Cancer is a leading cause of death globally and early detection is one of the most effective means to combat the disease. Recent clinical studies show that the number of cancer cells in cancer patients' blood can predict the disease development and treatment efficacy. Studying these cells may also lead to a better understanding of the disease. Moreover, getting access to blood samples is relatively easy and less invasive and painful than tumor biopsies.
Isolation and enumeration of circulating tumor cells (CTCs) in peripheral blood has clinical significance in combating cancer (J. M. Reuben, S. Krishnamurthy, W. Woodward, M. Cristofanilli, Expert Opin. Med. Diagnostics 2, 339 (2008); S. Urtishak, R. K. Alpaugh, L. M. Weiner, R. F. Swaby, Biomarkers Med. 2, 137 (2008)). Deaths resulting from cancer are mainly due to late diagnosis of the disease and when metastasis has occurred (G. P. Gupta, J. Massague, Cell 127, 679-695 (2006); P. S. Steeg. Nat. Med. 12, 895 (2006)). To ensure patients receive timely treatment, enumerating CTCs in blood can complement existing early detection methods. Furthermore, blood samples being a routinely extracted body fluid in any health test can be easily attained to check for CTCs. CTCs are found in patients with metastatic carcinomas (W. J. Allard, J. Matera, M. C. Miller, M. Repollet, M. C. Connelly, C. Rao, A. G. Tibbe, J. W. Uhr, L. W. Terstappen, Clin. Cancer Res. 10, 6897 (2004); S. Steen, J. Nemunaitis, T. Fisher, J. Kuhn. Proc 21, 127 (2008), Bayl Univ Med. Cent.) and are associated with the disease progression (M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. Terstappen, D. F. Hayes, N. Engl. J. Med. 351, 781 (2004); S. Mocellin, D. Hoon, A. Ambrosi, D. Nitti, C. R. Rossi, Clin. Cancer Res. 12, 4605 (2006); G. Wiedswang, B. Naume, Nat. Clin. Pract. Oncol. 4, 154 (2007)). The effectiveness of therapeutic treatments can also be measured by the number of CTCs in blood (F. Nole, E. Munzone, L. Zorzino, I. Minchella, M. Salvatici, E. Botteri, M. Medici, E. Verri, L. Adamoli, N. Rotmensz, A. Goldhirsch, M. T. Sandri, Ann. Oncol. 19, 891 (2008); A. Rolle, R. Gunzel, U. Pachmann, B. Willen, K. Hoffken, K. Pachmann, World J. Surg. Oncol. 3, 18 (2005)). Thus, there is much interest in isolating, quantifying and studying these cells obtained from peripheral blood.
CTCs are of very low concentration in blood which poses the technical difficulty to detect these rare cells (J. E. Losanoff, W. Zhu, W. Qin, F. Mannello, E. R. Sauter, Breast 17, 540 (2008); V. Zieglschmid, C. Hollmann, O. Bocher, Crit. Rev. Clin. Lab. Sci. 42, 155 (2005)). The absolute number of CTCs in blood of cancer patients varies and depends on the conditions of the patients. Leading techniques to enumerate CTCs include immuno-magnetic separation followed by immunocytochemistry detection, such as the CellSearch® system sold by Veridex LLC (a Johnson & Johnson company) of Raritan, N.J., U.S.A. (M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. Terstappen, D. F. Hayes, N. Engl. J. Med. 351, 781 (2004); H. Yagata, S, Nakamura, M. Toi, H. Bando, S. Ohno, A. Kataoka, Int J Clin Oncol 13, 252 (2008)). A further leading technique is RT-PCR to indicate the presence of CTCs in peripheral blood (L. A. Mattano Jr., T. J. Moss, S. G. Emerson, Cancer Res. 52, 4701 (1992); C. P. Schroder, M. H. Ruiters, S. de Jong, A. T. Tiebosch, J. Wesseling, R. Veenstra, J. de Vries, H. J. Hoekstra, L. F. de Leij, E. G. de Vries, Int. J. Cancer 106, 611 (2003)). These methods have been successfully demonstrated on various cancer types (S. Dawood, K. Broglio, V. Valero, J. Reuben, B. Handy, R. Islam, S. Jackson, G. N. Hortobagyi, H. Fritsche, M. Cristofanilli, Cancer 113, 2422 (2008); R. Szatanek, G. Drabik, J. Baran, P. Kolodziejczyk, J. Kulig, J. Stachura, M. Zembala, Oncol. Rep. 19, 1055 (2008); C. S. Wong, M. T. Cheung, B. B. Ma, E. Pun Hui, A. C. Chan, C. K. Chan, K. C. Lee, W. Cheuk, M. Y. Lam, M. C. Wong, C. M. Chan, J. K. Chan, and A. T. Chan, Int. J. Surg. Pathol. 16, (2008)). Alternative methodologies such as a direct visualization assay (H. J. Kahn, A. Presta, L. Y. Yang, J. Blondal, M. Trudeau, L. Lickley, C. Holloway, D. R. McCready, D. Maclean, A. Marks, Breast Cancer Res. Treat. 86, 237 (2004)), fluorescent activated cell sorter (FACS) (J. G. Moreno, S. M. O'Hara, S. Gross, G. Doyle, H. Fritsche, L. G. Gomella, L. W. Terstappen, Urology 58, 386 (2001)), fibre-optic array scanning technology (FAST) cytometer (R. T. Krivacic, A. Ladanyi, D. N. Curry, H. B. Hsieh, P. Kuhn, D. E. Bergsrud, J. F. Kepros, T. Barbera, M. Y. Ho, L. B. Chen, R. A. Lerner, R. H. Bruce, Proc. Natl. Acad. Sci. USA 101, 10501 (2004)) and anti-EpCAM coated microfabricated structures (S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber, M. Toner, Nature 450, 1235 (2007)) have also been used to enumerate CTCs in blood samples.
Complex procedures, tedious inspections and long processing time are the limiting factors associated with most existing techniques. Furthermore, viability of the isolated cells are lost as fixing of the samples is required by most existing techniques. There is much to understand about the condition of CTCs whilst in circulation (K. Pantel. R. H. Brakenhoff, B. Brandt, Nat. Rev. Cancer 8, 329 (2008)) and having viable cells after isolation would allow studies to be carried out on CTC sub-populations. This may provide valuable insights to the biological characteristics of the disease such as the link between cancer stem cells and metastasis. Although a recent study isolated CTCs (S. Nagrath, L. V. Sequist, S. Maheswaran, D. W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E. L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U. J. Balis, R. G. Tompkins, D. A. Haber, M. Toner, Nature 450, 1235 (2007)), the isolated cells are likely difficult to retrieve due to the binding of the tumor associated antigens to the device. Retrieving these cells may require high mechanical forces or biochemical agents and the integrity of these cells might be affected as a result (S. F. Chang, C. A. Chang, D. Y. Lee, P. L. Lee, Y. M. Yeh, C. R. Yeh, C. K. Cheng, S. Chien, J. J. Chiu, Proc. Natl. Acad. Sci. USA 105, 3927 (2008)). In addition, most methodologies will require functional modifications which is less desirable (0. Lara, X. Tong, M. Zborowski, J. J. Chalmers, Exp. Hematol. 32, 891 (2004)).
