OPTIMIZING THE PURITY OF MICROFILTERED CIRCULATING TUMOR CELLS USING LIVE IMAGING K. Turner 1,2 , A. Sanati Nezhad 1,2 , J. Alejandro Hernández-Castro 1,2 , and D. Juncker 1,2,3 1 McGill University and Genome Quebec Innovation Centre 2 Department of Biomedical Engineering, McGill University 3 Department of Neurology and Neurosurgery, McGill University This work is supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the NSERC-CREATE program in Integrated Sensor Systems. Our experimental setup involves: 1. Collecting mouse blood and staining blood cells red with fluorescent CellTracker™ dye 2. Culturing MDA-MB-231 breast cancer cells and staining green with fluorescent Vybrant™ dye 3. Spiking blood sample with MDA cells 4. Assembling the MFC with selected filter and injecting diluted blood sample with a syringe pump 5. Washing the filter with PBS to reduce contamination by leukocytes We have identified three parameters which affect the specificity of filtration: the sample dilution, the filter pore size, the flow rate of filtration. Fig. 3 illustrates the extremes of either (a) maximizing efficiency or (b) maximizing purity. REFERENCES CONCLUSION ANTI FOULING TECHNIQUES OPTIMIZING PURITY INTRODUCTION MICROFILTRATION SETUP Figure 3: The effect of filtration parameters on the efficiency of cell capture and the purity of isolated cells. (a) “Slow flow” filtration conditions and representative result. 2:1 blood dilution, 0.5 mL/min, and an 8 μm pore. (b) “Fast flow” filtration conditions and representative result. 10:1 blood dilution, 2.0 mL/min, and a 20 μm pore. With the aid of live imaging, we were able to characterize the effect of the PBS wash on filter specificity. Fig. 4 shows the reduction of background contamination with the increasing volume of PBS wash. We accomplish this without the loss of captured cancer cells. Figure 4: The improvement of filter specificity with PBS wash. (a) Mean red emission RFU values were taken from images of the filter at each point in the wash. Particle analysis was conducted on images from the green emission channel to determine the number of captured cancer cells at each point in the wash. (b-d) Representative composite images from (b) before PBS wash, (c) after 8 ml of wash, and (d) after 18 ml of wash. Our custom design for the MFC allows sample to be applied to the bottom of the filter, so that live imaging can occur with an inverted fluorescent microscope (Fig. 2a). The working distances of standard 4x or 10x objectives are compatible with the cartridge. The MFC was laser-cut from acrylic (ALine, Inc). 1 cm diameter silicon filters (Fig. 2b) containing up to 600,000 uniform pores from 6-20 μm in diameter (Fig. 2c) were fabricated by photolithography and deep reactive ion etching. These filters can be sandwiched between the top and bottom layers of the cartridge during assembly (Fig. 2d). O-rings and a silicone gasket are used to prevent leaks. The cartridge is placed on the microscope stage and a syringe pump is used to inject the blood sample (Fig. 2e). Figure 2: The MFC experimental setup. (a) A schematic of the custom cartridge for live imaging. (b) The silicon filter, patterned with (c) uniform pores. (d) The disassembled MFC, showing placement of the silicone gasket, an o-ring, and the silicon filter. (e) Microfiltration setup using a syringe pump to inject a blood sample into the cartridge, which is placed on the stage of an inverted microscope. Waste beaker is shown on the far left. Circulating tumor cells (CTCs) are recognized as a powerful indicator for cancer prognosis [1]. Microfiltration is commonly used to isolate and enrich CTCs from blood [2], but the purity of isolated cells is often very low due to filter fouling by leukocytes [3]. We propose a novel microfiltration cartridge (MFC) for live- imaging of filtration and use it to study the purity of isolated cell populations. Figure 1: Microfiltration cartridge (MFC) concept. A blood sample is collected from the patient and filtered using the MFC. Cancer cells which may express multiple different markers can be enriched using this label-free method. However, since cancer cells are of similar size to the far more prevalent white blood cells, microfiltration has historically been unspecific and CTC identification must be achieved with downstream staining and additional analysis. Figure 5: Back pulses improve the reduction in contamination. (a) Back pulses (indicated by dotted lines) reduce filter contamination without affecting the count of cancer cells on the filter. (b-d) Representative composite images from (b) before wash, (c) after 8 