MAGNETIC MICROPALLETS FOR SINGLE ADHERENT CELL RECOVERY AND ANALYSIS

This paper presents a novel microtechnology, magnetic micropallets, for the recovery of single adherent cells from large mixed populations. This work significantly advances the base technology, the micropallet array, which itself is effective for the analysis of adherent cell populations and isolation and selection of individual cells. We greatly im- proved the method of recovery of individual micropallets by incorporating ferromagnetic iron oxide nanoparticles into their structure such that single micropallets can be manipulated and collected magnetically. THEORY Micropallet arrays consist of hundreds of thousands of microscale polymer pedestals on which single adherent cells are held. Micropallets are fabricated from a transparent and biocompatible photopolymer using standard photolitho- graphic techniques. Any micropallet can be released from the glass substrate on demand using a pulsed laser, carrying the adhered cell with it and coming to rest on the array's surface. Collection of released micropallets was previously accomplished using a simple inversion process that caused any released micropallets to fall into an array of collection wells (2). This method has critical drawbacks including the requirement to search the collection wells to locate the re- covered micropallet, the large volume of collection fluid in which the cell is recovered (which is ill-suited for single cell analysis methods), very low throughput capacity, and increased rates of contaminating loosely adherent cells that de- tach from non-targeted micropallets of the array. A micropallet that is responsive to magnetic fields eliminates these drawbacks by enabling its collection and manipulation via magnetic means subsequent to its release. We proposed that a magnetic micropallet could be fabricated using a composite material that is both ferromagnetic and photopatternable, that the final micropallet would itself be magnetically responsive, and that single, viable cells could be collected and precisely delivered using the magnetic micropallet platform. EXPERIMENTAL Magnetic micropallets were fabricated using standard photolithographic techniques from a photopatternable poly- mer that was made in-house by dissolving EPON 1002F resin in solvent and adding a photoinitiator (3). The resultant 1002F photopolymer is similar to SU-8 photoresist, but exhibits much lower autofluorescence (3). The photopolymer was made magnetic by the addition of ferromagnetic iron (II, III) oxide nanoparticles. Microstructures made from this material exhibited ferromagnetism. The iron oxide nanoparticles were mixed into the 1002F photopolymer by mechan- ical stirring and centrifugation was used to remove aggregates of nanoparticles and thereby improve overall homogenei- ty of the composite material. The photopatterning of the ferromagnetic photopolymer was accomplished using a colli- mated light source and SU-8 developer following the same recipe as prescribed for standard 1002F photopolymer (3). NIH/3T3 cells were maintained in culture and seeded onto micropallet arrays that had been coated with human fi- bronectin. Cells were incubated for 3 h at 37 °C/10% CO2 to allow them to adhere to the micropallets. Individual mi- cropallets holding single cells were released using a pulsed laser focused at the micropallet and glass substrate inter- face, as previously described (2). Released micropallets were collected using a collection probe based on a 1 mm diameter permanent magnet. For clonal expansion experiments to demonstrate viability of recovered cells, micropallets holding single cells were transferred into individual wells of a 96-well tissue culture plate containing cell culture media and cultured for a period of 1 week. For RT-qPCR analyses, single 3T3 cells stably transfected with rat neu were col- lected, lysed and analyzed for mRNA expression of mouse s-actin and rat neu using a TaqMan PreAmp Cells-to-Ct Kit.