In accordance with an embodiment of the invention, there is provided a microfluidic device for isolating cells from a biological fluid. The device comprises an inlet receiving the biological fluid flowed into the device; and at least one array of a plurality of isolation wells receiving the biological fluid from the inlet, at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a cell within the cell trap, the cell trap comprising at least one gap of a size and shape suitable to prevent passage of the cells to be isolated but to permit passage of other components of the biological fluid through the cell trap.
In further, related embodiments, the biological fluid may comprise blood, the cells to be isolated may comprise circulating tumor cells, and the other components may comprise blood cells. The device may further comprise a cell collection point receiving isolated circulating tumor cells from the array of isolation wells; and a waste outlet receiving waste blood cells from the isolation wells. The cell trap may comprise a crescent-shaped structure. The cell trap may comprise at least one of a “U” shaped structure, a “V” shaped structure and/or a “C” shaped structure. The at least one array of the plurality of isolation wells may comprise a plurality of rows of isolation wells, which may be offset from each other, for example by about 25 μm. The at least one array of the plurality of isolation wells may comprise at least one row of isolation wells, the isolation wells of the at least one row being spaced apart from each other by a distance sufficient to prevent clogging of the at least one array by blood cells, for example by about 50 μm. A pressure differential between the inlet and a waste outlet of the device may be within the range of physiological pressure differences found in circulating whole blood.
In further related embodiments, the device may further comprise a pre-filter receiving the biological fluid from the inlet and flowing pre-filtered biological fluid to the at least one array, the pre-filter being linked to a waste outlet of the device. The pre-filter may comprise filter gaps of about 20 μm. The device may comprise at least two sections of arrays of a plurality of isolation wells, the at least two sections being separated by a flow passage to a cell collection point receiving isolated cells from at least one of the sections of arrays. The device may comprise at least one section of arrays of a plurality of isolation wells. A pressure differential between the inlet and a waste outlet of the device may produce flow of the biological fluid through the at least one array of isolation wells to isolate the cells to be isolated. A reversed pressure differential between the inlet and a waste outlet of the device may produce a reversed flow of fluid in the device that permits retrieval of isolated cells. The cell trap may comprise an open flow side permitting a reversed flow of fluid to free an isolated cell from the trap. The cell trap may comprise a left or right tilted orientation, the at least one array of the plurality of isolation wells comprising a plurality of rows of isolation wells, and the plurality of rows of isolation wells comprising alternating left and right tilted orientations of the cell traps in successive rows of the plurality of rows of isolation wells. The cell trap may comprise at least three microstructures separated by at least two gaps between the at least three microstructures, the at least three microstructures forming a shape that includes a wider open side of the shape that is opposite a side of the shape in which the at least two gaps are situated.
In other related embodiments, the device may isolate the cells to be isolated from the biological fluid based solely on biorheological property differences between the cells to be isolated and the other components of the biological fluid. The device may permit retrieval of viable isolated circulating tumor cells from a blood sample. The cells to be isolated may comprise at least one of breast cancer cells, colorectal cancer cells, kidney cancer cells, lung cancer cells, gastric cancer cells, prostate cancer cells, ovarian cancer cells, squamous cell cancer cells, hepatocellular cancer cells and nasopharyngeal cancer cells. A pressure differential between the inlet and a waste outlet of the device may be within the range from about 5 kPa to about 15 kPa. Each of the at least one gaps may be about 4 μm to about 5 μm in width. The cell trap may be of a size and shape suitable to mechanically isolate a cell of between about 5 μm and about 40 μm in diameter. The cell trap may be crescent shaped and between about 15 lam and about 40 μm in its longer dimension, the longer dimension being a distance measured from the outermost edge of a tip of one horn of the crescent to the outermost edge of a tip of the other horn of the crescent, the outermost edges being the edges that are on an outer side of the crescent, which has a larger radius, as opposed to an inner bowl of the crescent, which has a smaller radius. The cell trap may comprise at least two gaps of about 4 μm to about 5 μm width. The cells to be isolated may comprise diseased cells. The cells to be isolated may comprise at least one of fetal cells, malaria infected cells, sickle anemia cells, dengue cells and stem cells. The cells to be isolated may comprise a diameter of between about 6 μm and about 25 μm. The device may be mounted on a microscope slide. The device on the microscope slide may be mounted on an inverted microscope. The device on the microscope slide may be mounted on an upright microscope. The device may permit an isolation efficiency of at least about 80% for the cells to be isolated. The cells to be isolated may comprise circulating tumor cells. The device may permit cell integrity of isolated cells to be preserved after isolation; and may permit retrieval of cancer cells at a prevalence on the order of about 1 cancer cell in about 1 ml of blood. No functional biochemical modification of the device or the cells to be isolated may be necessary to maintain integrity of isolated cells. The device may isolate cells based solely on physical properties of the cells to be isolated, the physical properties comprising at least one of shear modulus, stiffness, size and deformability. The cell trap may be of a size and shape suitable to mechanically isolate most often a single cell within the cell trap. The device may permit real time visualization of isolation of the cell. The device may further comprise an imaging system to capture images from the device to permit real time visualization of isolation of the cell. The device may permit real time enumeration of isolated cells. The device may further comprise an imaging system to capture images from the device to permit real time enumeration of isolated cells. The device may permit enumeration of isolated cells.
In another embodiment according to the invention, there is provided a method for isolating cells from a biological sample (e.g., a biological fluid). The method comprising flowing the biological fluid into an inlet of a microfluidic device; flowing the biological fluid from the inlet through at least one array of a plurality of isolation wells to isolate the cells to be isolated from the biological fluid, the cells being isolated by at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a cell within the cell trap; and permitting components of the biological fluid, other than the cells to be isolated, to pass through the at least one array, the components being permitted to pass by at least one gap in the cell trap of a size and shape suitable to prevent passage of the cells to be isolated but to permit other components of the biological fluid to pass through the cell trap. The method may further comprise obtaining a biological sample from an individual.
