Patent Application
20060051265
The present invention relates in general to apparatuses and methods for sorting or fractionating microstructures such as free cells, viruses, bacteria, macromolecules, or minute particles. More particularly, the present invention relates to apparatuses and methods for sorting such microstructures in suspension in a fluid medium, including the sorting and isolation of rare microstructures within a fluid medium.
The sizing, separation, and study of microstructures such as free cells, viruses, bacteria, macromolecules and minute particles are important tools in many fields, including molecular biology. For example, the fractionation process, when applied to DNA molecules, is useful in the study of genes and ultimately in planning and implementation of genetic engineering processes. The fractionation of larger microstructures, such mammalian cells, promises to afford cell biologists new insights into the functioning of these basic building blocks of living creatures.
Isolating rare cells from biological fluids, including whole blood or bone marrow, is a further interesting biological application. For example, characterization of a few metastatic cells from cancer patients for further study is clearly desirable for prognosis/diagnosis. Traditional methods have not proven adequate for this particular application due to the compositional complexity of blood, with its large number of cell types.
In view of the above, further enhancements in microstructure sorting apparatuses and methods are deemed desirable.
The shortcomings of the prior art are overcome and additional advantages are provided through the provision of an apparatus for sorting microstructures in a fluid medium. The apparatus includes a receptacle and N regions of columns positioned in the receptacle between an inlet and an outlet thereof. Fluid medium introduced into the receptacle through the inlet passes sequentially through the N regions of columns before exiting through the outlet, wherein N≧2. Each region i (i=1 . . . N) of columns of the N regions of columns includes at least one row of columns spaced to define multiple fluidic channels of a respective minimum width Wi, with the minimum widths Wi of the multiple fluidic channels of the at least one row of each region of columns (1 . . . N) varying between adjacent regions of columns of the N regions of columns and decreasing in size in the receptacle between regions from the inlet to the outlet thereof.
In another aspect, a method of sorting microstructures in a fluid medium is provided. The method includes: providing a receptacle having N regions of columns positioned in the receptacle between an inlet and an outlet thereof, wherein N≧2 and fluid medium introduced into the receptacle through the inlet passes sequentially through the N regions of columns before exiting through the outlet, and wherein each region i (i=1 . . . N) of columns of the N regions of columns comprises at least one row of columns spaced to define multiple fluidic channels of a respective minimum width Wi, and wherein the minimum widths Wi of the multiple fluidic channels of the at least one row of each region of columns (1 . . . N) vary between the N regions of columns and decrease in size in the receptacle between regions from the inlet to the outlet thereof; and employing the receptacle to sort microstructures in a fluid medium by introducing the fluid medium with the microstructures therein into the receptacle through the inlet and allowing the fluid medium to pass through the N regions of columns before exiting through the outlet, wherein differently sized microstructures separate in different regions of the receptacle dependent, in part, on physical characteristics thereof.
Additional features and advantages are realized through the techniques of the present invention. Other embodiments and aspects of the invention are described in detail herein and are considered a part of the claimed invention.
One or more aspects of the present invention are particularly pointed out and distinctly claimed as examples in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which:
Generally stated, provided herein are apparatuses and methods for sorting microstructures such as cells, viruses, bacteria, macromolecules and minute particles (or their components) suspended in a fluid medium. The sorting apparatuses and methods disclosed herein, which are principally described below with reference to sorting or fractionation of cells in a fluid medium, separate cells based on physical characteristics and/or time to propagate through the channels of the apparatus.
In one aspect, the apparatus includes a receptacle comprising a first region of columns and a second region of columns positioned in the receptacle between an inlet and an outlet thereof. Fluid medium introduced into the receptacle through the inlet passes through the first region of columns and then the second region of columns before exiting through the outlet. The first region of columns includes at least one row of columns spaced to define multiple fluidic channels of minimum width W1 and the second region of columns includes at least one row of columns spaced to define multiple fluidic channels of minimum width W2, wherein W1>W2. Thus, in accordance with an aspect of the present invention, the minimum width Wi of the multiple fluidic channels defined in each region decreases in size in the receptacle between regions from the inlet to the outlet. Numerous enhancements to this basic apparatus are described and claimed herein.
