Patent Application
20190210019
Microfluidic devices are provided that are useful for, inter alia, concentrating cells and/or separating cells according to size and deformability.
The background description includes information that may be useful in understanding the systems and methods described herein. It is not an admission that any of the information provided herein is prior art, or that any publication specifically or implicitly referenced is prior art.
Biological samples obtained from patients contain a variety of different types of cells in a relatively large volume of fluid. Often it is desirable to concentrate cells, change buffers, or concentrate particular types of cells in order to perform various biological assays or otherwise study the cells. However, current techniques for performing these types of processes typically rely on heavy equipment such as centrifuges, utilize time consuming assays involving dialysis or ion-exchange, require multi-step complex processes, or render the cells non-functional for downstream biological assays. Additionally, these approaches often require relatively large sample sizes and have low cell recovery rates.
In some cases, the sample may contain a variety of cell types, including but not limited to, red blood cells, white blood cells, and various other cellular components (e.g., circulating tumor cells (CTCs), metastatic cells, normal cells, etc.). It can be difficult to isolate a particular cell type, e.g., CTCs, especially when these cells occur at a low frequency relative to other, more abundant, cell types. For example, it is estimated that about one CTC is present in a blood sample for every billion normal or blood cells.
Current approaches to separating a population of cells from a blood sample may include techniques such as fluorescence activated cell sorting, magnetic activated cell sorting, or immunomagnetic colloid sorting. These approaches are laborious, require operator expertise, may lead to significant cell loss (poor recovery) and/or are expensive to perform. Other techniques to isolate a population of cells lack separation specificity, for example, charge flow separation. New techniques to rapidly and easily concentrate cells, and isolate cells based on size are needed.
Various systems and methods for manipulating cells using a microfluidics device are presented herein. The present techniques allow for rapid concentration of cells, for isolating cells based on size or a size range, for buffer exchanges, for adding labels, etc., and these techniques can be integrated into other workflows. The present methods provide novel techniques of changing buffers and achieving a high cell recovery rate. These techniques allow for concentrating cells when the cells are present at a low frequency in a solution, as well as isolating cells of a particular size or a range of sizes from among a sample of heterogeneously sized cells. For example, systems and methods for concentrating cells from a sample can be used to separate tumor cells from a blood sample, while systems and methods for separating cells based on size can be used to separate activated T cells from non-activated T cells. In some aspects, the devices are manually operable. In other aspects, the devices are automated or partially automated.
In one aspect, a microfluidics device is presented, comprising: an input mechanism for introducing a solution comprising cells; a microfluidics chamber in fluid communication with the input mechanism, wherein the microfluidics chamber comprises a plurality of rows of posts, wherein each row comprises a plurality of posts distributed along a line having a slope, wherein the slope is determined relative to a side of the microfluidics chamber; and at least two output mechanisms in fluid communication with the microfluidics chamber. In general, the first output mechanism has a larger cross sectional area than the second output mechanism.
Methods are also provided for introducing a solution comprising cells into a microfluidics chamber via an input mechanism, wherein the chamber is in fluid communication with the input mechanism; applying pressure to cause the cells to flow through the microfluidics chamber, wherein the microfluidics chamber comprises a plurality of rows of posts, wherein each row comprises a plurality of posts distributed along a line having a slope, and wherein the slope is determined relative to a side of the microfluidics chamber; deflecting the cells to a side of the chamber by the rows of posts to deplete cells from the solution exiting a first output mechanism and enrich cells in the solution exiting a second output mechanism, wherein the second output mechanism has a smaller cross sectional area than the first output mechanism.
Thus, in one aspect, pressure is applied to cause cells to flow though the matrix of posts and to be deflected laterally towards a side of the chamber, resulting in a local increase in the concentration of the cells. The concentrated cells exit the chamber through an output mechanism, and are collected. In other aspect, pressure is applied to cause the cells to flow through the matrix of posts, wherein cells larger than a cutoff size are retained in a path while cells smaller than a cutoff size pass into adjacent rows via gaps between the posts. The larger sized cells exit the chamber through an output mechanism and are collected; the smaller sized cells exit the chamber through another output mechanism and may also be collected.
