Differences in the Requirements for Cryopreservation of Porcine Aortic Smooth Muscle and Endothelial Cells

Abstract

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me₂SO) in these cells at 2–4° and 22°C. The following results were obtained:

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Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me₂SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me₂SO. Following cooling at 0.3, 1, or 10°C/min and storage at less than –160°C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3°C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10°C/min.