Fluid medium flow through the receptacle from the inlet to the outlet, referred to herein as “main axial flow”, can be electrophoretic, electro-osmotic, and/or pressure driven. In the structures depicted in the figures, main axial flow is assumed for purposes of example to be left-to-right. In addition to the main axial flow, a cross-flow intersecting the main axial flow, e.g., at any angle between 10 degrees and 90 degrees, can be established for a period of time, or through the entire operational period of the apparatus, or after a sample has stopped flowing through the apparatus along the main axial flow.
Defining the channels of the apparatus using discrete columns is favored over an array of continuous channels without gaps for multiple reasons. For example, having regions of discrete columns as disclosed herein: (1) allows cross-flow to be established to manipulate cells or inject a biochemical reagent in situ; (2) allows cells to migrate around a blocked area; and (3) allows cells to deform and reform as the cells traverse the apparatus, with larger, more rigid cells taking longer than smaller, more flexible cells, introducing time as an additional dimension for the sorting of cells (i.e., in addition to cell size, mobility, and mechanical properties such as rigidity).
Information from retained cells can be extracted in various ways. For example, a device can be examined (e.g., visually or through fluorescence) for presence or absence of retained cells in an expected region of the apparatus. If cells are found retained in the apparatus, then the cells can simply be counted. By way of example, chemotherapy is monitored by enumerating the number of circulating tumor cells. If the number decreases, therapy is working. If cells do not decrease in number, a different chemotherapy or an entirely different treatment can be applied. Further, cells can be lysed in situ and the released nucleic acids and proteins can be collected at the outlet. Still further, retained cells (or lysed components from cells) can be extracted intact by cross-flow through one or more cross-flow openings in the apparatus. Alternatively, main axial flow through the apparatus can be reversed, and the retained viable cells can be collected at the original inlet side of the apparatus.
Thus, cells of interest can be retained inside the device, while all other cell types migrate to the output, as the case for cancer, sperm and other applications, or cells of interest can be the cells that migrate through the entire apparatus, while other cell types become retained inside the apparatus, as would be the case for fetal cell separation. Further, surfaces of the apparatus can be treated or coated to improve fluid flow through the apparatus, or to prevent or even promote specific cell adherence within the apparatus.
As an overview, the following applications for the apparatuses and methods described herein are contemplated:
Various embodiments of the apparatuses and methods for sorting microstructures in a fluid medium in accordance with aspects of the present invention are depicted in
Referring to
Upper receptacle portion 120 of apparatus 100 is sized and configured to mate with lower receptacle portion 110, and includes a first, inlet end 122 having an inlet plenum 124 provided therein and a second, outlet end 126, having an outlet plenum 128 provided. Inlet plenum 124 is fed via one or more inlets 125 through which fluid medium with microstructures disposed therein is introduced into the apparatus. Outlet plenum 128 is in fluid communication with at least one outlet 129 through which fluid medium and any microstructures passing through the apparatus are withdrawn. Upper receptacle portion 120 further includes, in this embodiment, multiple inlet cross-flow openings 160, 162 and multiple outlet cross-flow openings 170, 172. Openings 160, 162, 170 & 172 facilitate the introduction and removal of a cross-flow fluid through the apparatus. For example, these openings may be aligned to facilitate the removal of microstructures sorted into the different regions, or the introduction of a biochemical agent during passage of fluid medium through the apparatus from the inlet plenum to the outlet plenum. If desired, cross-flow openings (as well as main inlets and outlets) could be provided in lower receptacle portion 110 as well.
