Optimizing a Protocol for Cryogenic Preservation of Hepatocyte Encapsulates.

摘要

Lack of specific and established methodology for maintaining long-term cell viability in an engineered liver cell encapsulate limits its lifespan and feasibility for clinical application. Controlled rate cryogenics is an attractive option for maximizing cell viability and preserving liver functions. The solution holds numerous potential benefits because primary hepatocytes of a liver experience expedited loss of metabolic function in ex-vivo. This is due largely in part to the oxidative stresses incurred during isolation and the complexity of reproducing the host environment in a micro-engineered tissue environment. The lack of specific methodology for the cryopreservation of engineered constructs poses a challenge in creating a sustained post-thaw model.;In this research, alginate based hydrogel encapsulates of hepatocytes were developed for the subsequent design an execution of an improved cryopreservation protocol. The cryopreservation process is founded upon known physical, chemical, and kinetic cryogenic principles. Using primary rat hepatocyte models, we supplemented traditional media with 3 novel sugars and performed an expanded post-thaw protocol which included dynamic maintenance of encapsulate culture. Viability assessments were performed through fluorescent microscopy intensity profiles, and MTT assay. We hypothesized that the dual alterations to traditional cryopreservation practices would greatly improve post-cryopreservation viability and functionality. This project sought to improve previously achieved viability through the combined use of novel carbohydrate compounds in pre-incubation culture and rocker sustainment of post thaw culture. Our results demonstrated the benefits of two of our novel compounds (dexamethasone & a proprietary sugar) in post-thaw viability. Furthermore, we have found supporting evidence that post-thaw rocker culture greatly improved long-term sustained viability. Successful preservation of the primary function of hepatocytes in our hydrogel encapsulate model holds useful application to the mass production of bioengineered tissue models for clinical application and drug toxicity studies.