The overall goal of this procedure is to assemble and use a semi-automated microfluidic based platform to cultivate and stimulate small populations of cells and recover cell lysates for molecular analyses. This is accomplished by first establishing microscale cell cultures in fibronectin coated micro capillaries referred to as cell perfusion chambers. The second step is to connect the perfusion chambers to a digital microfluidics or DMF module, which provides a flexible means by which reagents are routed to and from the microscale cell cultures.
Next, the DMF module delivers the stimulus of interest to the perfusion chambers and the cells are incubated with the stimulus for a predetermined time of exposure. Finally, the cells are lysed by chemical means and the lysates are recovered onto the DMF module for molecular analysis. The recovered lysates are then analyzed using quantitative R-T-P-C-R in order to characterize the cell population's transcriptional response to the stimulus.
The main advantage of this technique over conventional cell culturing methods, it is possible with this technique that you can do cell stimulation studies using small amounts of cells, and also use small amounts of reagents. Our research has demonstrated that platform's precise handling of cells and reagent at microscale minimizes experiment to experiment variability. The platform is very versatile as well.
The digital microflow module can route cells and reagents to and from the perfusion chambers with great flexibility such that experiments can be customized and reconfigured quickly and easily. To begin, prepare a sterile solution of 50 microgram per milliliter fibronectin and use a multi-port syringe pump to draw 30 microliters of the solution into each micro capillary to accelerate coating of the micro capillary inner surfaces. Incubate at 37 degrees Celsius for two hours, then withdraw and discard the fibronectin solution and allow the micro capillaries to dry at room temperature for six hours.
Next, connect the fiber nin coated micro capillaries or perfusion chambers to polycarbonate tubing and the tubing to the syringe pump using capite fittings. The setup shown here uses an aid port syringe pump supporting six perfusion chambers. Using tape, secure the body of each perfusion chamber to a heat block and set the heat blocks temperature to 37 degrees Celsius.
Then immerse the open ends of the perfusion chambers in a reservoir containing cells, suspended in fresh growth, medium evenly dispersed at a concentration of 1 million cells per milliliter. Instruct the syringe pump to pull 10 microliters of the cell suspension into each perfusion chamber. Then remove the open ends of the perfusion chambers from the cell suspension and instruct the syringe pump to pull enough air to move the liquid plugs into the sections secured to the heat block.
Allow the cells to adhere to the fibronectin coated interior surfaces of the perfusion chambers by incubating them at 37 degrees Celsius for one to two hours. In the meantime, transfer the open ends of the perfusion chambers to a new reservoir containing fresh growth medium. After the adhesion period, instruct the syringe pump to send the liquid plugs to waste and then pull 10 microliters of fresh medium into each profusion chamber, followed by enough air to position the fresh medium over the adhered cells.
Continue to incubate the cells at 37 degrees Celsius, exchanging the medium every two hours until the cultures are sufficiently equilibrated and expanded.Pattern. The bottom plate of the DMF module with 46 indium tin oxide electrodes. Using standard photolitographic techniques coat the bottom plate of the DMF module with five microns of Lene C via chemical vapor deposition, and then spin coat with 50 nanometers of Teflon af.
Next spin coat and un patterned indium tin oxide glass substrate with 50 nanometers of Teflon AF to be used as the top plate. Use metal compression frames to fix the plates in a polymer cast with recesses that maintain a 400 micron spacing between the plates. This spacing combined with the actuation electrode size of 2.5 millimeters squared defines the droplet volume as 2.5 microliters per electrode.
Then insert Teflon coated transfer micro capillaries into the space between the DMF plates. Positioning each so that it extends to the edge of its cognate actuation electrode to the opposite end of each transfer. Micro capillary affix a capite fitting.
Next, engage the electrical connectors to supply voltage to the Indium 10 oxide electrodes. Electrode activation sequences are automated through use of a computer controlled electronic interface running predetermined scripts. Then fill the DMF modules onboard reservoirs with the appropriate reagents for cell stimulation, washing and lysis.
In some cases, it's best to load the stimulus itself at a later step just before its use drawing from the loaded onboard reservoirs, dispense micro droplets onto the DMF module. Test, actuation of the micro droplets, including their transport between the DMF module and the transfer micro capillaries.Blais. First, prepare the stimulus in fresh medium with 0.1%onic F1 27 at a final concentration of 100 micrograms per milliliter.
Then load 80 to 100 microliters of the stimulus into its designated onboard reservoir. Next, remove the open ends of the perfusion chambers from the medium reservoir and insert them into the capite fittings affixed to the ends of the transfer Micro capillaries then instruct the syringe pump to send the liquid plugs within the perfusion chambers to the waste container. Next, instruct the DMF module to generate 10 microliter droplets of stimulus and to deliver them to the perfusion chambers via the transfer micro capillaries.
Incubate the cells with the stimulus at 37 degrees Celsius for one to four hours if required. Exchange for fresh stimulus at regular intervals following the stimulation period. Instruct the syringe pump to send the stimulus to the waste container and instruct the DMF module to deliver a 10 microliter droplet of wash buffer to each perfusion chamber.
Incubate the cells with the wash buffer for five minutes. Then exchange the wash buffer for 10 microliters of lysis solution and incubate for an additional five minutes. Next, instruct the syringe pump to send each cell lysate to the DMF module.
Remove the top plate of the DMF module taking care not to tilt the plate sideways as this can result in cross-contamination between droplets. Finally, collect the lysates from the open DMF module using a pipette man for off-platform analysis, such as gene expression, profiling via quantitative R-T-P-C-R to prepare the platform for the next experiment. First, immerse the transfer micro capillaries, CAPITE fittings, and DMF module plates in 10%bleach for 10 minutes at room temperature, the DMF module plates are rinsed with isopropanol followed by deionized water and dried using nitrogen gas, and then baking at 160 degrees Celsius for 10 minutes.
The seeding methods described in this protocol resulted in adherence of approximately 500 miren macrophages per perfusion chamber. After incubating the cells with growth medium at 37 degrees Celsius for 16 hours, the population size doubled to approximately 1000 cells per perfusion chamber. Shown here are results from quantitative R-T-P-C-R analysis of four genes known to play important roles in innate immune responses to bacteria.
The macrophages were grown in either conventional or microscale cultures and stimulated with e coli bio particles for either one or four hours. The blue bars indicate the average fold increase in gene expression upon stimulation of conventionally cultured cells, whereas the red bars indicate induced expression in cells grown and stimulated. In microscale cultures, the error bars indicate standard deviations between biological replicates from three independent experiments.
The gene expression profiling results were very similar, even though the conventional cultures used 96 well plates with approximately 50, 000 cells per well, whereas the microscale cultures used micro capillaries with approximately 1000 cells per perfusion chamber experiment to experiment variability was at least comparable and often reduced in microscale cultures as indicated by the shorter error bars working at the microscale. Reduced consumption of cells by fivefold and reduced consumption of reagents by three to 50 fold. Relative to the conventional experiments.
The method could be used to culture, virtually any adherence cell type, in some cases using a different extracellular matrix component or growth. Medium non-adherence cell types can be challenging, but cell tethering strategies have shown to be successful in similar contexts. The approach described here supports microscale culture for up to 24 hour.
The platform includes all the function to do a long term culture as well, but a cell passaging protocol needs to be developed. Additional modules may be integrated into digital microfluidic cub through the micro capillary interconnects. In this way, the DMF serves as a central hub for connecting the peripheral modules to carry out complex workflows.
We primarily focused on characterizing host transcriptional responses to infectious agents, but the platform is designed to be extremely versatile and so it should support a wide variety of research applications.
