Robotic Fluid Dispensing to Microfluidic Chips
Microscale systems that enable measurements of oncological phenomena at the single cell level have a great capacity to improve therapeutic strategies and diagnostics. Such measurements can reveal unprecedented insights into cellular heterogeneity and its implications into the progression and treatment of complicated cellular disease processes such as those found in cancer.
Typically assays involve multiple fluid manipulation steps including delivery of cells, growth media, drugs, wash solutions, and agents for readout of protein expression via immunocytochemistry (i.e., fixatives, membrane permeabilizers, stains, etc.). Though many microfluidic platforms have been developed that are suitable for studying populations of cells, significant improvements in automation and interfacing to the macroscale world are needed in order for these microfluidic platforms to find routine use by personnel in biology or clinical pathology laboratories. Automation is expected to increase repeatability, eliminate human error, and enable increased throughput, especially for sophisticated, multistep assays such as multiparameter quantitative immunocytochemistry.
We developed a simple approach based on conventional laboratory robotics to bridge this gap (see Figure 1). The low-cost microfluidic chip contains an array of chambers, each with an inlet port at one end and an outlet at the other. A pair of needles connected to the robotic pipetting system interfaces with each microfluidic chamber to exchange its liquid contents. One needle dispenses fresh reagent (aspirated from a standard microwell plate) into the chamber, while the second needle simultaneously aspirates the previous contents out of the chamber. Adherent cells are loaded after conditioning the chambers and remain affixed to the channel walls during subsequent operations. After completion of all assay steps, the chips can be imaged using standard fluorescence-based immunocytochemistry and microscopy tools, and and quantitatiely analyzed to determine, for example, drug response based on differences in protein expression and/or activation of cellular targets on an individual cell level.
Figure 1: (a) Photograph of automated platform showing custom dispensing head on 3-axis motion actuator and two plate nests for microfluidic chips and reagents. (b) Custom chip holder in plate nest #1 with three microfluidic chips installed. (c) Standard microwell plate containing reagents in plate nest #2.
To ensure repeatable delivery to microfluidic chips, the original PDMS chips in which proof of concept was demonstrated had to be modified due to variable dimensional change that occurred during curing of the PDMS. The redesigned chips consisted of a glass substrate, a channel layer in which slots were cut, a rigid plastic layer containing inlet and outlet ports, and a film acting as an evaporation barrier (see Figure 2). The rigid layer also contained features to align the chip to the robotic platform to ensure repeatable positioning of the fluidic ports.
Viability of cells delivered robotically to the new chips was compared to that of cells manually delivered to the PDMS chip. Results were comparable. Carryover during fluid exchange was also characterized in the platform and found to be comparable. Finally, a target inhibition assay was conducted on the two platforms. Histograms of the expression level of pS6 in each cell were shifted similarly in the two platforms upon treatment with different concentrations of rapamycin.
Figure 2: Schematic of individual microfluidic assay chip
Current Status and Next Steps
We are currently developing improved microfluidic chips and a simplified fluid delivery platform.
- Sun, J.; Masterman-Smith, M. D.; Graham, N. A.; Jiao, J.; Mottahedeh, J.; Laks, D. R.; Ohashi, M.; DeJesus, J.; Kamei, K.; Lee, K. B.; Wang, H.; Yu, Z. T.; Lu, Y. T.; Hou, S.; Li, K.; Liu, M.; Zhang, N.; Wang, S.; Angenieux, B.; Panosyan, E.; Samuels, E. R.; Park, J.; Williams, D.; Konkankit, V.; Nathanson, D.; van Dam, R. M.; Phelps, M. E.; Wu, H.; Liau, L. M.; Mischel, P. S.; Lazareff, J. A.; Kornblum, H. I.; Yong, W. H.; Graeber, T. G.; Tseng, H. R., A microfluidic platform for systems pathology: multiparameter single-cell signaling measurements of clinical brain tumor specimens. Cancer Res 2010, 70 (15), 6128-38.
- J. Ly, M. Masterman-Smith, R. Ramakrishnan, J. Sun, B. Kokubun, and R. M. van Dam, “Automated Reagent-Dispensing System for Microfluidic Cell Biology Assays,” J. Lab. Autom., vol. 18, no. 6, pp. 530–541, Dec. 2013.
- Jimmy Ly (graduate student)
- Seunghyun "Noel" Ha (graduate student)
- Michael Masterman-Smith (collaborator from Kornblum lab ; later collaborator from Harmony Biosciences, Inc.)
- Ravichandran Ramakrishnan (collaborator from Graeber lab)
- Brent Kokubun (undergraduate student)
- Jing Sun (collaborator from HR Tseng lab)