Application of Bioassays for Suspended 293 Cell Enumerations in Chemically Defined Medium (CDM)

Abstract

Suspended 293 cells grown in chemically defined medium (CDM) are difficult to accurately enumerate using the hemacytometer. Since 293 cells tend to form aggregated clumps of various sizes as they grow, inaccurate hemacytometer cell counts are likely due to errors in attempting to count aggregated cell clusters as well as uneven distribution of the aggregates in the capillary loaded counting chambers. For our work, it was imperative that we develop two different fluorescence based bioassays to 1) ascertain an accurate 293 cell enumeration and to 2) determine the cell vitality. We applied these bioassays in the development of CDM for single-cell suspension culture of 293 cells. Moreover, these fluorescence bioassays can be used as tools in monitoring the cells during viral and transient protein production. One of the bioassays measures the metabolic activity by Alamar Blue staining and the other measures the cell numbers by Sytox nucleic acid staining. Alamar Blue (AB) is a water soluble fluorometric/colormetric growth indicator based on detection of metabolic activity (REDOX). Sytox is a green nucleic acid stain that easily penetrates cells with compromised plasma membranes. These bioassays can be formatted from 96 well to 6 well plates. Both the Alamar Blue and Sytox assays have proved to be powerful tools in new media development and in the monitoring of spinner suspension cell growth kinetic experiments.