Capture and Release of Viable Circulating Tumor Cells from Blood

Summary

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.

Abstract

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.

Introduction

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 cancer^1-3. However, recent advances in CTC field has indicate....

Protocol

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)

1. Weigh out PIPAAm to prepare a 10% w/v solution in butanol. Mix using a vortex until the solution is clear.
2. Cut plastic microscope slides into roughly 12 mm x 12 mm squares either with a pair of scissors or sharp blade. Alternatively use a guillotine.

Results

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 significantly lower (7%.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Slot Filter	Circulogix Inc.	MSF-01	Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)
poly(N-iso-propylacrylamide) (PIPAAm)	Ploysciences Inc.	21458	Non-Hazardous. Store at room temp.
1-Butanol	Sigma Aldrich	B7906	Use in well ventilated area
Plastic Microscope Slides	Cole-Parmer	48510-30	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
Spin Coater	Specialty Coating Systems	SCS G3 Spin Coater	Instrument
Polyimide Tape	Uline	S-7595	Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)
HBSS- Hank's Balanced Salt Solution	Gibco	14025-092
1x PBS	Gibco	10010-023
Falcon Petri dishes 35 x 10 mm	VWR	25373-041
Microfilter Cassette	Circulogix Inc.	FC-01	Custom catridges are avilable based on filtration for CTC from blood or urine
Syringe 20 ml	BD Scientific	302830
Syringe Pump	KD scientific	78-0100V	Any syringe pump capable of holding a 25 ml syringe may be used
Cellstar 50 ml Centrifuge tube	VWR	82050-322
Greiner Bio One 6 well plate	VWR	89131-688	Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant
SKBR3 Cells	ATCC	HTB-30
Live Dead Assay	Life Technologies	L3224	Any assay that can provide a reasonable analysis to evaluate live cells will work
Cell Culture Incubator	VWR	98000-368	Any incubator that can be used for cell culture will suffice