In further, related embodiments, the biological fluid includes blood, lymph, spinal fluid, amniotic fluid, urine and the like. In a particular embodiment, the biological fluid may comprise blood, the cells to be isolated may comprise circulating tumor cells and the other components may comprise blood cells. The method may further comprise collecting isolated circulating tumor cells from the array of isolation wells at a cell collection point of the device; and passing waste blood cells from the isolation wells to a waste outlet of the device. The cell trap may comprise a crescent-shaped structure. The cell trap may comprise at least one of a “U” shaped structure, a “V” shaped structure and/or a “C” shaped structure. The method may comprise passing the biological fluid through a plurality of rows of isolation wells. The isolation wells of the plurality of rows of isolation wells may be offset from each other, for example by about 25 μm. The method may comprise passing the biological fluid through at least one row of isolation wells, the isolation wells of the at least one row being spaced apart from each other by a distance sufficient to prevent clogging of the at least one array by whole blood cells. The isolation wells of the at least one row may be spaced apart by about 50 μm.
In further, related embodiments, the method may comprise operating the inlet and a waste outlet of the device at a pressure differential that is within the range of physiological pressure differences found in circulating whole blood. The method may further comprise pre-filtering the biological fluid received from the inlet, flowing the pre-filtered biological fluid to the at least one array, and flowing waste from the pre-filtering to a waste outlet of the device. The pre-filtering may comprise flowing the biological fluid through filter gaps of about 20 μm. The method may comprise passing the biological fluid through at least two sections of arrays of a plurality of isolation wells, the at least two sections being separated by a flow passage to a cell collection point receiving isolated cells from at least one of the sections of arrays. The method may comprise passing the biological fluid through at least one section of arrays of a plurality of isolation wells. The method may comprise operating the inlet and a waste outlet of the device at a pressure differential that produces flow of the biological fluid through the at least one array of isolation wells to isolate the cells to be isolated; and may comprise reversing a pressure differential between the inlet and a waste outlet of the device to produce a reversed flow of fluid in the device that permits retrieval of isolated cells. The method may comprise reversing flow of fluid through the device to free an isolated cell from an open flow side of the cell trap.
In further, related embodiments, the cell trap may comprise a left or right tilted orientation, the at least one array of the plurality of isolation wells comprising a plurality of rows of isolation wells, and the plurality of rows of isolation wells comprising alternating left and right tilted orientations of the cell traps in successive rows of the plurality of rows of isolation wells. The cell trap may comprise at least three microstructures separated by at least two gaps between the at least three microstructures, the at least three microstructures forming a shape including a wider open side of the shape that is opposite a side of the shape in which the at least two gaps are situated. The method may comprise isolating the cells to be isolated from the biological fluid based solely on biorheological property differences between the cells to be isolated and the other components of the biological fluid. The method may comprise retrieving viable isolated circulating tumor cells from a blood sample. The method may comprise isolating viable circulating tumor cells comprising at least one of breast cancer cells, colorectal cancer cells, kidney cancer cells, lung cancer cells, gastric cancer cells, prostate cancer cells, ovarian cancer cells, squamous cell cancer cells, hepatocellular cancer cells and nasopharyngeal cancer cells.
In further, related embodiments, the method may comprise operating a pressure differential between the inlet and a waste outlet of the device within the range from about 5 kPa to about 15 kPa. Each of the at least one gaps may be about 4 μm to about 5 μm in width. The cell trap may be crescent shaped and between about 15 μm and about 40 μm in its longer dimension, the longer dimension being a distance measured from the outermost edge of a tip of one horn of the crescent to the outermost edge of a tip of the other horn of the crescent, the outermost edges being the edges that are on an outer side of the crescent, which has a larger radius, as opposed to an inner bowl of the crescent, which has a smaller radius. The cell trap may comprise at least two gaps of about 4 μm to about 5 μm width. The method may further comprise using the isolated cells to perform at least one of diagnosing and monitoring of a disease or condition of an individual in need of the at least one of diagnosis and monitoring. The isolated cells may comprise diseased cells. The isolated cells may comprise at least one of fetal cells, malaria infected cells, sickle anemia cells, dengue cells and stem cells. The cells to be isolated may comprise a diameter of between about 6 μm to about 25 μm. The method may further comprise performing an enumeration of the isolated cells. The method may comprise determining a cell type of the isolated cells. Determining the cell type may comprise staining the isolated cells within the microfluidic device. The stained isolated cells may be viewed under a microscope. The microfluidic device may be mounted on a microscope slide. The stained cells may comprise circulating tumor cells. The microfluidic device may be mounted on a microscope slide on an inverted microscope. The microfluidic device may be mounted on a microscope slide on an upright microscope. The method may comprise isolating the cells to be isolated with an isolation efficiency of at least about 80%. The cells to be isolated may comprise circulating tumor cells. The method may comprise preserving cell integrity of the cells to be isolated after isolation. The method may comprise retrieving cancer cells that have a prevalence on the order of about 1 cancer cell in about 1 ml of blood. The method may comprise performing no functional biochemical modification of the device or cells to be isolated while maintaining integrity of isolated cells. The method may comprise isolating cells based solely on physical properties of the cells to be isolated, the physical properties comprising at least one of shear modulus, stiffness, size and deformability. The cell trap may be of a size and shape suitable to mechanically isolate most often a single cell within the cell trap. The method may further comprise permitting real time visualization of isolating the cells. The method may further comprise capturing images from the device with an imaging system to permit real time visualization of isolating the cells. The method may comprise permitting real time enumeration of isolated cells. The method may further comprise capturing images from the device with an imaging system to permit real time enumeration of isolated cells.
In another embodiment according to the invention, there is provided a method of diagnosing cancer in an individual in need of diagnosis thereof. The method comprises flowing blood from a sample of the blood of the patient into an inlet of a microfluidic device; and flowing the blood from the inlet through at least one array of a plurality of isolation wells to isolate circulating tumor cells from the blood, wherein if circulating tumor cells are present the circulating tumor cells are isolated by at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a circulating tumor cell within the cell trap. Blood cells are permitted to pass through the at least one array, the blood cells being permitted to pass by at least one gap in the cell trap of a size and shape suitable to permit blood cells to pass through the cell trap but to prevent passage of the circulating tumor cells to be isolated. The individual may be diagnosed with cancer based at least in part on the isolated circulating tumor cells.
In a further embodiment according to the invention, there is provided a method of monitoring progression of cancer in an individual in need of monitoring thereof. The method comprises flowing blood from a sample of the blood of the individual into an inlet of a microfluidic device; and flowing the blood from the inlet through at least one array of a plurality of isolation wells to isolate circulating tumor cells from the blood, the circulating tumor cells being isolated by at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a circulating tumor cell within the cell trap. Blood cells are permitted to pass through the at least one array, the blood cells being permitted to pass by at least one gap in the cell trap of a size and shape suitable to permit blood cells to pass through the cell trap but to prevent passage of the circulating tumor cells to be isolated. Progression of the cancer in the individual may be monitored based at least in part on the isolated circulating tumor cells. The individual may be undergoing treatment for the cancer.
