Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging

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...

Full description

Saved in:
Bibliographic Details
Published in:Biotechnology progress 2015-09, Vol.31 (5), p.1172-1178
Main Authors: Evans, Krista, Albanetti, Thomas, Venkat, Raghavan, Schoner, Ronald, Savery, James, Miro-Quesada, Guillermo, Rajan, Bhargavi, Groves, Christopher
Format: Article
Language:English
Subjects:
Animals
Antibodies, Monoclonal - biosynthesis
cell imaging
Cell Line
cell line development
Cell Separation
CHO Cells
Clone Cells
Cricetinae
Cricetulus
Flow Cytometry
Image Processing, Computer-Assisted - methods
mammalian cell culture
Models, Molecular
Recombinant Proteins - biosynthesis
single-cell cloning
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 31:1172–1178, 2015
ISSN:8756-7938
1520-6033
DOI:10.1002/btpr.2145