A cell microdispenser for accurate positioning of single cell

A CELL MICRODISPENSER FOR ACCURATE POSITIONING OF SINGLE CELL Vincent Haguet1, Florence Rivera2, Urban Seger2, Nathalie Picollet-D’hahan1, Philippe Renaud2 and François Chatelain1 1 2 CEA Grenoble, Biochip Laboratory, FRANCE EPFL, Microsystems Laboratory, STI-LMIS, Lausanne, SWITZERLAND ABSTRACT A new microdevice and its related equipment were specifically designed for the jetbased deposition of single cells. The microdispenser is a cell-specific printhead that integrates microfluidic inlets and channels for the injection and transport of a cell suspension, on-chip microelectrodes for the flow-through impedance-based detection of individual cells, and a miniaturized solenoid valve. Hydraulic pressure pulses driven by the microvalve push the cells towards an orifice to achieve ejections of nanoliter-sized droplets. The goal is to spot a well-controlled number of cells and to use them as microarray for high-throughput cell analysis. Keywords: Cell printing, microdispenser, high-throughput screening, microdroplet 1. INTRODUCTION The question of accurately positioning cells onto substrates is currently carried out by means of microwells, chemical surface patterning, dielectrophoresis or aspiration through microholes, although these techniques require specifically manufactured substrates to operate. Jet-based printing technology applied to cell dispensing could represent a more flexible approach for producing various cell patterns on a wide range of samples and applications. The cell microdispenser presents several advantages compared to previous work performed with commercial non-contact spotters [1] and ink-jet printers [2,3]. Commercial equipments suffer from a lack of spot-to-spot reproducibility in the local amount of cells, absence of single-cell resolution, frequent clogging of the fluidic supplying system by cells and a volume disproportion of the spotted droplet relative to the cell dimension. To overcome these limitations, microelectrodes were assembled in our dispensing microdevice in order to individually detect and count cells before the ejection with real-time electronic processing. Moreover, permanent movement of cells inside the microdispenser prevents cellular adhesion on the inner walls of the printhead. 2. PRINCIPLE OF MICRODISPENSING Figure 1 shows the microdispenser and the target substrate. The dispenser is composed of a microfluidic part for transporting the cell suspension and ejecting droplets containing cells, and an electronic part for signal processing. Cells enter into a microfluidic channel where they are flowing, and are individually detected in the close vicinity of the nozzle via differential impedance spectroscopy [4]. A miniature solenoid valve (The Lee Company, Westbrook, USA) is assembled onto the microfluidic chip in front of the orifice. When a cell is detected, the microvalve is opened in order to create a hydraulic pressure pulse that allows pushing the cell towards the orifice and forming a microdroplet containing the detected cell. The sub-elements of the microfluidic chip are shown in Figure 2: the cell flowing in the microchannel (a) encounters the microelectrodes (b) about 50 µm before the nozzle channel, is then deviated by a pressure pulse due to the microvalve opening (c) and is finally ejected through the orifice (d). Electronic connector Injection of cell suspension Target substrate Microfluidic chip Figure 1. Photograph of the microdispenser, composed of a microfluidic chip inserted in a plastic holder, a printed circuit board for electronic connection, a supply of cell suspension and a solenoid valve (backside of the microdispenser). (c) (b) (a) (d) Figure 2. Close-up of the microfluidic chip with the main elements: (a) microchannel, (b) microelectrodes, (c) location of the solenoid microvalve, (d) orifice. 3. EXPERIMENTAL RESULTS AND DISCUSSION The cells are suspended in Phosphate Buffered Saline (PBS). Pressure-driven flow of the cell suspension in the microchannel is performed by a pressure regulator in the cell injection inlet and by a vacuum pumping in the opposite outlet. The electrovalve is also supplied with PBS to avoid dilution during ejection. In theory, different liquids could be considered for supplying the microchannel and the electrovalve in order to achieve simultaneously cell deposition and mixing with chemical reagents. The electrovalve flow is driven by a pressure regulator. When the valve is at rest, flowing-out of the liquid through the orifice may appear, but this can be regulated by modulating the vacuum pump. Addition of nanoliter-sized droplets on every sites of the target substrate results in a cellcontaining droplet microarray (Figure 3). The relation of the droplet volume made through a 20 x 50 µm2 orifice to the pressure pulse characteristics is shown in Figure 4. A delivery of <4-nl droplets is achieved by triggering the ejection with short valve opening durations and hydraulic pressure of ~1 bar. Valve opening duration: 260 µs 255 µs 250 µs 16 14 droplet volume (nL) 12 10 8 6 4 2 0 1 mm Figure 3. Sideview of the microdispenser and of the target substrate. Series of 190 nL spots are made by dispensing 50 droplets of 3.8 nL. 1,00 1,05 1,10 1,15 1,20 Pressure (bar) Figure 4. Volume of the spotted droplet (in nL) vs. pressure applied on the microvalve (in bar), for three valve opening durations. The resulting automation of jet-based cell dispensing may be significant for manufacturing complex substrates with a high spatial resolution, reproducibility and wide pattern architecture. Relevant fields of application include basic cell biology research, cell culture microarrays, cell-based biosensors, cell-based diagnostics and tissue engineering. The production of cell culture droplets containing a precise number of cells is particularly interesting to facilitate toxicological studies, a field for which controlling the amount of patterned cells is fundamental [5]. REFERENCES [1] P. Cooley, D. Wallace, B. Antohe, "Applications of ink-jet printing technology to bioMEMS and microfluidic systems", Proceed. SPIE Conference on Microfluidics and BioMEMS, San Francisco, USA, 21-24 October 2001. [2] W.C. Wilson, T. Boland, "Cell and organ printing 1: protein and cell printers", Anat. Rec. Part A, 272A(2), 491-496 (2003). [3] H. Ben Hsieh, J. Fitch, D. White, F. Torbes, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, S. Elrod, "Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors", J. Biomol. Screening, 9(2), 85-94 (2004). [4] S. Gawad, L. Schild, Ph. Renaud, "Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing", Lab Chip, 1(1), 76-82 (2001). [5] http://toxdrop.vitamib.com Vincent Haguet, CEA Grenoble, Life Science Division, Biochip Lab., 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Phone: +33 (0)4 38 78 23 86; Fax: +33 (0)4 38 78 59 17; [email protected]