title: An automated system for delivery of an unstable transcription factor to hematopoietic stem cell cultures.
abstract: An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT-HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single-use, closed-system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 +/- 0.2 h) and extracellular (3.7 +/- 1.8 h and 78 +/- 27 h at 37 degrees C and 4 degrees C, respectively) half-lives of TAT-HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which was used to predict the dynamic intracellular concentration of TAT-HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT-HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem cell culture systems.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, TD-CCBR Rm. 1116, 160 College Street, Toronto, Ontario, Canada M5S 3E1.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Csaszar
authors.forename: Geneviève
authors.initials: G
authors.lastname: Gavigan
authors.forename: Mark
authors.initials: M
authors.lastname: Ungrin
authors.forename: Cynthia
authors.initials: C
authors.lastname: Thérien
authors.forename: Pascale
authors.initials: P
authors.lastname: Dubé
authors.forename: James
authors.initials: J
authors.lastname: Féthière
authors.forename: Guy
authors.initials: G
authors.lastname: Sauvageau
authors.forename: Denis Claude
authors.initials: DC
authors.lastname: Roy
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D001331:Automation / Q000379:methods*; D001709:Biotechnology / Q000379:methods*; D002478:Cells, Cultured; D006207:Half-Life; D006412:Hematopoietic Stem Cells; D006801:Humans; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors