ATTACHMENT STATE SHIFTS VIABILITY VERSUS COOLING RATE (INVERTED U CURVE) DURING FREEZING FOR HUMAN DERMAL FIBROBLASTS

摘要

A large number of studies in cryobiology have focused on understanding the underlying biophysics at the cellular level to help predict survival outcome after cryopreservation or cryosurgery. While this behavior is increasingly well studied and understood in cells gaps remain in our understanding of how cells in tissues behave which can hamper freezing applications in tissues. This study compares freezing behavior in cells in suspension vs. attached (a model tissue) state to investigate any differences in cellular behavior in these two states. Cellular level injury to freezing is often ascribed to two biophysically mediated factors: solution effects injury at slow cooling rates and intracellular ice formation (IIF) at fast cooling rates [1]. While freezing of isolated and cultured cells has been extensively studied for various cell types [2-5] relatively fewer studies have been performed in native tissues due to measurement difficulties. In part to circumvent this some studies have focused on cells in monolayer or artificial tissue constructs [6-8]. Previous work has shown that cells in artificial tissues do dehydrate and form intracellular ice and that an "inverted U" survival curve as a function of cooling rate can be expected for cells in monolayer and artificial tissue constructs [9]. However, the connection of biophysics to viability within artificial tissue systems and whether this connection changes when the cell is suspended vs. in an artificial tissue are relatively unexplored. In this work we show that the connection of biophysics to viability for human dermal fibroblasts (HDFs), a commonly used cell in artificial tissues, changes considerably between suspension and attached states. Specifically we study 1) biophysical response of HDFs to freezing in suspension, monolayer and artificial tissue constructs (i.e. fibrin and collagen) and obtaining the biophysical (water transport and IIF) model parameters for the different systems and 2) measuring and comparing the post-thaw viability changes to HDFs cooled under similar conditions in suspension vs. attached (monolayer or fibrin) systems. Resulting differences are discussed.