ml of wash and one pulse, and (d) after 18 ml of wash and two pulses. (e) Contamination can be significantly reduced using back pulses, to below the point of “fast flow” conditions, but without sacrificing efficiency to do so. The optimized wash from Figure 4 is also compared. Back pulses are typically used in macrofiltration to reduce filter cake formation, however to our knowledge they have not been implemented in microfiltration. Following real-time visualization, we observed that adding two short pulses of back flow during washing can further improve the purity of captured cells (Fig. 5a). Using back pulses, we were able to reduce the contamination by blood cells to below that of both fast flow conditions and previous washing conditions (Fig.5e). We have used a novel MFC for live-imaging the filtration of cancer cells from whole mouse blood in order to optimize filtration and washing steps. Using real-time monitoring with the MFC, we can: • Increase the purity of trapped cancer cells without compromising recovery • Explore the use of back pulses to further reduce contamination • Improve the specificity of simple, rapid filtration By improving the purity of cells isolated using a label-free method, we can facilitate easier downstream analysis. 1. V. Plaks et al. 2013. Circulating Tumor Cells. Science 341: 1186-88. 2. C. Jin et al. 2014. Technologies for label-free separation of circulating tumor cells. Lab Chip 14: 32-44. 3. D.L. Adams et al. 2014. The systematic study of circulating tumor cell isolation using lithographic microfilters. RSC Adv. 4: 4334-42. Patient blood sample White blood cells Cancer cell 0 5000 10000 15000 20000 Fast flow Optimized wash Back pulses Contamination in Mean Red RFU (averaged) 1 cm LIVE IMAGING OF FILTRATION a b e b d e Cancer cells Blood cells/ debris O-ring Gasket Silicon filter Syringe pump MFC Microscope a c 0 20 40 60 80 100 120 140 0 1000 2000 3000 4000 5000 6000 0 2 4 6 8 10 12 14 16 18 Number of Cancer Cells Contamination in Mean Red RFU mL PBS Wash Background Contamination Cancer Cells a b c d Back pulses 0 10 20 30 40 50 60 70 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 2 4 6 8 10 12 14 16 18 Number of Cancer Cells Contamination in Mean Red RFU mL PBS Wash Background Contamination Cancer Cells a b c d
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
OPTIMIZING THE PURITY OF MICROFILTERED CIRCULATING TUMOR CELLS USING LIVE IMAGING
K. Turner1,2, A. Sanati Nezhad1,2, J. Alejandro Hernández-Castro1,2, and D. Juncker1,2,3
1McGill University and Genome Quebec Innovation Centre 2Department of Biomedical Engineering, McGill University 3Department of Neurology and Neurosurgery, McGill University
This work is supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, and the NSERC-CREATE program in Integrated Sensor Systems.
Our experimental setup involves: 1. Collecting mouse blood and staining blood cells red with fluorescent
CellTracker™ dye
2. Culturing MDA-MB-231 breast cancer cells and staining green with fluorescent Vybrant™ dye
3. Spiking blood sample with MDA cells
4. Assembling the MFC with selected filter and injecting diluted blood sample with a syringe pump
5. Washing the filter with PBS to reduce contamination by leukocytes
We have identified three parameters which affect the specificity of filtration: the sample dilution, the filter pore size, the flow rate of filtration. Fig. 3 illustrates the extremes of either (a) maximizing efficiency or (b) maximizing purity.
REFERENCES
CONCLUSION
ANTI FOULING TECHNIQUES OPTIMIZING PURITY INTRODUCTION
MICROFILTRATION SETUP
Figure 3: The effect of filtration parameters on the efficiency of cell capture and the purity of isolated cells. (a) “Slow flow” filtration conditions and representative result. 2:1 blood dilution, 0.5 mL/min, and an 8 µm pore. (b) “Fast flow” filtration conditions and representative result. 10:1 blood dilution, 2.0 mL/min, and a 20 µm pore.
With the aid of live imaging, we were able to characterize the effect of the PBS wash on filter specificity. Fig. 4 shows the reduction of background contamination with the increasing volume of PBS wash. We accomplish this without the loss of captured cancer cells.
Figure 4: The improvement of filter specificity with PBS wash. (a) Mean red emission RFU values were taken from images of the filter at each point in the wash. Particle analysis was conducted on images from the green emission channel to determine the number of captured cancer cells at each point in the wash. (b-d) Representative composite images from (b) before PBS wash, (c) after 8 ml of wash, and (d) after 18 ml of wash.
