Culture substrate dependence of mouse fibroblasts survival at 4 degrees C

When L-929 mouse fibroblasts grown in Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) were stored in a monodisperse suspension at 4 degrees C, the viability decreased rapidly from the beginning of storage. The viability in this study was determined by counting electronically the number of cells with the capacity to attach to glass substrate and with the membrane boundary resistant to a proteolytic digestion. When, however, the dissociated cells were preincubated briefly at 37 degrees C, and subsequently stored at 4 degrees C as they were attaching on a glass substrate, the rapid loss of viability could be reduced effectively. A biphasic survival profile consisting of an initial phase of slowly decreasing viability and the subsequent phase of rapidly decreasing viability were than observed. The rapid viability loss occurred not only when the cell suspension was prepared by mechanical dislodging but also after trypsinization or dispase treatment. Such viability loss was also observed when the dissociated cells were not stored at 4 degrees C directly but preincubated in a monodisperse suspension at 37 degrees C in a siliconized plate and then stored at 4 degrees C. The above results show that the rapid loss of viability is associated closely with the fact that the cells were not attached to the substrate but in suspension.