In a further embodiment, there is provided a method of monitoring treatment of cancer in an individual in need of monitoring thereof. The method comprises flowing blood from a sample of the blood of the individual into an inlet of a microfluidic device; and flowing the blood from the inlet through at least one array of a plurality of isolation wells to isolate circulating tumor cells from the blood, the circulating tumor cells being isolated by at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a circulating tumor cell within the cell trap. Blood cells are permitted to pass through the at least one array, the blood cells being permitted to pass by at least one gap in the cell trap of a size and shape suitable to permit blood cells to pass through the cell trap but to prevent passage of the circulating tumor cells to be isolated. Treatment of the cancer in the individual may be monitored based at least in part on the isolated circulating tumor cells.
In a further, related embodiment, the method may further comprise determining the efficacy of the treatment based on the number of circulating tumor cells isolated by the microfluidic device.
In another embodiment according to the invention, there is provided a method of providing a prognosis of cancer in an individual in need of prognosis thereof. The method comprises flowing blood from a sample of the blood of the individual into an inlet of a microfluidic device; and flowing the blood from the inlet through at least one array of a plurality of isolation wells to isolate circulating tumor cells from the blood, the circulating tumor cells being isolated by at least one isolation well of the plurality of isolation wells comprising a cell trap of a size and shape suitable to mechanically isolate a circulating tumor cell within the cell trap. Blood cells are permitted to pass through the at least one array, the blood cells being permitted to pass by at least one gap in the cell trap of a size and shape suitable to permit blood cells to pass through the cell trap but to prevent passage of the circulating tumor cells to be isolated. Prognosis of the cancer in the individual may be provided based at least in part on the isolated circulating tumor cells.
In further, related embodiments, the method may further comprise retrieving the isolated circulating tumor cells from the micro fluidic device. The method may also further comprise identifying the isolated circulating tumor cells when they are situated within the microfluidic device.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
A description of example embodiments of the invention follows.
Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between disease development and the number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients.
In accordance with an embodiment of the present invention, there is provided a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is on average at least 80% for tests performed on breast cancer, colon cancer and other types of cells. Viable isolated cells are also obtained which may give further insights to enhance the understanding of the metastatic process. Contrasting with conventional biochemical techniques, a microdevice according to an embodiment of the invention provides a mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting.
In accordance with an embodiment of the present invention, there is provided a device that presents a means to enumerate and isolate viable CTCs from peripheral blood with a high throughput and high efficiency. Isolation and quantification of CTCs presents an alternate disease marker that aid in monitoring cancer progression and determine overall survival. In accordance with an embodiment of the invention, retrieving viable CTCs assists in the further study of key biological determinants to the disease which might provide insights to the lethality of the disease.
Compared with most existing methodologies, the ability to separate CTCs from blood in fewer steps in accordance with an embodiment of the invention reduces the complexity of obtaining vital disease information. Most existing methodologies require multiple enrichment and identification procedures which is tedious and time-consuming. A device in accordance with an embodiment of the invention is able to take whole blood samples for processing directly.
Furthermore, viability of the cells is compromised in most existing approaches. Even in existing techniques using anti-EpCAM coated microstructures that are able to isolate viable CTCs, retrieval is a challenge because high shear flows are required to break the affinity of the biochemical bonds.
Microfluidic devices provide an alternative technique compared to conventional biochemical separations. Devices utilizing dielectrophoretic forces to separate and manipulate cells are advantageous as they do not require functionalization of the sample or the microdevice (P. Y. Chiou, A. T. Ohta, M. C. Wu, Nature 436, 370-372 (2005); D. S. Gray, J. L. Tan, J. Voldman, C. S. Chen, Biosens. Bioelectron. 19, 771-780 (2004); A. Rosenthal, J. Voldman, Biophys. J. 88, 2193-2205 (2005); J. Voldman, Curr Opin Biotechnol 17, (2006)). However, efficient cancer cell separation may be difficult due to the low concentration of CTCs in blood and the relative similar dimensions of leukocytes with cancer cells.
In accordance with an embodiment of the present invention, through a biomechanical means of isolation that utilizes CTCs' distinct biorheological difference with blood constituents, the retrieval of CTCs is made relatively easy to achieve by controlling the input/output conditions. In addition, no functional biochemical modification of the device or CTCs is necessary to maintain the integrity of CTCs. The microsystem has a high throughput and is able to process the blood samples within minutes. Further, an embodiment according to the invention permits real time visualization of the isolation process. Further, a system may provide a simple means of enumerating cells (such as CTCs) using a uniform array. In addition, a device may permit real time enumeration of isolated cells.
A device in accordance with an embodiment of the invention has potential application to aid clinicians in cancer disease monitoring and retrieval of viable CTCs for further analysis. The ease of use and portability of the device presents an attractive replacement to current methodologies. A high throughput ensures fast recovery time of diagnosis results which can aid in timely drug administering to better the chances of survival.
A device in accordance with an embodiment of the invention is fabricated in accordance with the biorheology of cells and depends on the deformability and dimensions of the cells. Studies of various different types of cells' rheology may be used to determine the dimensions of the device. In such studies, fresh cell samples may be used to ensure consistent rheological properties of the cells.
In accordance with an embodiment of the invention, there is provided a microfluidic device to isolate viable cancer cells of breast and colon origins from whole blood using solely the biorheological property differences of cancer cells and blood constituents. No functional modifications of the microdevice are required as isolation is solely dependent on the biorheological property differences of cancer cells and blood constituents. Past studies have revealed that the shear modulus, stiffness, size and/or deformability of diseased cells (L. Weiss, Adv Cancer Res 54, (1990); L. Weiss, D. S. Dimitrov, J. Theor. Biol. 121, 307 (1986)) is distinctively different from blood constituents (H. Mohamed, L. D. McCurdy, D. H. Szarowski, S. Duva, J. N. Turner, M. Caggana, IEEE Trans. Nanobioscience 3, 251 (2004); J. P. Shelby, J. White, K. Ganesan, P. K. Rathod, and D. T. Chiu, Proc. Natl. Acad. Sci. U.S.A. 100, 2003). The adopted approach draws upon this dissimilarity to achieve high purity in isolating cancer cells in blood. A feasibility study has also been successfully conducted to separate samples based on biorheological differences (S. J. Tan., L. Yobas, G. Y. H. Lee, C. N. Ong, C. T. Lim, International Conference on Biocomputation, Bioinformatics, and Biomedical Technologies, 2008. BIOTECHNO '08 (2008)). The isolation process is achieved in a single step, preserving the integrity and viability of these cells. Retrieval of the isolated cells is also straightforward by controlling and manipulating the flow conditions in a device according to an embodiment of the invention. The microdevice may be used to capture and isolate circulating tumor cells from peripheral blood of cancer patients for diagnostic and prognostic purposes. The microdevice is attractive for applications in oncology research, particularly prognostication and prediction of drug response.