Advantages of present invention embodiments include high throughput, high yield of concentrated cells, flow rate independence, configurability with regard to concentrating or separating cells of varying sizes by varying the gap between posts and/or the slope/rotation of each post and/or the shape/geometry of each post, rotation of the posts, and spacing of the posts, and compatibility with manual or automated operation. In general, the performance of the microfluidics device in the context of concentrating cells and size-based cell separation is independent of the flow rate under normal conditions of laminar flow. The device performance is similar under different flow rates, but may be dependent on a sheath-to-sample flow rate ratio. Accordingly, the cells will be deflected laterally towards a side of the chamber, independently of the rate of fluid flow through the microfluidics device. Additional advantages include the ability to manufacture the microfluidics device for a low cost. In some embodiments, a solution comprising cells may be loaded into a syringe, and the syringe may be connected to an input mechanism of the microfluidics device. An operator of the syringe depresses the plunger manually (e.g., hand injection) to cause the solution containing the cells to flow through the device. Further, the embodiments disclosed herein do not rely on complex technology (e.g., centrifuges, magnetic forces, etc.) in order to achieve cell concentration. In some embodiments, the microfluidic devices are single use devices. In other embodiments, volumes of solutions that are injected into the microfluidics device range from 1 mL to 10 mL or more.
Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments.
As used herein, the term “antibody” generally refers to immunoglobulin molecules and immunologically active portions or fragments thereof of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., on the surface of the cell). Unless the context dictates otherwise, the term “antibody” or “antibodies” includes but is not limited to all isotypes and subtypes of antibodies (e.g., IgA, IgD, IgE, IgG, IgM, etc.), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule, as well as all active fragments (having immunological activity) thereof. It is also understood that any heavy chain (e.g., IgA, IgD, IgE, IgG, IgM) may be paired with any light chain (e.g., kappa or lambda forms).
Antibodies also include, but are not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, murine antibodies, conjugated antibodies (e.g., to a chemotherapeutic agent, to a radionuclide, to another protein, etc.), synthetic antibodies, bi-specific antibodies, chimeric antibodies, single chain antibodies, antibody fragments produced by a Fab expression library, and antibody fragments produced by mRNA display or phage display. Antibodies also include but are not limited to monovalent immunoglobulins (e.g., IgG), and fragments, e.g., F(ab′)2, Fab2, Fab′, Fab, Fv, single-chain Fv (scFv), scFv-Fc, VhH, disulfide-linked Fvs (sdFv), etc. or any active fragment thereof.
In some embodiments, the antibody is labeled with a colorimetric probe, a fluorescent tag, a chemiluminescent tag, or a radiolabel, and incubated with the solution comprising cells. A variety of techniques are suitable for detecting binding of an antibody to a substrate (https://www.rndsystems.com/resources/protocols/detection-visualization-antibody-binding). Such techniques, including various fluorescently tagged, colorimetric, chemiluminescent, or radiolabeling immunoassays, are well known to a skilled artisan.
As used herein, the term “binding molecule” refers to a molecule capable of specifically binding to a cell, e.g., by binding to a marker on the surface of the cell. A “binding moiety” includes but is not limited to antibodies or fragments thereof, aptamers, protein scaffolds, affimers, small molecules, peptides, proteins, phage display scFvs, mRNA display peptides or mRNA display scFvs, etc.
As used herein, the term “cell” refers to the smallest structural and functional unit of a living organism. Human cells range in size, e.g., provided herein as a volume, from about 30 μm3 to about 4,000,000 μm3. A typical cell size for a red blood cell is about 100 μm3 and for a megakaryocyte is about 30,000 μm3.
