Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers.

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access.

Circulating tumor cells: clinically relevant molecular access based on a novel CTC flow cell.

BACKGROUND: Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available. METHODOLOGY/PRINCIPAL FINDINGS: Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL. CONCLUSIONS/SIGNIFICANCE: The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.

Integrated microfluidics platforms for investigating injury and regeneration of CNS axons.

We describe the development of experimental platforms to quantify the regeneration of injured central nervous system (CNS) neurons by combining engineering technologies and primary neuronal cultures. Although the regeneration of CNS neurons is an important area of research, there are no currently available methods to screen for drugs. Conventional tissue culture based on Petri dish does not provide controlled microenvironment for the neurons and only provide qualitative information. In this review, we introduced the recent advances to generate in vitro model system that is capable of mimicking the niche of CNS injury and regeneration and also of testing candidate drugs. We reconstructed the microenvironment of the regeneration of CNS neurons after injury to provide as in vivo like model system where the soluble and surface bounded inhibitors for regeneration are presented in physiologically relevant manner using microfluidics and surface patterning methods. The ability to control factors and also to monitor them using live cell imaging allowed us to develop quantitative assays that can be used to compare various drug candidates and also to understand the basic mechanism behind nerve regeneration after injury.

Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection.

We describe the integrated use of pulsed laser microbeam irradiation and microfluidic cell culture methods to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, lambda = 532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 mum and 50 mum in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interferes with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a micropatterned cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.

Microfluidic-based strip assay for testing the effects of various surface-bound inhibitors in spinal cord injury.

This paper describes a novel microfluidic-based assay for spinal cord injury (SCI) research. Conventional methods such as neurite outgrowth and strip assays cannot recapitulate the organized structure of the spinal cord and thus poorly simulate the injury microenvironment. In addition, it is difficult to obtain quantitative results to compare subtle differences on a chemical's effect on normal growth and regeneration. In SCI, the cell bodies are often located away from the immediate lesion, while the damaged and regenerating axons are exposed to the inhibitory milieu of the scar-tissue. We combined micropatterning and microfluidics to selectively place high purity CNS neurons on favorable substrate but allow only axons to interact with permissive (i.e. polylysine) and inhibitory substrates (i.e. aggrecan) presented in alternating strips. On patterned surfaces, axons were confined on permissive lanes and consistently avoided inhibitory strips. Since processes are expected to proceed in a pre-defined direction/geometry, even small deviations, indicative of the drug's effectiveness, can be readily detected. To demonstrate the potential utility of the method in drug screening for SCI, we used chondroitinase-ABC as a model drug to overcome the inhibitory effects of aggrecan. Enzymatic treatment promoted axons to cross onto the nerve-inhibitory strips and extend randomly across the pattern. Such effects can be easily observed and confidently quantitated to obtain objective comparison. This approach is amenable for high throughput screening and may be used to study the effects of pharmaceuticals that suppress inhibitors of neuronal growth/regeneration.

Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.

We have developed a simple microfluidic device for generating stable concentration gradients in 2D and 3D environments. The device, termed the Ladder Chamber, uses a two-compartment diffusion system to generate steady state gradients across flow-free channels that connect the source and sink channels. To demonstrate the utility of the Ladder Chamber for cell migration, neutrophil chemotaxis was successfully observed in soluble chemoattractant (IL-8) gradient. The Ladder Chamber's simple design and experimental implementation make it an attractive approach for investigating cell migration and other biological experiments in well-defined gradients in 2D surfaces as well as in 3D gels.

Fabrication of a microfluidic device for the compartmentalization of neuron soma and axons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80 degrees C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

Non-plasma bonding of PDMS for inexpensive fabrication of microfluidic devices.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding. In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass. This could be due, perhaps, to a plasma cleaner not being available. In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device. Some researchers, however, still prefer to remove the device. In this case, reversible bonding of the device to the cover glass makes that possible. There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed. In some cases axons may grow under the grooves rather than through them. Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device. Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded. The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds. For non-plasma bound devices, liquid flow through the devices takes several minutes. It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

Microfluidic culture platform for neuroscience research.

This protocol describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) and peripheral nervous system neurons for neuroscience applications. This method uses replica-molded transparent polymer parts to create miniature multi-compartment cell culture platforms. The compartments are made of tiny channels with dimensions of tens to hundreds of micrometers that are large enough to culture a few thousand cells in well-controlled microenvironments. The compartments for axon and somata are separated by a physical partition that has a number of embedded micrometer-sized grooves. After 3-4 days in vitro (DIV), cells that are plated into the somal compartment have axons that extend across the barrier through the microgrooves. The culture platform is compatible with microscopy methods such as phase contrast, differential interference microscopy, fluorescence and confocal microscopy. Cells can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration. This protocol can be completed in 1-2 days.