Photoablative dilution with pre-enrichment for the clonal isolation of rare cancer cells

摘要

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to cell biology. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce clones by photoablative dilution. The LEAP instrument performs automated fluorescence imaging and real-time image analysis to classify cells. The instrument also features a pulsed laser that gives it the ability to purify a sample by eliminating unwanted cells via laser ablation or UV-induced apoptosis. In photoablative dilution, rare cells are deposited into a multiwell plate at 10 cells per well. Then one cell is chosen to clone, and the other cells present in the well are eliminated by laser ablation. We have successfully used LEAP to produce single cell clones in 95% of wells (originally containing 5±2.1 cells/well). While photoablative dilution is a very effective way of producing clonal cultures, it has a fundamental limitation in the low number of cells that can be processed. This can be overcome by performing a pre-enrichment to increase the frequency of the rare cells to be cloned. Another enrichment strategy is flow cytometry based cell sorting. Flow sorting can provide greater than 10~4 fold enrichment and cells can be sorted directly into a multiwell plate. With pre-enrichment, photoablative dilution can be used to clonally isolate rare cells. This is especially important in cases where the total number of potentially rare cells recovered by first stage enrichment sorting is only 10-200 cells. Such a situation which would normally preclude second pass sorting for purity by the high-throughput first stage cell separation technology.