GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process
GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process
复制标题
DOI：
10.1089/ten.tec.2013.0571
复制
发表时间：
2014-09-01
影响因子：
3
通讯作者：
Morris, G. John
中科院分区：
文献类型：
--
作者：
Massie, Isobel;Selden, Clare;Morris, G. John
在再生医学应用中越来越需要冷冻保存方案，但必须在临床规模上提供功能性产品并符合良好生产工艺（GMP）。虽然使用基于斯特林制冷机的可控速率制冷机（CRF） （EF600）可以实现小规模的GMP冷冻保存，但由于传热问题和冰核控制，成功的大规模GMP冷冻保存更具挑战性，这两个复杂的事件都会影响成功。我们已经开发了一种大型的基于低温冷却器的CRF (VIA Freeze)，可以处理更大的体积，并使用海藻酸盐包裹的肝细胞（HepG2）球体（ELS）对其进行了评估。预计ELS将包含生物人工肝脏的细胞成分，并且临床使用需要类似2l的体积。样品温度和斯特林制冷机功耗记录整个冷却运行小（500亩L）和大（200毫升）体积样品。利用活力（FDA/PI染色及图像分析）、细胞数量（细胞核计数）和功能（蛋白质分泌），以及冷冻扫描电镜和冷冻替代技术来确定可能的损伤机制，对ELS恢复进行评估。缓慢冷却模式成功应用于EF600和VIA Freeze中的样品，并评估了许多冷却和升温模式。EF600和VIA Freeze都采用了优化的冷却方案，从冰成核到-60摄氏度的非线性冷却剖面。在VIA Freeze中，通过控制软件检测冰的成核，既可以对成核事件进行无创检测，以达到质量控制的目的，也可以主动修改冰成核后的冷却剖面。在VIA冷冻活力为93.4%+/- 7.4%的条件下处理200 mL的ELS，得到活细胞数为14.3 +/- 170万个细胞核/mL海藻酸盐，蛋白分泌量为10.5 +/- 1.7 μ g/mL/24 h，与对照ELS（活力-98.1%+/- 0.9%，活细胞数-18.3 +/- 100万个细胞核/mL海藻酸盐，蛋白分泌-18.7 +/- 1.8 μ g/mL/24 h）相比，均有显著提高。使用VIA冷冻，大容量GMP冷冻保存ELS是可能的，功能恢复良好，也可以应用于其他再生医学应用。
Cryopreservation protocols are increasingly required in regenerative medicine applications but must deliver functional products at clinical scale and comply with Good Manufacturing Process (GMP). While GMP cryopreservation is achievable on a small scale using a Stirling cryocooler-based controlled rate freezer (CRF) (EF600), successful large-scale GMP cryopreservation is more challenging due to heat transfer issues and control of ice nucleation, both complex events that impact success. We have developed a large-scale cryocooler-based CRF (VIA Freeze) that can process larger volumes and have evaluated it using alginate-encapsulated liver cell (HepG2) spheroids (ELS). It is anticipated that ELS will comprise the cellular component of a bioartificial liver and will be required in volumes of similar to 2 L for clinical use. Sample temperatures and Stirling cryocooler power consumption was recorded throughout cooling runs for both small (500 mu L) and large (200 mL) volume samples. ELS recoveries were assessed using viability (FDA/PI staining with image analysis), cell number (nuclei count), and function (protein secretion), along with cryoscanning electron microscopy and freeze substitution techniques to identify possible injury mechanisms. Slow cooling profiles were successfully applied to samples in both the EF600 and the VIA Freeze, and a number of cooling and warming profiles were evaluated. An optimized cooling protocol with a nonlinear cooling profile from ice nucleation to -60 degrees C was implemented in both the EF600 and VIA Freeze. In the VIA Freeze the nucleation of ice is detected by the control software, allowing both noninvasive detection of the nucleation event for quality control purposes and the potential to modify the cooling profile following ice nucleation in an active manner. When processing 200 mL of ELS in the VIA Freeze-viabilities at 93.4%+/- 7.4%, viable cell numbers at 14.3 +/- 1.7 million nuclei/mL alginate, and protein secretion at 10.5 +/- 1.7 mu g/mL/24 h were obtained which, compared well with control ELS (viability -98.1%+/- 0.9%; viable cell numbers -18.3 +/- 1.0 million nuclei/mL alginate; and protein secretion -18.7 +/- 1.8 mu g/mL/24 h). Large volume GMP cryopreservation of ELS is possible with good functional recovery using the VIA Freeze and may also be applied to other regenerative medicine applications.