How to Sort Circulating Tumor Cells Part IV: Electrokinetic Separation

x-posted at Erica Pratt’s Blog

Keywords

Anion:a negatively charged ion.asophil, Lymphocyte, Neutrophil: Different types of white blood cells.
Biofouling: Fouling of surface with biological material.
Cation: A positively charged ion.
Conductivity: Measure of a substance’s ability to conduct electricity.
Cytoplasm: Inner contents of the cell, which holds everything outside the nucleus.
CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Erythrocyte: Red blood cell.
PBMCs: Peripheral mononuclear blood cells, aka blood cells that have a nucleus (e.g. white blood cells). Cartoon of the different kinds of white blood cells here.
MDA-MB-***: Human breast carcinoma immortalized cell lines.
Phenotype: Observable characteristics of a cell.

This is the last major sorting technique in this series (for now), and I will be using the review paper I co-wrote with Charlie Huang as the framework for my description of this technique1. Charlie works extensively on electrokinetic manipulation of cells, and his half of the review paper lends itself well to explaining how electrokinetics can be used to sort CTCs.

Why use electrokinetic separation?

Most CTC sorting devices target some observed cancer cell phenotype that was determined from studying tumor tissue directly, or from using immortalized cancer cell lines. This means that active sorting techniques, like size-based selection and immunocapture, require some level of a priori knowledge about CTCs before you can engineer a device to capture them. Microscopic characterization is one CTC identification method that circumvents this problem, fixing (killing) the cells, and then using imaging in combination with rapid scanning to look at almost everything present in the blood sample. Electrokinetic separation of cancer cells is another, but enables live cell isolation without knowing its physical or biochemical properties beforehand.

What types of electrokinetic techniques are used?

There are two commonly used types of electrokinetic manipulation for mammalian cells, electrophoresis (EP) and dielectrophoresis (DEP). Electrophoresis involves applying a uniform electric field across a charged particle, causing it to polarize (i.e. free charge aligns with the electric field), inducing a net particle migration. However, if a uniform electric field is applied to an electrically neutral particle, the charges will polarize to form a dipole, but there is no actuation because the force on each anion is cancelled out by the force on its respective cation, and vice versa. To induce actuation, a non-uniform electric field must be applied (Dielectrophoresis), causing the charge on one side of the particle to feel the electrical force more strongly than the other, resulting in particle migration—as shown in the example below (thanks to the Kirby Lab DEP subgroup for the great schematic!).

Electrophoresis is good for moving charged particles around; however, the net charge from cell type-to-cell type is often not distinct enough to sort cells with high resolution. In contrast, dielectrophoresis is excellent for sorting cells because motion is dependent not on net charge, but on cell membrane and cytoplasm electrical properties as well as cell size, as dictated by this equation:

Where a is the particle radius, E0 is the applied electric field magnitude, and Re[fCM] is the real part of the Clasius-Mossotti factor, which is a measure of effective polarizability of the particle, and is dependent on the frequency of the applied electric field.

DEP can be operated in two modes depending on the sign of Re[fCM]. Positive DEP (pDEP) is when Re[fCM] is positive in sign, causing particles migrate in the direction of high electric field gradients. Negative DEP (nDEP) is when cells are repulsed from high electric field gradients because Re[fCM] is negative. Gascoyne et al. measured Re[fCM] in cancer cells and PBMCs and the data to the left shows there is a frequency range where the cancer cells experience pDEP while PBMCs are simultaneously undergoing nDEP.

Most devices then use interdigitated array (IDA) electrodes for binary sorting2,3,4, or continuous field-flow fractionation (DEP-FFF)5,6,7, to separate cancer cells from PBMCs. An example of DEP-FFF of CTCs from ApoStream technology is shown on the left, where tumor cells experience pDEP and are pulled towards the electrodes, trapping them there. In contrast, PBMCs experience nDEP, and are repelled from the electrodes (DEP levitation), allowing them to be washed away with the fluid flow. The tumor cells are then released when the electric field is turned off. More recently, researchers have used “contactless” DEP to sort cancer cells while minimizing biofouling8,9.

What are the drawbacks for sorting using electrokinetic properties?

Very few DEP sorters have been used to isolate actual CTCs from patient samples. But all have performed device characterization using cancer cell lines, which can be spiked at known concentrations in blood, to measure efficiency and purity of isolation. The data show that device performance decreases dramatically as the cancer cell:PBMC ratio approaches levels found in vivo (1-10 CTCs per billion blood cells)1. This means blood samples must be preprocessed to enrich CTCs for most DEP devices to have optimal performance.

Additionally, most DEP-based cancer cell sorters must extract the cancer cells and PBMCs and from blood (usually via centrifugation), and then resuspend them in some other buffer solution before processing1. This is because whole blood is a very high conductivity medium—much higher than that of the cell cytoplasm—causing cells to experience nDEP at all frequencies10, which makes pDEP separation techniques, like the one I showed above, impossible. Devices that do process blood, must dilute it several times with another buffer in order to isolate cancer cells, drastically increasing sample processing time.

When should you sort CTCs using electrokinetics?

Electrokinetic based sorting is ideal if:

  • You want a sorting technique unbiased by cell morphology or protein expression.
  • There is a pre-enrichment step so there are more CTCs for every PBMC.
  • You want to characterize electrical properties of the cell membrane and cytoplasm.
  • Lower throughput and blood pre-processing is not a concern.
  • You want a sorting method with a simple and easy whole cell release step.

References

1. Pratt E.D., Huang C., Hawkins B.G., Gleghorn J.P. & Kirby B.J. (2011). Rare cell capture in microfluidic devices, Chemical Engineering Science, 66 (7) 1508-1522. DOI:

2. Becker et al. Separation of human breast cancer cells from blood by differential dielectric affinity. Proceedings of the National Academy of Sciences of the United States of America 1995; 92:860-864

3. Kim et al. Selection of mammalian cells based on their cell-cycle phase using dielectrophoresis. Proceedings of the National Academy of Sciences of the United States of America 2007; 104:20708-20712

4. Cristofanilli et al. Dielectric cell separation of fine needle aspirates from tumor xenografts. Journal of Separation Science 2008; 31(21):3732-9

5. Gascoyne et al. Isolation of rare cells from cell mixtures by dielectrophoresis. Electrophoresis 2009; 30(8):1388-98

6. Shim et al. Dynamic physical properties of dissociated tumor cells revealed by dielectrophoretic field-flow fractionation. Integrative Biology 2011; 3(8):850-62

7. Yang et al. Dielectrophoretic Separation of Prostate Cancer Cells. Technology in Cancer Research and Treatment 2012.

8. Shaifee et al. Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP). Lab on a Chip 2010; 10:438–445

9. Sano et al. Modeling and development of a low frequency contactless dielectrophoresis (cDEP) platform to sort cancer cells from dilute whole blood samples. Biosensors and Bioelectronics 2011; 30(1):13-20

10. Electromanipulation of Cells by Ulrich Zimmermann, Ph.D. (Editor), Garry A Neil, M.D. (Editor), 1996.

All Kirby Lab publications are available on our webpage.