As used herein, “solution of cells” includes a biological sample comprising a plurality of cells. In some cases, the solution of cells may optionally include additional components, including but not limited to, buffers, preservatives, diluents, and other molecules to facilitate processing by the microfluidics device. As an example, a blood sample may contain a plurality of red blood cells and white blood cells as well as other types of cells, such as CTCs, metastatic cells, and/or normal cells.
As used herein, the term “chamber” refers to a region having an associated length, width and height, e.g., a rectangular component, comprising a matrix of posts. The matrix of posts refers to a configuration, e.g., a grid or grid-like, in which multiple rows of posts are present, with each row having a plurality of posts. Thus, the posts may be described as a plurality of rows, each row containing a plurality of posts (as used herein). Alternatively, the posts may be described as a plurality of columns, each column containing a plurality of posts. In some aspects, each row of the posts or at least a portion of each row of the posts is arranged along a line having a slope, wherein the slope is calculated using standard definitions in mathematics (e.g., m=(y2−y1)/(x2−x1), and wherein the x,y positions refer to the locations of posts). In this regard, slope is determined relative to a side of the chamber, which has zero slope. Thus, the posts are not arranged in lines that are parallel to the sides of the chamber. The chamber has one or more input mechanisms (e.g., to introduce cells, fluids, binding agents, buffers into the chamber, etc.) and one or more output mechanisms (e.g., to facilitate collection of concentrated cells, to facilitate collection of size separated cells, to connect to another input mechanism, to connect to another chamber for further processing, etc.).
As used herein, the term “path” or “channel” refers to an area between two adjacent rows of posts through which cells pass through (see,
When the device is configured to separate cells based on size, the posts are arranged to retain cells larger than a threshold and to permit cells smaller than a threshold to cross one or more rows of posts thereby entering two or more paths. It is noted that the configuration of posts disclosed herein is distinct from systems that employ configurations which result in particles zig-zagging back and forth (e.g., path 1 to path 2, path 2 to path 1) between two or more paths. The present techniques employ directional movement (e.g., path 1 to path 2, path 2 to path 3), to steer the cells towards a side of the chamber for collection. Thus, while the microfluidics device can be used for cell concentration and cell separation, both of which deflect cells laterally toward a side of the chamber, it is understood that in the context of cell concentration, cells predominantly remain in a single path, and in the contact of cell separation, cells above a threshold value of size predominantly remain in a single path.
As used herein, the term “concentration” refers to a measure of an amount of a component in a solution, e.g., how many cells, how many cells of a particular type of cell, etc. are present in a particular volume of a solution.
As used herein, the term “flow rate” refers to a volume of fluid (e.g., a solution comprising cells) that is moving through a given cross sectional area per unit time.
As used herein, the term “in fluid communication with” or “fluidly coupled to/with” refers to two spatial regions being configured such that a liquid may flow between the two spatial regions. In some embodiments, the two spatial regions are in fluid communication through one or more valves, restrictors, mixers or other components, configured to control the flow of a solution comprising cells through the microfluidic device. It is noted that two spatial regions may be in fluid communication even if the fluid is not able to flow freely into the spatial region with which it is in communication with. For example, an input mechanism may be in fluid communication with a chamber, even when a valve is present such that fluid flows into the chamber only upon application of pressure. In other embodiments, an input mechanism may be in fluid communication with a chamber into which a fluid (e.g., a solution comprising cells) may flow freely into.
As used herein, the term “linear” refers to a straight line or substantially a straight line. For example, for posts distributed along a straight line, some posts may be slightly above the line (e.g., within about a 10%, a 5%, a 2%, or a 1% deviation), and other posts may be positioned slightly below the straight line (within about a 10%, a 5%, a 2%, or a 1% deviation). However, taken as a whole, the posts are essentially distributed along a straight line. In other embodiments, the row of posts may be associated with a curvature, to more efficiently direct cells to a side of the microfluidics device.