An embodiment according to the invention overcomes the technical challenges posed by the low cancer cell count in blood combined with large sample volumes. By utilizing the biomechanical property differences of cancer cells from blood, an embodiment according to the invention achieves an effective isolation of cancer cells.
In accordance with an embodiment of the invention, the sample inlet 402 (see
In an experiment in accordance with an embodiment of the invention, the arrays of crescent-shaped structures were created using soft lithography (J. C. McDonald, G. M. Whitesides, Acc. Chem. Res 35, 491 (2002)). The photo mask was drawn in Cadence (Cadence Design Systems, Inc., San Jose, Calif., USA) and produced on glass with critical dimensions of 2 μm (Infinite Graphics Inc., Minneapolis, Minn., USA). Fabrication of the masters was done by spin coating (3200 rpm, 45 seconds) SU-8 2025 (MicroChem Corp., Newton, Mass., USA) on an 8-inch silicon wafer. Depth of the microdevice was 18 μm and it was cast from polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning Corp., Midland, Mich., USA). Following manufacturer's instructions, pre-cured PDMS was degassed in a desiccator and cured in an oven preset at 80° C. for 2 hours. After peeling the PDMS off the master, fluidic ports were punched using a flat tip needle. Irreversible bonding of the PDMS microdevice to a glass slide was achieved using oxygen plasma treatment.
It will be appreciated that other techniques of fabrication may be used in accordance with an embodiment of the invention.
In accordance with an embodiment of the invention, a 3D computational model of the microdevice was developed to better understand the flow characteristics as well as to help in the design of the microdevice. A simplified model of the microdevice consisting of fourteen isolation structures was created using Gambit (Ansys Inc., Lebanon, N.H., USA) and simulated using Fluent (Ansys Inc., Lebanon, N.H., USA). An optimized mesh number of 579,820 was used. Mesh independence was ascertained by increasing the mesh number and observing the difference in velocities to be less than 1%. Adopting a no-slip wall boundary condition and fluid properties of pure H2O (density at 998.2 kg/m3 and viscosity 0.001003 kg/ms), the study was carried out by analyzing the velocity profiles and shear stresses at the irregular isolation structures under different initial conditions.
With the computational results, simulated particles could be traced in the flow to check upon the isolation effectiveness of the microdevice. 25 simulated particles were placed uniformly at the inlet and the flow pathlines traced to check whether the particles intercept the crescent traps. Where the pathlines intercepted with the crescent trap, it was presumed that there was a possibility for cells in the sample solution to be impeded. This helped to determine if the design was able to fulfill its task effectively. When simulating in the reverse flow direction for cell retrieval, the flow pathlines of the simulated particles aided to determine whether the isolation structures obstruct the recovery. This helps to ensure minimal damage to the isolated cells. The shear stresses and flow patterns in the microdevice at various operating conditions were extracted from the computational analysis and compared to physiological conditions to aid in optimizing the design and determining the operating conditions for the microdevice.
In accordance with an embodiment of the invention, three different cell lines MCF-7, MDA-MB-231 (human breast adenocarcinoma) and HT-29 (human colon adenocarcinoma) of human origin were tested in the microdevice. Culturing of cells was done in a 25 cm2 tissue culture flask and maintained with Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, Mo., USA) supplement with 10% fetal bovine serum (FBS) (Hyclone, Logan, Utah, USA) and 1% penicillin G/streptomycin/amphotericin B (Gibco, Carlsbad, Calif., USA). For each experiment, cells were grown to confluence, trypsinized and resuspended in culture media. A portion of the suspended cells were taken as control in the experiment and cultured normally. The rest were diluted to a concentration of approximately 100 cells per milliliter and the sample solution was injected into the device for characterizing its isolation efficiency. In the cell proliferation analysis, isolated cells were retrieved from the microdevice and reseeded into the culture flask to observe their proliferation status over a period of 5 days under normal culturing conditions.
For cell size measurements of the cancer cell lines, the diameter was an average reading obtained from images of 100 suspended cells and determined using an image processing software (NIS-Elements AR, Nikon Corp., Singapore). Cancer cell counting was done with a hemocytometer and serial diluted to achieve the desired concentration of 100 cells per milliliter of 1× phosphate buffered saline (PBS).
In an experiment in accordance with an embodiment of the invention, all apparatus including the microdevices, tubes and fluidic connectors, were thoroughly sterilized with 70% ethanol prior to use. Subsequently, sterile 1×PBS was injected to wash out any traces of ethanol. The microdevice was then mounted in a custom made fixture with the fluidic connections attached (see the embodiment of
To ascertain the isolation purity, each of the cancer cell types were added into whole blood donated from healthy donors at concentration of approximately 100 cells per milliliter. The sample solution was further diluted with sterile 1×PBS in a 1:2 ratio to reduce the sample viscosity so that it could be processed easily. Isolated cells in the microdevice were stained with fluorescence (see below) to distinguish between cancer cells and WBCs. For cell proliferation experiments, isolated cells were retrieved by reversing the pressure differential and directing the isolated cells to the collection point. The collected solution was then centrifuged at 1200 rpm for 5 minutes with the cell pellet resuspended later in culturing media DMEM and reseeded in a T25 culture flask. Their proliferative rates were compared to normal cultures which acted as controls in the experiment. A further part of the analysis involved testing the microdevice at lower concentrations of cancer cells in sterile 1×PBS (1-2 cells per milliliter). Cells were picked out manually using a pipette with a 200 μl pipette tip from a cell suspension of approximately 100 cells per milliliter under the microscope. The cells were then directly added to 1×PBS and injected into the microdevice.
In an experiment in accordance with an embodiment of the invention, immunofluorescence staining allowed visual examination to characterize cancer cell isolation purity in the microdevice. For the control experiment, the premixed sample of blood and cancer cells (200 μl) was incubated onto a 12 mm coverslip (polylysine coated) for 30 minutes. The sample was subsequently fixed with 4% paraformaldehyde (PFA) for 30 min and washed with 1×PBS for 15 minutes. It was then permeabilized by 0.1% Triton X-100 in 1×PBS for 10 minutes, followed by a PBS wash for 15 minutes and adding 10% goat serum for 30 minutes to block out non-specific bindings. The sample was then stained for Epithelial Cell Adhesion Molecule (EpCAM) (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA) for 1 hour followed by the secondary antibody (1:500, goat anti-mouse AlexaFluor 568, Invitrogen Corp., Carlsbad, Calif., USA) for another hour to identify cancer cells. Fluorescein isothiocyanate (FITC) conjugated CD45 (1:50, Santa Cruz Biotechnology Inc., Santa Cruz, Calif., USA) was then used (1 hour) to identify WBCs and 4′,6-diamidino-2-phenylindole (DAPI) for nuclei visualization. The coverslip was then washed with 1×PBS for 10 minutes and mounted.