One approach for fabricating an apparatus in accordance with the present invention is depicted in
Those skilled in the art will note that there are many variations to the above-outlined fabrication protocol. Different polymers will have different process conditions, and certain polymers cannot be used directly on, for example, silicon wafers, so that an intermediate molding step may be required. In such a case, the silicon wafer might have the exact device structure, not the complementary image, since two molding steps will take place.
As a more specific example, intact cancer cells can be removed from the apparatus by reversing the fluid medium flow after cancer cells have been retained and all blood cells have been flushed through the apparatus. In order to ensure that only retained cancer cells are extracted, the inlet tubing can be changed after cell loading, but prior to cell reversal (or a different inlet could be used). Additionally, the apparatus can be flushed for a period of time (for example, 20 minutes) using medium only, prior to flow reversal. Reversing the flow, while keeping both ends of the apparatus wet at all times, can be achieved by filling the output plenum with fluid medium one or two seconds prior to reversing the flow. In cell lysis, removal of cell lysed components from captured cells can be accomplished by flowing water or a buffer through the apparatus, with the desired component being collected via the output plenum.
In the apparatus examples described above, it is assumed that fluid medium flows through the apparatus via a pressure differential between the inlet and outlet, for example, through the application of a vacuum pressure at the outlet of the apparatus.
Electrodes 940 & 942 can also be added to the sides of apparatus 900 to facilitate movement of microstructures in a direction other than the main axial flow direction. For example, a cross-flow fluid can be introduced through a side inlet side 960 and removed through a side outlet 970, with cross-electrophoresis employed in extracting isolated cells at the interface between first region 910 and second region 920. Again, the use of electrode driven cross-flow could be an alternative to a pressure driven cross-flow, or in combination therewith.
Electrodes can be added to the sorting apparatus by inserting thin platinum wires during a final molding step. These wires could be externally mounted onto the apparatus, with a simple fixture used to position the electrodes, or they could be deposited in the apparatus using additional mask and photolithography steps to transfer the electrode pattern into the apparatus and then deposit the electrodes.
In the embodiment of
In
Those skilled in the art will note from the embodiments of
In the embodiment of
If all regions cannot practically be accommodated in one receptacle, then it is possible to group two or more receptacles in parallel, as depicted in
Those skilled in the art will note from the above discussion that an apparatus in accordance with aspects of the present invention requires no prior information about a cell's surface marker or genetic abnormality. The apparatus can retrieve live cells for further analysis, where many existing apparatuses cannot. Further, the apparatus described is less laborious and expensive than existing approaches and can be widely implemented. For example, an apparatus such as depicted in
To summarize, a new technology has been develop to isolate cells, e.g., one or two or more per milliliter of volume, from biological fluids such as blood, bone marrow, amniotic fluid, ascites, sputum, sweat, urine, feces, cerebrospinal fluid, edema, semen and fluid from the female genital tract. The apparatus presented has applications in a wide variety of areas where there is a need to isolate, remove, manipulate and/or monitor individual cells. The isolation of cells is based on their physical properties requiring no prior knowledge about surface markers or gene expression. By implementing specific versions of the device, the application areas become many fold and examples include: non-invasive prenatal diagnostic testing; cancer detection; bone marrow purging; and other applications involving the measurement, sorting or removal of individual biological cells. The apparatus is simple to use and includes a design for clinical testing applications using standardized testing protocols. For testing applications, cells can be isolated in less than one hour with only one sample manipulation step prior to apparatus loading (i.e., dilution of fractionation). No operator time is required during a test and the receptacle or cartridge is disposable.
The apparatus fabrication process described herein allows for high volume production at low cost, permitting the device to be disposable, and thereby eliminating contamination concerns. In addition, the flexibility of the technology platform lends itself to custom cartridge type applications for specific solution concerns. The flexible device design and its micro-scale size also lend it to being interfaced with external devices for additional downstream applications. For example, the modular design allows it to be connected to existing genetic testing devices once cells of interest are isolated.
The following examples are based on cartridges specific to various applications.
