Published: October 28th, 2016
A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients' blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.
We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 °C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 °C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.
Metastatic disease is responsible for most cancer deaths. Developing prognostic and companion diagnostic biomarker for metastasis is crucial in cancer management and treatment. Circulating tumor cells (CTC) play a central role in tumor dissemination and metastasis. Furthermore, being easily accessible as a 'liquid biopsy' biomarker, CTC in cancer patients' peripheral blood has been rising as a 'hotbed' for cancer biomarker research. CTC have been well validated as a prognostic biomarker in various cancer settings, including breast, prostate and colorectal cancer1-3. However, recent advances in CTC field has indicate....
Ethics Statement: To protect the rights of human subjects, blood samples were obtained following an informed consent under protocols approved by the University of Miami institutional review boards under IRB 20150020.
NOTE: Blood to be filtered for CTC capture should be collected in an EDTA tube to prevent coagulation.
1. Coating the Microfilter with Poly (N-iso-propylacrylamide) (PIPAAm)
Using healthy donors' blood (obtained under a protocol approved by the University of Miami IRB 20150020 following an informed consent) spiked with cultured cancer cells, the thermoresponsive technique for release of viable circulating tumor cells (CTC),achieved capture, release and retrieval efficiency of 94% ± 9%, 82% ± 5% and 77% ± 5% respectively (Table 1)15. By comparison, the release and retrieval efficiency of uncoated filters were sign.......
The process of capturing viable CTC from whole blood and releasing them from the microfilter is relatively straightforward; however a few critical points are worth mentioning. It is imperative, as with all cell culture that a sterile condition is maintained through the whole process. The initial step of coating the filter with PIPAAm is critical, as the basis for the technique of releasing the cells from the filter is based on exploiting PIPAAm's temperature responsive interfacial properties. To ensure the filter has.......
We thank all the patients who have donated blood samples to support this work. We thank Drs. Guiseppe Giaconne, Ritesh Parajuli, and Marc E. Lippman for their assistance in clinical sample acquirement, and Drs. Carmen Gomez, Ralf Landgraf, Stephan Züchner, Toumy Guettouche, Diana Lopez for their insightful discussions. Zheng Ao thanks partial support and assistance from the Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center.....
|Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)
|Non-Hazardous. Store at room temp.
|Use in well ventilated area
|Plastic Microscope Slides
|Any plastic slides or alternatively any sort of square (Metal, Acrylic etc.) can be used if it will be bale to hold the 8mmx8mm filter square
|Specialty Coating Systems
|SCS G3 Spin Coater
|Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)
|HBSS- Hank's Balanced Salt Solution
|Warm in 37 ⁰C water bath before use. McCoys was used for SKBr3 cells, if you use different cell lines or patient blood, please use media that would be optimal for that particular case
|Falcon Petri dishes 35x10 mm
|Custom catridges are avilable based on filtration for CTC from blood or urine
|Any syringe pump capable of holding a 25mL syringe may be used
|Cellstar 50mL Centrifuge tube
|Greiner Bio One 6 well plate
|Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant
|Live Dead Assay
|Any assay that can provide a reasonable analysis to evaluate live cells will work
|Cell Culture Incubator
|Any incubator that can be used for cell culture will suffice
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