For staining in the microdevice, a pressure differential of 5 kPa was used to induce flow into the microdevice. The value of 5 kPa was chosen as it best preserves the state of the isolated cells in the microdevice as compared to using higher pressure differentials. Lower pressure conditions will increase the processing time. Captured cells were first fixed by flowing 4% PFA for 30 minutes, permeabilized by 0.1% Triton X-100 in 1×PBS for 10 minutes, followed by washing with 1×PBS for 15 minutes and adding 10% goat serum to block out non-specific bindings for 30 minutes. To identify cancer cells, 0.2 ml of EpCAM antibodies were injected for 15 minutes, left to stand for another 45 minutes and followed by PBS washing. The procedures of antibody injection and PBS wash were repeated for the secondary antibody (1:500, goat anti-mouse AlexaFluor 568, Invitrogen Corp., Carlsbad, Calif., USA). For the identification of WBCs, 0.2 ml of FITC conjugated CD45 antibodies were injected for 15 minutes, left to stand for another 45 minutes and followed by washing with PBS. Staining was completed by flowing DAPI for 15 minutes at 5 kPa followed by washing with PBS.
The prognostic values of enumerating CTCs in peripheral blood have been widely reported (P. D. Beitsch, E. Clifford, Am. J. Surg. 180, 446 (2000); G. T. Budd, M. Cristofanilli, M. J. Ellis, A. Stopeck, E. Borden, M. C. Miller, J. Matera, M. Repollet, G. V. Doyle, L. W. Terstappen, D. F. Hayes, Clin. Cancer Res. 12, 6403 (2006); M. Cristofanilli, K. R. Broglio, V. Guarneri, S. Jackson, H. A. Fritsche, R. Islam, S. Dawood, J. M. Reuben, S. W. Kau, J. M. Lara, S. Krishnamurthy, N. T. Ueno, G. N. Hortobagyi, V. Valero, Clin. Breast Cancer 7, 471 (2007); H. Gogas, G. Kefala, D. Bafaloukos, K. Frangia, A. Polyzos, D. Pectasides, D. Tsoutsos, P. Pangiotou, J. Ioannovich, D. Loukopoulos, Br. J. Cancer 87, 181 (2002); D. F. Hayes, M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, M. C. Miller, J. Matera, W. J. Allard, G. V. Doyle, L. W. Terstappen, Clin. Cancer Res. 12, 4218 (2006); D. S. Hoon, Nat. Clin. Pract. Oncol. 1, 74 (2004)). This method is also minimally invasive as compared to traditional biopsies. The number of CTCs in blood is directly associated with the disease progression and can help in evaluating cancer treatment efficacy (F. Nole, E. Munzone, L. Zorzino, I. Minchella, M. Salvatici, E. Botteri, M. Medici, E. Verri, L. Adamoli, N. Rotmensz, A. Goldhirsch, M. T. Sandri, Ann. Oncol. 19, 891 (2008)). Thus, it is important to have a high cancer cell isolation efficiency for the microdevice to count these rare cells in blood samples accurately. An approach in accordance with an embodiment of the invention draws mainly upon the highly deformable nature of erythrocytes (J. P. Shelby, J. White, K. Ganesan, P. K. Rathod, and D. T. Chiu, Proc. Natl. Acad. Sci. U.S.A. 100, 2003) and leukocytes (B. Yap, and R. D. Kamm, J. Appl. Physiol. 98, (2005)) that enable these cells to traverse capillaries as small as 2-5 μm whose cell diameters can range from 8 to 25 μm. On the other hand, cancer cells are more likely to be arrested in capillaries of similar dimensions (L. Weiss, D. S. Dimitrov, Cell Biophys. 6, 9 (1984)). Average sizes of MCF-7, MDA-MB-231 and HT-29 cells used in the experiments in accordance with an embodiment of the invention were tabulated to be 16.2±1.80 μm, 17.9±2.94 μm and 15.5±1.25 μm respectively. It should be noted that these size variances were for cell lines; actual clinical samples typically have higher variation in size. For example, renal cancer cells may have a size of from about 5 μm or about 6 μm to about 15 μm.
In an experiment in accordance with an embodiment of the invention, in order to understand the flow profile around the irregular shaped structures and ascertain minimal damage on isolated cancer cells due to hydrodynamic forces, the fluid velocity and shear stress profiles were simulated for the operating pressure differentials applied.
In an experiment in accordance with an embodiment of the invention, for characterizing cell isolation efficiency, low concentration of cancer cells (100 cells per milliliter) spiked in 1×PBS was injected into the microdevice at various pressure differentials. Small numbers of cancer cells in the sample solution mimicked the rarity of CTCs in peripheral blood. By visually counting the number of trapped and escaped cancer cells, the efficiency of cancer cell capture could be determined using Equation (1).
Isolation Efficiency(%)=((Trapped Cells)/(Trapped Cells+Escaped Cells))×100% (1)
In an embodiment according to the invention, the main factor affecting cell isolation efficiency was the pressure applied, since the input condition directly alters the flow conditions in the device. Although higher flow rates mean less sample processing time, the larger shear forces on the cells are undesirable as they can cause cell behavior modifications due to mechanical activated signal pathways or cell death (S. F. Chang, C. A. Chang, D. Y. Lee, P. L. Lee, Y. M. Yeh, C. R. Yeh, C. K. Cheng, S. Chien, J. J. Chiu, Proc. Natl. Acad. Sci. USA 105, 3927 (2008)). In the experimental investigation, the selected pressure differentials included 5 kPa, 10 kPa and 15 kPa which are comparable to physiological conditions. However, it should be appreciated that other pressure differentials may be used depending on the types of cells to be isolated, which may be cancer cells or non-cancer cells.
Other leading techniques to enrich cancer cells from peripheral blood have efficiency ranging from 20% to 90% (W. J. Allard, J. Matera, M. C. Miller, M. Repollet, M. C. Connelly, C. Rao, A. G. Tibbe, J. W. Uhr, L. W. Terstappen, Clin. Cancer Res. 10, 6897 (2004); M. Balic, N. Dandachi, G. Hofmann, H. Samonigg, H. Loibner, A. Obwaller, A. van der Kooi, A. G. Tibbe, G. V. Doyle, L. W. Terstappen, T. Bauernhofer, Cytometry B Clin. Cytom. 68, 25 (2005); O. Lara, X. Tong, M. Zborowski, J. J. Chalmers, Exp. Hematol. 32, 891 (2004)). However, there are also numerous restrictions and complex preparation procedures. For example, there is limited purity when detecting low concentrations of CTCs (D. A. Smirnov, D. R. Zweitzig, B. W. Foulk, M. C. Miller, G. V. Doyle, K. J. Pienta, N. J. Meropol, L. M. Weiner, S. J. Cohen, J. G. Moreno, M. C. Connelly, L. W. Terstappen, S. M. O'Hara, Cancer Res. 65, 4993 (2005)) in peripheral blood and various preparatory steps such as centrifugation, incubation and functional modifications which can be tedious and time consuming. A microdevice in accordance with an embodiment of the present invention is comparable to other leading biochemical techniques in terms of cancer cell enumeration from blood and is done without any functional modifications. Removing isolated cells for further analysis can also be achieved with ease by altering the flow conditions to induce the cells to flow towards the cell collection point.
