title: Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.
abstract: Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low-density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality.
authors.affiliation: Cell Culture and Fermentation Sciences, Biopharmaceutical Development, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Krista
authors.initials: K
authors.lastname: Evans
authors.affiliation: Cell Culture and Fermentation Sciences, Biopharmaceutical Development, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Thomas
authors.initials: T
authors.lastname: Albanetti
authors.affiliation: Cell Culture and Fermentation Sciences, Biopharmaceutical Development, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Raghavan
authors.initials: R
authors.lastname: Venkat
authors.affiliation: Cell Culture and Fermentation Sciences, Biopharmaceutical Development, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Ronald
authors.initials: R
authors.lastname: Schoner
authors.affiliation: Data Management and Quantitative Sciences, MedImmune, Gaithersburg, MD , 20878.
authors.forename: James
authors.initials: J
authors.lastname: Savery
authors.affiliation: Data Management and Quantitative Sciences, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Miro-Quesada
authors.affiliation: Respiratory, Inflammation and Autoimmunity, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Bhargavi
authors.initials: B
authors.lastname: Rajan
authors.affiliation: Respiratory, Inflammation and Autoimmunity, MedImmune, Gaithersburg, MD , 20878.
authors.forename: Christopher
authors.initials: C
authors.lastname: Groves
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016466:CHO Cells; D002460:Cell Line; D002469:Cell Separation; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins
keywords: cell imaging; cell line development; flow cytometry; mammalian cell culture; single-cell cloning