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Keywords:

  • cell purification;
  • cell sorting;
  • image cytometry;
  • laser;
  • cloning

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 108 W/cm2 at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 109 W/cm2 at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 1010 W/cm2 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 (104-106/cm2). 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 101 to 108 cells. Throughput ranged from approximately 103 to 105 total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively. © 2004 Wiley-Liss, Inc.