In an experiment in accordance with an embodiment of the invention, in order to ascertain the capture purity, cancer cells mixed with blood samples collected from healthy human volunteers (−100 cancer cells per milliliter) were diluted with 1×PBS to reduce sample viscosity and injected into the microdevice. To differentiate between hematologic and cancer cells, immuno-fluorescence staining of the isolated cells was carried out. It is reported that the EpCAM is over-expressed in human carcinoma (P. A. Baeuerle, O. Gires, Br. J. Cancer 96, 417 (2007); W. A. Osta, Y. Chen, K. Mikhitarian, M. Mitas, M. Salem, Y. A. Hannun, D. J. Cole, W. E. Gillanders, Cancer Res. 64, 5818 (2004)) which makes this an ideal marker to identify the cancer cells. MCF-7, MDA-MB-231 and HT-29 have been reported to be positive for EpCAM (D. Flieger, A. S. Hoff, T. Sauerbruch, I. G. Schmidt-Wolf, Clin. Exp. Immunol. 123, 9-14 (2001); W. A. Osta, Y. Chen, K. Mikhitarian, M. Mitas, M. Salem, Y. A. Hannun, D. J. Cole, W. E. Gillanders, Cancer Res. 64, 5818 (2004)). CD45, a trans-membrane glycoprotein is expressed among hematologic cells and was used to distinguish WBCs.
In an experiment in accordance with an embodiment of the invention, the conditions of cancer cells after isolation were of interest given that tumor cells in circulation are likely to be responsible for the progression of the disease cancer. Preserving the native state of cells after isolation will help to determine their exact nature and allow a detailed study of CTC sub-populations. Retrieval of the isolated cells in the microdevice was achieved by altering the inlet conditions with the pressure regulators. The waste reservoir was cleared first to prevent backflow of waste materials, followed by closing of the sample inlet fluidic port. The valve leading to the cell collection point was then opened and the pressure differential between the waste outlet and cell collection point quickly increased to 20 kPa. Isolated cells were then dislodged and the recovery rate and standard deviation were determined to be (95±8.0) % for MCF-7, (97±2.6) % for MDA-MB-231 and (96±4.4) % for HT-29. The recovery rate was calculated based on the number of cells that were dislodged to the initial number of trapped isolated cells. The process of cell recovery was repeated for 5-8 cycles to obtain enough cell number and the retrieved cells were then reseeded to a culture flask.
In clinical reports, a CTC count of 5 cancer cells per 7.5 ml of blood is significant to represent the severity of the disease (M. Cristofanilli, G. T. Budd, M. J. Ellis, A. Stopeck, J. Matera, M. C. Miller, J. M. Reuben, G. V. Doyle, W. J. Allard, L. W. Terstappen, D. F. Hayes, N. Engl. J. Med. 351, 781 (2004); F. Nole, E. Munzone, L. Zorzino, I. Minchella, M. Salvatici, E. Botteri, M. Medici, E. Verri, L. Adamoli, N. Rotmensz, A. Goldhirsch, M. T. Sandri, Ann. Oncol. 19, 891 (2008)).
In an experiment in accordance with an embodiment of the present invention, in order to test the effectiveness of the microdevice to function at this concentration, 1 or 2 cancer cells were manually picked out and added into 1 ml of PBS. The difficulties in getting reproducible cell numbers and the need for enough cells for the proliferation analysis restrict the characterization with such small cell numbers.
A microfluidic platform in accordance with an embodiment of the invention has successfully demonstrated the enumeration of cancer cells of breast and colon origin in blood samples by utilizing cancer cells' inherently lower cell deformability and larger size as compared to blood constituents. This has potential in CTCs applications that can help to monitor the disease progression and treatment efficacy in contrast with biochemical techniques that are usually employed. The microdevice also achieves significant cancer cell isolation purity while preserving the integrity and state of these cells. With isolation efficiency of at least 80% for MCF-7, MDA-MB231 and HT-29, the microdevice can provide a potential useful assessment of the disease status. High cancer cell isolation purity for the microdevice will also minimize false positive results. The microdevice requires neither functional modification nor complex enrichment procedures, simplifying operation procedures. For clinical blood tests which usually handle larger sample volumes, parallel setups using multiple microdevices simultaneously can be adopted. Flowing 5 ml of sample solution through 3 microdevices at the same time at 5 kPa took no more than 2.5 hours which is considerably shorter than most leading techniques. These unique features make a microdevice in accordance with an embodiment of the invention attractive for possible CTC studies and potential clinical monitoring of the disease cancer.
In accordance with a further embodiment of the invention, a microdevice similar to those described above may be used to isolate cells to diagnose and/or monitor a disease or condition of a patient. In particular, a method may include diagnosing cancer, monitoring progression of cancer, monitoring treatment of cancer and/or providing a prognosis of a cancer in a patient. The diagnosis, monitoring or prognosis may be based on the isolated cells alone or made based on the isolated cells in conjunction with other tests. The isolated circulating tumor cells may be retrieved from the microfluidic device, or may be identified in situ within the microfluidic device. Where the microdevice is used in monitoring treatment, it may be used, for example, to monitor a patient's response to chemotherapy, drug treatment or other therapy. For example, if the number of CTCs isolated by the microdevice decreases (increases, or remains constant), this may reflect positively (or negatively) on the efficacy of the treatment.
In accordance with an embodiment of the invention, it will be appreciated that although isolation of cells from cell lines are referred to herein, a microdevice similar to those described above may be used to isolate cells from actual clinical samples. Further, a microdevice similar to those described above may be used to trap types of cells other than circulating tumor cells, including other diseased cells and non-cancer cells. For example, a microdevice similar to those described above may be used to isolate fetal cells, malaria infected cells, sickle anemia cells, dengue cells, stem cells and other types of cells from biological fluids, such as blood. When used with such non-cancer cells, the isolated cells may have a diameter of, for example, between about 6 μm and about 25 μm. It will be appreciated that other dimensions of such non-cancer cells may be isolated, depending on the size and type of cell to be isolated. Further, the microdevice may be used to isolate cells from biological fluids other than blood; for example, lymph, spinal fluid, urine, amniotic fluid or other biological fluids may be flowed through the device. In addition, the microdevice may be used to isolate cells from a separated component of whole blood, such as to isolate cells from a plasma component, a platelet component or a red blood cell component of blood. Further, the microdevice may be used to enrich samples.
