Temperature Control of a Microscope Freezing Stage for the Cryopreservation of Living Cells: Comparison of Two Different Concepts

Abstract

Procedures for the cryopreservation of living cells are often developed in cryo micro scope freezing stages. In a cryomicroscope the temperature of the sample has to be controlled with a precision of ΔT≤1°C within the range from the physiological temperature (37°C) and the storage temperature (< -80°C). At the beginning of an optimization process for a new cell species the survival rates can be very low (<1%). Therefore the area of observation must be large (here: 20 x 15 mm²) to freeze and thaw a large number of cells (here: > 1000) under uniform conditions. A freezing stage was developed to cryopreserve isolated heart muscle cells from adult rats. Two chambers form the stage. One is made of biocompatible materials and contains the cells. The second one is used to control the temperature of the first chamber by a nitrogen gas flow. The chambers are separated by a sapphire disc (thickness 1 mm), which generates a homogeneous temperature field and couples the two chambers thermally. The temperature is measured by a thermocouple on the disc and recorded on a small computer (PC). Cooling rates up to -15°C/min and heating rates up to 60°C/min are realised by the nitrogen gas flow through the stage. Two different concepts to control the temperature of the stage were tested. First, one single gas flow is cooled to -180°C in liquid nitrogen and then heated by a PID controlled electrical fan (compensation principle). The second concept is working with two alternative (180°C /-180°C) gas flows. Four valves to switch the gas flows are PC controlled (two-point controller). Higher precision and quicker response times were achieved with the second concept.