title: High-throughput laser-mediated in situ cell purification with high purity and yield.
abstract: BACKGROUND
  Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield.
  
  
  METHODS
  An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility.
  
  
  RESULTS
  Photothermal purification required approximately 10(8) W/cm(2) at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10(9) W/cm(2) at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 10(10) W/cm(2) independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5-100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1-99%) and cell densities (10(4)-10(6)/cm(2)). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated.
  
  
  CONCLUSIONS
  In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 10(1) to 10(8) cells. Throughput ranged from approximately 10(3) to 10(5) total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively.
authors.affiliation: Cyntellect, Inc., 6199 Conerstone Court, Suite 111, San Diego, CA 92121, USA. fkoller@cyntellect.com
authors.forename: Manfred R
authors.initials: MR
authors.lastname: Koller
authors.forename: Elie G
authors.initials: EG
authors.lastname: Hanania
authors.forename: Janine
authors.initials: J
authors.lastname: Stevens
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Eisfeld
authors.forename: Glenn C
authors.initials: GC
authors.lastname: Sasaki
authors.forename: Annabeth
authors.initials: A
authors.lastname: Fieck
authors.forename: Bernhard Ø
authors.initials: BØ
authors.lastname: Palsson
mesh_terms: D001331:Automation; D001402:B-Lymphocytes / Q000166:cytology; D002460:Cell Line; D003001:Cloning, Molecular; D003201:Computers; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods*; D007834:Lasers; D008027:Light; D012666:Semiconductors; D013601:T-Lymphocytes / Q000166:cytology; D013997:Time Factors; D014162:Transfection
chemical_list: D049452:Green Fluorescent Proteins