For this application, maternal blood is drawn intravenously and the apparatus is used to isolate the fetal nucleated red blood cells (fNRBCs), thus eliminating the current invasive procedures such as amniocentesis or chorionic villus sampling. The test can be performed starting from the sixth week of gestational age, and has the potential to help support a clinical diagnosis or screen for a particular health problem throughout the duration of the pregnancy. Theoretically, testing would not be limited to certain gestational periods. In addition, the technology is likely to reduce the cost associated with the prenatal diagnostic testing.
In this application, the apparatus is implemented to isolate metastatic cells from a cancer patient's blood to monitor treatment and detect relapse at an earlier point than is currently possible. Blood can be sampled more frequently, leading to more detailed follow-up and improved patient management, including “personalized” drug regimens. Further research may confirm that testing can be performed on cells isolated from the peripheral circulation, instead of using more invasive procedures such as needle aspiration and testing of non-metastatic cells. Additionally, isolating metastatic cells may provide a means to study cell tumor gene expression in order to identify efficacious drugs at the time of diagnosis and to aid in prognosis and staging.
Prior to transplantation, the apparatus can be used to “filter” cancer cells from bone marrow. Passing the sample through the device retains the cancerous cells while allowing the healthy cells to migrate through where they are collected at the opposite end. This cancer free sample can then be transplanted back into the patient.
Experiments:
First-generation devices had channels that were 5, 10, 15 or 20 μm wide, and 5, 10, 15 or 20 μm deep. The channel width and depth were constant across the entire device. Experimentations with various combinations of channel widths and depths were performed. When the channel dimentions were larger than the neuroblastoma (NB) cell (≈10 μm in diameter), such cells passed through the entire device without resistance. (The used NB cell line was: SK-N-MC, and was purchased from ATCC, Manassas, Va.) A reproducible flow pattern could not be established when the channel dimensions are comparable to or smaller than the cellular dimensions; that is, cells adhered at times, particularly when a concentrated pure population was loaded, to the column walls at the entrance of the channel, due to high concentration, slow flow, and wall roughness reproduced from the etch process.
Second-generation devices were designed by integrating all four channel segments. Channels narrowed through the device with widths of 20 μm, then 15 μm, then 10 μm, and finally 5 μm wide. Channel depths, constant within a given apparatus, were 20 μm, 15 μm, 10 μm, or 5 μm. Each segment was composed of ˜1,800 channels of the same dimensions arranged in ˜375 parallel arrays. The first two regions were designed to ensure an even flow distributed over the entire width of the apparatus, while the two subsequent smaller regions were designed to optimize separation. The goal was to use the device to model separation of NB cells from human whole blood. In order to achieve this goal, the “critical dimensions” for blood were established first to better characterize the apparatus, and then, to extend its use to additional cell fractionation applications beyond the isolation of NB cells.
The output connector was connected to house vacuum (≈23 in Hg, +/−10% maximum variation) for each experiment. A wetting solution, 2% tetra (ethylene glycol)-dimethyl-ether solution (Sigma-Aldrich) in Eagle's minimal essential medium (EMEM), was used to enhance cell migration through the device. This solution was tested for its effects on cell viability, and was found to have little effect for short-term exposure; since cell are loaded in straight medium after wetting, and flow is established. The wetting and test solutions were pressure-driven, from the inlet reservoir to the outlet reservoir (negative pressure being applied to the output side). Once the complete apparatus was wet, the cell solution was introduced. All experiments were completed under a fluorescence microscope equipped with a digital video camera.
Experiments with human whole blood proved that the second generation of devices had gaps and depths larger than required for peripheral blood fractionation; as a result, all cells from healthy human whole blood crossed the entire apparatus without resistance.