As used herein, the term “input mechanism” refers to a port or other opening that is in fluid communication with the chamber comprising a matrix of posts. Fluid (e.g., the solution comprising cells) may be introduced into the microfluidics device via the input mechanism. In some embodiments, a syringe or pump (e.g., peristaltic pump, vacuum pump, syringe pump, displacement pump, etc.) may be connected to the input mechanism through tubing, e.g., via a twist screw cap that attaches to the input mechanism of the microfluidics device. Pressure may be applied to drive fluid flow through the microfluidics device. In some embodiments, the input port may be in fluid communication with an optional input chamber (a region without a matrix of posts), which is in fluid communication with the chamber comprising the matrix of posts.
As used herein, the term, “interval” or “spacing” refers to a unit of distance. Interval, in this context, includes both fixed intervals (e.g., the same distance) and varying intervals (e.g., varying distances). Intervals can refer to the distance between successive rows of posts (e.g., a vertical measurement), or the distance between successive posts in a respective row (e.g., a horizontal measurement).
As used herein, the term “microfluidics device” refers to a device designed to control the flow of small volumes of fluid through a 3D structure in order to perform a desired operation. In certain embodiments the microfluidics devices disclosed herein comprise a matrix of posts for concentrating cells, and may also perform other operations such as buffer change and cell type isolation. In still other embodiments, the microfluidics devices disclosed herein may sort cells according to a size or a range of sizes.
As used herein, the term “output mechanism” refers to a port or other opening that is in fluid communication with the chamber comprising a matrix of posts. Fluid (e.g., the solution comprising concentrated cells) may exit the microfluidics device via the output mechanism, into a reservoir. In some embodiments, the reservoir may be connected to the output mechanism through tubing, e.g., via a twist screw cap that attaches to the output mechanism of the microfluidics device. In some embodiments, the output port may be in fluid communication with an optional output chamber (also a region without a matrix of posts), which is in fluid communication with the chamber comprising the matrix of posts.
As used herein, the term “post” refers to a structure within the chamber having an associated in-plane dimension, an out-of-plane dimension, rotational angle, and shape. An in-plane dimension may refer to the length (l) and width (w) of the post, while the out-of-plane dimension may refer to the height (h) of the post. A tilting angle or a rotational angle (ϕ) refers to the rotation of the post with respect to the chamber. Shape refers to the 3-D characterization of the post, e.g., cylindrical, conical, pyramidal, cubic or cuboid, etc. In general, a post will be positioned such that its axis (out-of-plane dimension) is perpendicular to the surface to which it is affixed. In some embodiments, all posts within the chamber are of the same dimensions. In other embodiments, the dimensions of the posts vary as a function of space. In general, the post may be of any shape, and is not limited to a particular geometric shape presented herein. The post shape may be arbitrary, as shown in
Non-activated T cells have a diameter of about 6-8 μm and can be concentrated with this configuration. Cancer cells, occurring at a low frequency within a sample, can be concentrated with this configuration along with similarly sized or larger cells, e.g., including immune cells. Additionally, this configuration is compatible with buffer changes, and other processing steps, e.g., as set forth herein. In general, this type of geometric configuration has an increased overlapping area, a narrow gap, and is not symmetric.
As used herein, “regularly spaced interval” or “fixed interval” refers to an interval having a fixed distance. According to embodiments presented herein, posts may be distributed along a line at a fixed interval, e.g., a post may be positioned along a straight or substantially straight line e.g., at every 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, etc.
As used herein, the term “rotational angle” refers to the degree of rotation of a post with regard to its position in the chamber. In some embodiments, the post has a length that is greater than its width. In this embodiment, a rotational angle of zero means that the length of the post is aligned with an axis perpendicular to a side of the chamber. To describe different rotational angles, the post may be rotated in a clockwise or counter clockwise direction. As an example, the posts in
As used herein, the term “specifically binds” typically refers to non-covalent interactions between a target entity (e.g., a marker on a cell surface) and a binding agent and usually refers to the presence of such an interaction with a particular structural feature (e.g., such as an antigenic determinant) of the target entity with the binding agent. As understood by one of skill in the art, an interaction is considered to be specific if it occurs in the presence of other alternative interactions.
