升生物量值的1.00±0.05、1.19±0.10、1.23±0.18、2.03±0.04）。研究还表明，生物量升温速率的进一步提高可将恢复时间缩短至<48小时。这是如此体积的生物量在单个盒中成功冷冻保存并重新培养的首个实例。它证明了生物人工肝装置可以被冷冻保存，并在扩大大规模冷冻保存方面有更广泛的应用。
