Microfiltration-based sequential perfusion: A new approach for improved loading/unloading of cryoprotectants

In cryopreservation, the steps of loading and unloading of cryoprotectants (CPAs) are often time consuming and related to significant osmolality imbalance which causes osmotic damage to cells. Many attempts have been made but major challenges still remain. Here, we present an innovative strategy using microfiltration-based sequential perfusion (MSP) approach to achieve safe and efficient CPA loading /unloading. After successful fabrication of the prototype device, both simulation and experiments were conducted to evaluate the proposed MSP process. Device-level simulation and cell-free experiments demonstrated the device successfully generated sequential perfusion to cell suspension for both CPA loading and unloading purposes. Furthermore, cell-level simulation results displayed signs of reduced level of osmotic shock to cells in MSP when compared with conventional methods. In-vitro experiments with RBCs further demonstrated the presented MSP device is capable of performing efficient CPA introduction and removal with favorable cell recovery rates (> 88 %) at high throughput (1 ml/min for CPA loading and up to 0.8 ml/min for CPA unloading, respectively). In conclusion, the data obtained from this study has not only proven that distinct superiority of the presented MSP over the other conventional approaches in terms of CPA loading/unloading efficiency and cell survival, but also demonstrated the flexibility in device geometry customization to accommodate cryopreservation specimens with various sizes. The developed MSP-based CPA loading and unloading system with higher efficiency, higher cell safety, higher throughput and higher degree of device modification ability offers a promising solution to directly address one of today’s major challenges in cryopreservation.