Although transparent devices have been described herein, including those mounted on an inverted microscope, a microdevice in accordance with an embodiment of the invention may also be opaque, for example when mounted on silicon, in which case an upright microscope may be used instead of an inverted microscope.
As used herein, the term “biorheological property” means a property relating to the deformability and flow of a biological fluid or component thereof (such as a cell). A “biorheological property difference” means a difference between the biorheological properties of two or more biological fluids or components thereof (such as cells).
As used herein, where an isolation well comprises a cell trap of a size and shape suitable to mechanically isolate a cell within the cell trap, it should be appreciated that the size and shape of the cell trap may be determined by a variety of different possible techniques such as those described herein, based on the type of cell to be isolated. For example, the size and shape may be determined using experiments and/or computational fluid dynamics simulations, such as those provided above. As a result of such experiments, it may be determined that the cell trap is appropriately sized and shaped to isolate mostly single, or sometimes double, cells within a single cell trap. For instance, in the above experiments, a crescent, “U, “V” or “C” shaped cell trap with a longer dimension (the distance measured from the outermost edge of the tip of one “horn” of the crescent, “U,” “V” or “C” to the outermost edge of the tip of the other “horn” of the crescent, where outermost means the edge that is on the outer side of the crescent, which has a larger radius, as opposed to the inner bowl of the crescent, which has a smaller radius) of between about 15 μm to about 40 μm and a left or right tilt was determined to be of a size and shape suitable to isolate breast cancer cells. (For instance, in
Further, in accordance with an embodiment of the invention, an isolation well need not be crescent-shaped. Instead, an isolation well may be any size or shape suitable to mechanically isolate a separate individual cell within the isolation well. For example, “U” shaped, “V” shaped or “C” shaped isolation wells may be used instead of (or in addition to) crescent-shaped isolation wells.
Further, in accordance with an embodiment of the invention, it should be noted that live cells need not be used; fixed cells may be used instead, or cells that are in any state of health or viability. Further, pressure ranges and other numerical ranges provided herein should not be taken as limiting. For example, pressure ranges may be altered; if fixed cells are used rather than live cells, the pressure range may be higher because fixed cells are more sturdy than live cells. Further, it will be appreciated that dimensions given herein should not be taken as limiting, since the dimensions used are dependent on the size and type of cell to be isolated. For example, in one embodiment, cells (such as circulating tumor cells) of a diameter between about 5 μm to about 40 μm may be isolated, depending on the size and type of cell that is the target of the device. As used herein, it should be appreciated that a “diameter” of a cell may refer to a cell that is elongated or otherwise not spherical in shape, in which case “diameter” is intended to refer to the longest dimension of the cell. In clinical samples, for example, renal cancer cells may have sizes of about 5 μm or 6 μm to about 15 μm, cells of melanoma of the skin may have sizes of about 22 μm to about 28 μm, cells of a malignant synovioma may have sizes of about 21 μm to about 26 μm, cells of carcinoma of the bronchus may have sizes of about 13 μm to about 19 μm or larger, such as 25 μm to 30 μm; cells of carcinoma of the stomach may have sizes of about 30 μm to about 40 μm (R. F. Alexander and A. I. Spriggs, J. Clin. Path. 13, 414 (1960)). However, dimensions of clinical samples may vary widely, and it will be appreciated that other ranges of dimensions may be found for these and other cell types. Cell lines may have different dimensions than clinical samples. Any given fabricated device may be designed to isolate cells within a small range of sizes, for example varying by as little as 1-2 μm from a desired target cell size for the device; or may be designed to isolate cells over a larger range of sizes. Given the variation in dimensions of clinical samples, it will be appreciated that various different possible amounts of variation from a target cell size within a given fabricated device may be permitted. Further, more than one size of isolation well may be used within the same fabricated device. Further, it should be noted that renal cancer cells as small as close to 5 μm or 6 μm have been positively stained, which are smaller than a blood cell. However, because the cancer cells are stiffer than blood cells, they can still be isolated by a microdevice according to the invention. It should therefore be appreciated that other physical properties than size may be used to isolate cells, such as shear modulus, stiffness, size and/or deformability. Further, the cells to be isolated may be smaller than the blood cells or other components of a biological fluid in which the cells to be isolated are included.
Table 1 provides examples of cancer cell lines that have been tested, in accordance with an embodiment of the invention.
In addition, clinical samples of non-small cell lung carcinoma (NSCLC), nasopharyngeal carcinoma (NPC) and renal cell carcinoma have been tested, in accordance with an embodiment of the invention. An efficiency of 95.9% was achieved with 79 blood samples for the renal cell carcinoma. It will be appreciated that other types of clinical samples may be used.
Further, although examples of certain types of cancer cells are given above, it should also be appreciated that other types of cancer cells may be isolated; for example, breast cancer cells, colorectal cancer cells, kidney cancer cells, lung cancer cells, gastric cancer cells, prostate cancer cells, ovarian cancer cells, squamous cell cancer cells, hepatocellular cancer cells, nasopharyngeal cancer cells and other types of cancer cells may be isolated.
In addition, in accordance with an embodiment of the invention, the microdevice may be used to allow enumeration of cells, for example by counting the number of cells that have been isolated in the microdevice. Further, a microdevice may be used to determine a cell type, for example by permitting staining of cells within the microdevice. Such stained cells may be viewed by a microscope. The microdevice may be mounted on a microscope slide. For example, cells may be stained to determine whether the cells are CTCs or another type of cell. Further, an embodiment according to the invention may permit real time visualization of the isolation process; and/or may permit real time enumeration of isolated cells. For example, an imaging system may be connected to the device, to capture images from the device, and/or may receive light from the device, in order to permit real time visualization of the isolation process and/or to permit real time enumeration of isolated cells. In one example, the imaging system may view and/or digitize the image obtained through a microscope when the device is mounted on a microscope slide. For instance, the imaging system may include a digitizer and/or camera coupled to the microscope and to a viewing monitor and computer processor. The imaging system may, for example, allow a user to view stained cells, or cells that have not been stained. It will be appreciated that the imaging system may include appropriate light sources, microscopes, cameras, lenses, reflectors, detectors, digitizers, computers, processor devices, monitors and/or other appropriate imaging or optical devices to permit real time visualization of isolation and/or to permit real time enumeration of isolated cells. In addition, the imaging system may be used for other imaging of the device.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/172,250, filed on Apr. 24, 2009, the entire teachings of the which application are incorporated herein by reference.
Number | Date | Country | |
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61172250 | Apr 2009 | US |