(L-473) Large-Volume Droplet Vitrification Enables High-Throughput Storage of Primary Hepatocytes

Introduction:: End-stage liver disease claims over 50,000 lives each year in the U.S. Despite being the 9th most common cause of death, the only treatment currently available for individuals affected by this illness is orthotopic liver transplantation. This course of treatment lacks efficacy on the population scale due to the widespread organ shortage, with a yearly deficit of approximately 4000 livers. To bridge this gap, alternative hepatocellular-based treatments are in development including recellularized scaffolds and direct hepatocyte transplantation. These treatments require large stocks of primary hepatocytes, on the magnitude of approximately 10 billion cells per patient. This necessity poses a challenge, however, due to the fact hepatocytes are single passage cells, and cannot be grown, necessitating long-term storage. The current standard for hepatocyte storage is slow-freezing in the presence of DMSO, a cryoprotectant agent (CPA). This method is limited by a dramatic loss of viability and poor yield. To combat this issue, we developed an alternative method for hepatocyte storage: bulk-droplet vitrification. Using a high-concentration CPA cocktail, we use rapid mixing of cells and CPAs to enable vitrification of cell-laden "glass" droplets. Despite its success in maintaining viability and improving yield, this batch technique is labor-intensive, and limited in the number of cells that can be stored at a time. To fix this issue, we developed a large-volume alternative approach enabling single-step vitrification of whole-liver hepatocyte yields, while maintaining cellular viability. This approach promises to improve approaches to organ engineering and push the field of liver transplantation closer to organ parity.


Materials and Methods:: Prior to the vitrification process, vitrification solution 1 (V1) and vitrification solution 2 (V2) were prepared. V1 consists of UW solution supplemented with 2.4 ml/mL bovine serum albumin (BSA). V2 consists of UW solution supplemented with 2 mg/mL BSA, 800 mM sucrose, 32.5% v/v ethylene glycol (EG), and 32.5% v/v propylene glycol. Primary rat hepatocytes were isolated day of the experiment and counted using AOPI stain, after which the hepatocyte yield was spun down at 25G for 5 minutes. The hepatocytes were resuspended in V1 at 10,000,000 cells/mL. 3.75% v/v DMSO and 3.75% v/v were added with a 3-minute incubation period. Following resuspension, the cell solution was transferred to a 100 mL beaker with a magnetic stir bar set to the lowest possible speed to prevent sedimentation. Tubing connected to a peristaltic pump was placed in the cell suspension. V2 solution was added to a 50 mL syringe and placed on a syringe pump with tubing connected. The outflow of each tubing was connected to a mixing needle set above a full liquid nitrogen dewar with a 3D printed inset containing 4 50 mL conicals for droplet collection. Both pumps were set to flow of 1 mL/min until the full cell suspension had finished vitrifying. Following vitrification, the cells were added to DMEM media with 400mM sucrose at 37°C and spun down. The cells were resuspended, counted, and plated. 24 hours after plating, presto blue metabolic assay and live/dead staining were performed to obtain cell viability and functionality.


Results, Conclusions, and Discussions:: Cell suspensions containing upwards of 100 million cells were successfully vitrified in a single-step manner. Upon devitrification, the large-volume vitrification (LVV) cells showed a negligible loss in viability when compared to the batch droplet vitrification process, and improved viability when compared to the slow-freezing cryopreservation (SFC) method, although a loss in viability was seen when compared to freshly cultured cells. The cultures were kept alive for 24 hours and plating was compared, showing similar activity between LVV and batch, improved activity between LVV and SFC, and impaired activity between LVV and fresh. When looked at under the microscope, LVV showed healthy hepatocyte morphology, with cells spreading across the plate and forming bile canaliculi, indicating the preservation of baseline functionality. We have successfully shown vitrification of whole-liver hepatocyte yields. Upon devitrification, the cells showed improved viability and functionality when compared to standard slow freezing cryopreservation. This technique is the first instance of single-step bulk hepatocyte cryopreservation. Access to large hepatocyte stores will enable improvements across the fields of liver transplantation as new approaches to organ engineering will have access to bulk hepatocyte yields, enabling the creation of larger artificial grafts and higher capacity cellular therapeutics. The next step in this project will be to vitrify the full hepatocyte yield obtained from human livers, a 50x scale-up over rat livers. We expect that the completion of this project will open up access to the clinical translation of current experimental techniques in liver engineering and help mitigate the liver graft shortage.


Acknowledgements (Optional): : This material is partially based upon work supported by the National Science Foundation under Grant No. EEC 1941543. Support from the US National Institutes of Health (R01DK114506 ) is gratefully acknowledged.


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