(4bo) Systematic Design, Implementation and Evaluation of Vitrification as a Preservation Method for An Encapsulated Cell System | AIChE

(4bo) Systematic Design, Implementation and Evaluation of Vitrification as a Preservation Method for An Encapsulated Cell System

Authors 

Lawson, A. - Presenter, Georgia Institute of Technology
Sambanis, A. - Presenter, Georgia Institute of Technology


The primary goal of Tissue Engineering is to utilize current biological knowledge and engineering principles to ?produce functional replacement tissue for clinical use? (MacArthur, Nature 2005). In order to achieve large-scale clinical use, it will be necessary to manufacture, test, store, transport and therefore preserve the product. Preservation will be critical in bringing most tissue-engineered constructs from the benchtop to the clinic. Currently, most of the methods being used in Cryobiology are developed through trial-and-error. In order to better understand and design methods for cryopreservation, it is important to systematically investigate all components of cryopreservation. This includes the investigation of the effects of cryoprotectants (CPAs), alone and as cocktails in solution, and the investigation of CPA addition/removal and cooling/warming protocols. A tissue-engineered pancreatic substitute consisting of encapsulated murine insulinomas was chosen as the test-bed for these studies. The systematic approach was as follows. (1) A mathematical model was used to design addition and removal protocols for cryopreservation. (2) The effects of cryopreservation on the encapsulated cell system?both cellular viability and function and biomaterial integrity and function?were determined in vitro. (3) The effects of cryopreservation on the in vivo functionality were determined in an induced-diabetic mouse model. The most common method of cryopreservation in use today is conventional freezing. Conventional freezing utilizes low levels of CPAs, slow cooling and rapid warming to preserve cells, tissues or constructs. The relatively low concentrations of CPAs are usually minimally cytotoxic to the cells. Unfortunately, these low levels allow ice formation in the extracellular matrix. This ice formation may be detrimental to the integrity, strength or even overall function of a construct. Vitrification was developed in response to this and utilizes high concentrations of CPAs with rapid cooling and warming to achieve a vitreous, or glassy, state. Successful vitrification eliminates ice formation and may maintain the biomaterial better than conventional freezing. However, the addition of high concentrations of CPAs also increases the overall cytotoxicity and requires multi-step addition and removal as osmotic excursions must be maintained within the cell's tolerable limits. These two types of cryopreservation address different concerns in the preservation of a construct. In order to gain insight into the preservation of an encapsulated system, both were used to cryopreserve the tissue-engineered pancreatic substitute. A previously developed mathematical model that describes the mass transfer and cell permeability was used to simulate the osmotic excursions that the cells within the construct undergo. This allows the design of protocols that achieve vitrifiable concentrations throughout the construct while minimizing the cell's osmotic excursions and exposure to the CPAs. This mathematical model was expanded to include heat transfer and CPA cytotoxicity. The expanded model allows for investigation of more complex conditions that may be difficult to achieve experimentally but that warrant further exploration. This mathematical model was used to design addition and removal protocols for vitrification. Critical to the success of a preservation method is the maintenance of cellular viability and function as well as biomaterial integrity and function. Encapsulated cells were cryopreserved using conventional freezing or vitrification and the results compared with encapsulated cells that did not undergo a cryopreservation procedure. The results of in vitro experiments and of in vivo studies involving implantation of encapsulated cells at sub-therapeutic amounts in normal animals indicate that the alginate matrix is preserved during vitrification but appears also not to be functionally damaged by the ice formed during conventional freezing. However, encapsulated cells that are vitrified may retain their in vitro function better than those that are conventionally frozen. Finally, the cryopreserved pancreatic substitute is assessed for in vivo performance by implanting therapeutic amounts in mice with chemically induced diabetes. The results of this systematic design of cryopreservation protocols, their in vitro assessment, and the in vivo performance and efficacy of the cryopreserved capsules will be discussed.