When cultured NB cells were tested with the 5 μm deep device, cells were retained consistently in the first few rows of the 10 μm channels. NB cells were cultured in EMEM with 10% fetal bovine serum (FBS), 5% L-Glutamine, and 5% PENSTREP, at 37° C. and under 5% CO2. Cultured NB cells were first tested in medium only. A challenge with these cells was adhesion to the channel walls, particularly in earlier segments, upstream of where freely moving cells were retarded based on the limitations of channel size. The roughness of the column wall from the Bosch etching process, reproduced with high fidelity in the polyurethane, and the slow flow at the beginning of the device due to the device length and depth, contributed to the problem. A shorter device (3.5 cm), with deeper channels (20 μm), reduced the resistance to flow and alleviated the adhesion problem.
Based on the results from the second-generation device, a third-generation device with channel spacing at 15 μm, 10 μm, 5 μm, and 2.5 μm intervals was fabricated as described above, for additional experimentation with human blood. All channels were 5 μm deep. Either whole blood diluted in medium (1:10 v/v; blood was obtained from a healthy adult volunteer in our laboratory), or isolated mononuclear cells were used in the device. Mononuclear cells were isolated by 1:1 dilution (v/v) with phosphate buffered saline (PBS, pH 7.4) and centrifugation on a density gradient (Ficoll-Paque). After Ficoll separation, the mononuclear cell layer contains adult red blood cells at very low concentrations. When the mononuclear cell layer was used in the 5 μm deep devices, the white cells were retained at the beginning of the 2.5 μm wide channel segment, while red blood cells, identified by morphology under the microscope, traversed the entire device. The previous experiment was repeated, and the blood was stained with nucleic acid stain (SYTO Red, Molecular Probes, Inc., Eugene, Oreg.) to differentiate nucleated white blood cells from enucleated mature red blood cells. These results using fluorescence confirmed our bright field microscope results, the retained cells were indeed nucleated. Isolated red blood cells, when present in very low concentration in medium as described above, traversed the entire apparatus without resistance down to the 2.5 μm channel width. Here, they slowed down but still passed through to the output reservoir. Adult red blood cells in higher concentration, 1:100 v/v in medium encountered a higher resistance at the 2.5 μm wide channels and slowed down to the point that they aggregated at the channel exits, and were retained. Higher concentrations of adult red blood cells led to uncontrolled cell aggregation, even in upstream segments wider than the 2.5 μm channels.
Adult whole human blood diluted in medium (EMEM), at 1:10 v/v, and spiked with cultured NB cells, was tested in the apparatus. As expected, the NB cells were retained at the beginning of the 10 μm wide by 20 μm deep channels, while adult blood cells passed through the entire device without resistance, proving our hypothesis and duplicating the results obtained by testing the cell populations separately.
To summarize, the isolation of cultured NB cells has been demonstrated when mixed with whole blood, based solely on size and deformation characteristics. With the exception of dilution of the blood sample with medium, no other sample manipulation was required. Using the second-generation device, NB cells were consistently isolated in the 10 μm wide by 20 μm deep channels, while blood cells migrated to the output reservoir. Experiments using 2 mL of whole blood, before dilution, took approximately 2-3 hours. This method thus presents great advantages, in both cost and time, over existing methods such as FACS and MACS, which require 2 days to allow for manual manipulation, testing, and result assessment. The time frame can be reduced yet further by optimizing the microfluidics and/or increasing the width, by making the device even more massively parallel, and by decreasing the length. This device will be used to capture metastatic cells in the patient's peripheral circulation, for later characterization by molecular means. Additionally, this device will be used to capture tumor cells in bone marrow for molecular analysis and/or for purging.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims.
This application derives priority from U.S. Provisional Application No. 60/607,954, filed Sep. 8, 2004, entitled “Sparse Cell Isolation Device”. This Provisional Application is incorporated herein by reference in its entirety.
This invention was made with support from the United States Nanobiotechnology Center and the United States National Science Foundation under Contract Nos. 0142522-04 (1999-2003) and ECS 987-6771, respectively. Accordingly, the United States government may have certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60607954 | Sep 2004 | US |
