title: Use of reduced temperature cell pausing to enhance flexibility of cell-based assays.
abstract: Construction and supply of cell-based reagents for in vitro plate-based screens are often highlighted as a bottleneck within drug discovery. Recent years have seen the successful application of both cryopreservation and automation to increase the capacity and flexibility of cell provision. However, routine cell culture remains a fixed experimental process that requires cells to be prepared and used at specific times. We have investigated the potential of reduced temperature incubation to be used as a simple methodology for stopping and starting cell growth and introduce further flexibility into cell provision. Our results show that incubation of CHOK1, HEK293, and 1321N1 cells at 23 degrees C arrested growth while maintaining cell viability. Recovery of these paused cells at 37 degrees C resulted in resumption of normal cell growth and target protein function. Experiments demonstrated that paused cells, expressing either a recombinant G-protein-coupled receptor or an ion channel, performed comparably with the equivalent continuously cultured cells in a 384-well cell-based assay. This simple technique offers the potential to introduce flexibility into cell culture experiments and processes that were previously considered to be fixed.
authors.affiliation: Pfizer Global Research & Development, Sandwich, Kent, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Wise
authors.forename: Paul William
authors.initials: PW
authors.lastname: Abel
authors.forename: Darren
authors.initials: D
authors.lastname: Cawkill
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D049109:Cell Proliferation; D002470:Cell Survival; D002477:Cells / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D055808:Drug Discovery; D017930:Genes, Reporter; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D018004:Receptors, Bombesin / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D007473:Ion Channels; C546407:P2RX3 protein, human; D018004:Receptors, Bombesin; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3; D011994:Recombinant Proteins; D001618:beta-Lactamases; D002118:Calcium