Our custom design for the MFC allows sample to be applied to the bottom of the filter, so that live imaging can occur with an inverted fluorescent microscope (Fig. 2a). The working distances of standard 4x or 10x objectives are compatible with the cartridge. The MFC was laser-cut from acrylic (ALine, Inc). 1 cm diameter silicon filters (Fig. 2b) containing up to 600,000 uniform pores from 6-20 µm in diameter (Fig. 2c) were fabricated by photolithography and deep reactive ion etching. These filters can be sandwiched between the top and bottom layers of the cartridge during assembly (Fig. 2d). O-rings and a silicone gasket are used to prevent leaks. The cartridge is placed on the microscope stage and a syringe pump is used to inject the blood sample (Fig. 2e).
Figure 2: The MFC experimental setup. (a) A schematic of the custom cartridge for live imaging. (b) The silicon filter, patterned with (c) uniform pores. (d) The disassembled MFC, showing placement of the silicone gasket, an o-ring, and the silicon filter. (e) Microfiltration setup using a syringe pump to inject a blood sample into the cartridge, which is placed on the stage of an inverted microscope. Waste beaker is shown on the far left.
Circulating tumor cells (CTCs) are recognized as a powerful indicator for cancer prognosis [1]. Microfiltration is commonly used to isolate and enrich CTCs from blood [2], but the purity of isolated cells is often very low due to filter fouling by leukocytes [3]. We propose a novel microfiltration cartridge (MFC) for live-imaging of filtration and use it to study the purity of isolated cell populations.
Figure 1: Microfiltration cartridge (MFC) concept. A blood sample is collected from the patient and filtered using the MFC. Cancer cells which may express multiple different markers can be enriched using this label-free method. However, since cancer cells are of similar size to the far more prevalent white blood cells, microfiltration has historically been unspecific and CTC identification must be achieved with downstream staining and additional analysis.
Figure 5: Back pulses improve the reduction in contamination. (a) Back pulses (indicated by dotted lines) reduce filter contamination without affecting the count of cancer cells on the filter. (b-d) Representative composite images from (b) before wash, (c) after 8 ml of wash and one pulse, and (d) after 18 ml of wash and two pulses. (e) Contamination can be significantly reduced using back pulses, to below the point of “fast flow” conditions, but without sacrificing efficiency to do so. The optimized wash from Figure 4 is also compared.
Back pulses are typically used in macrofiltration to reduce filter cake formation, however to our knowledge they have not been implemented in microfiltration. Following real-time visualization, we observed that adding two short pulses of back flow during washing can further improve the purity of captured cells (Fig. 5a). Using back pulses, we were able to reduce the contamination by blood cells to below that of both fast flow conditions and previous washing conditions (Fig.5e).
We have used a novel MFC for live-imaging the filtration of cancer cells from whole mouse blood in order to optimize filtration and washing steps. Using real-time monitoring with the MFC, we can:
• Increase the purity of trapped cancer cells without compromising recovery
• Explore the use of back pulses to further reduce contamination
• Improve the specificity of simple, rapid filtration
By improving the purity of cells isolated using a label-free method, we can facilitate easier downstream analysis.
1. V. Plaks et al. 2013. Circulating Tumor Cells. Science 341: 1186-88.
2. C. Jin et al. 2014. Technologies for label-free separation of circulating tumor cells. Lab Chip 14: 32-44.
3. D.L. Adams et al. 2014. The systematic study of circulating tumor cell isolation using lithographic microfilters. RSC Adv. 4: 4334-42.
Patient blood sample
White blood cells
Cancer cell
0
5000
10000
15000
20000
Fast flow Optimized wash Back pulses Cont
amin
atio
n in
Mea
n Re
d RF
U
(ave
rage
d)
1 cm
LIVE IMAGING OF FILTRATION
a b
e
b
d e
Cancer cells
Blood cells/ debris
O-ring
Gasket
Silicon filter Syringe pump
MFC
Microscope
a
c
0
20
40
60
80
100
120
140
0
1000
2000
3000
4000
5000
6000
0 2 4 6 8 10 12 14 16 18
Num
ber
of C
ance
r Ce
lls
Cont
amin
atio
n in
Mea
n Re
d RF
U
mL PBS Wash
Background Contamination Cancer Cells
a
b c d
Back pulses
0
10
20
30
40
50
60
70
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 2 4 6 8 10 12 14 16 18
Num
ber
of C
ance
r Ce
lls
Cont
amin
atio
n in
Mea
n Re
d RF
U
mL PBS Wash
Background Contamination
Cancer Cells
a
b c d
Related Documents
