Inexpensive technologies enabling widespread utilization of image-predicated cell sorting

The most mature, widespread sorting technology, fluorescence-activated cell sorting (FACS), offers high throughput and sorts predicated on a wide range of phenotypes that can be conveyed through average cellular fluorescence or light scattering levels. However, FACS cannot detect a wide range of phenotypes easily observable through microscopy, such as details of cell morphology, sub-cellular localization, and temporal fluorescence expression with single-cell resolution. Presently, there is no widespread, user-friendly technique to sort cells following microscopy. Commercial solutions offering this capability are prohibitively expensive to individual labs, and are most likely to be found only in well-resourced core facilities.

This thesis presents the design, implementation, and testing of three new technologies for simple, inexpensive, viable, image-predicated cell sorting that can be disseminated to individual labs. For anchorage-dependent cells, we have implemented two technologies. The first is a method termed polymerization-activated cell sorting, or PACS, that permits the selective hydrogel-mediated photo-encapsulation of undesired cells in a culture dish and the retrieval of desired cells using enzyme-mediated release. The second is a method termed radical-activated cell sorting, or RACS, that permits the light-mediated killing of undesired cells in a culture dish through photo-patterning of radical toxicity. For non-anchorage-dependent cells, we have developed optofluidic cell sorting, or optoflucs. In this approach, cells are injected into a microfluidic device where they are allowed to settle into an array of cell-sized microwells. A laser is then used to optically levitate desired cells out of the array and into a flow stream for collection.

This thesis demonstrates > 100-fold enrichment of target cell populations and up to 89% output purity following a single round of image-predicated, viable cell sorting. In addition, this thesis demonstrates examples of sorts predicated on fluorescence localization, a phenotype that cannot be sorted using FACS. These results, coupled with the minimal expense and simplicity of these technologies make these techniques appealing for widespread adoption by individual biology labs. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)