The examples presented herein are not intended to be limiting. It is understood that many different variations of these examples are disclosed within the application, and that all such embodiments fall within the scope of the embodiments disclosed herein.
Systems and methods of concentrating cells or separating cells based on size from a biological sample (e.g., a bodily fluid) are provided. Bodily fluids may comprise a variety of cells obtained from a patient. Bodily fluids include but are not limited to blood, serum, plasma, saliva, urine, tear, sweat, interstitial fluid, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, or other bodily secretions or exudates.
In some embodiments, the bodily fluid undergoes additional processing, before being brought into contact with the microfluidics device. In other embodiments, it is noted that the present techniques may be used directly with whole blood, or with diluted whole blood. Processing steps may also include adding one or more additional ingredients (e.g., buffers, physiologically acceptable carriers, diluents, excipients, stabilizers, diluents, emulsifiers, preservatives, etc.) to the bodily fluid. Additionally, one or more other ingredients may be added, including but not limited to, antioxidants, bacteriostats, buffers, carbohydrates, chelating agents such as EDTA or glutathione, blocking reagents such as bovine serum albumin, coloring, flavoring and/or aromatic substances, lubricants, pH buffering agents, salts for influencing osmotic pressure, polypeptides (e.g., glycine), proteins, solubilizers, wetting agents, substances to reduce cell adhesion, etc., which do not deleteriously react with the cells. Buffers include but are not limited to saline, neutral buffered saline, phosphate buffered saline, etc. Carbohydrates include but are not limited to dextrans, glucose, mannose, mannitol, sucrose, etc. Thus, the solution comprising cells that is provided to the microfluidics device may comprise the biological fluid as obtained from the patient or the biological fluid that has undergone additional processing. It is presumed that the flow of fluid through the microdevice occurs predominantly as laminar flow.
In this example, two output mechanisms are shown for the chamber. The solution that exits the chamber via output mechanism 120 is depleted of cells, while the solution that exits the chamber via output mechanism 122 is enriched in cells. Depletion refers to a state in which the concentration of cells in the solution exiting the chamber via output mechanism 120 has been reduced by 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to the concentration of cells in the solution entering the chamber at input mechanism 110. Enrichment or concentration refers to a state in which the concentration of cells in the solution exiting the chamber via output mechanism 122 has been increased by 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more as compared to the concentration of cells in the solution entering the chamber at input mechanism 110. For the cells to be directed to output mechanism 122, the deflection point 130 of the chamber should be situated such that exiting cells are routed to output mechanism 122 (and not output mechanism 120). In general, output mechanism 122 is configured to have a larger cross sectional area than output mechanism 120. In general, the output mechanisms will have a cross sectional area large enough for fluid flow at the flow rates described herein, without producing high pressures which would damage the cells or the device.
In some embodiments, the width of output mechanism 120 is greater than the width of output mechanism 122. For example, the width of output mechanism 120 is 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5× the width of output mechanism 122. In other embodiments, the sum of the cross-sectional areas of the output mechanisms may be equal to or greater than the sum of the cross-sectional areas of the input mechanisms.
In some embodiments, the microfluidics device is configured to allow a standard pump or a syringe or the like to be connected to the microfluidics device to flow a solution comprising cells through the device. Parameters that can influence the flow of a solution though a device include the dimensions of the chamber 105, the in plane and out of plane dimensions of the posts 115, the spacing of the posts within a row (dx), the rotation of the posts (0, the distance between rows of posts (dy), the diameter of the input mechanism, and the diameters of the output mechanisms (see,
In general, cells may flow into the microfluidic device in a series (one at a time and into a particular path) or in multiples (multiple cells that flow into multiple paths).
In some embodiments, the width of the chamber is 1 mm, 2.5 mm, 5 mm, 7.5 mm, 10 mm, 12.5 mm, 15 mm, 17.5 mm, 20 mm, 30 mm, 40 mm, 50 mm, or more, or any size in between. The width of the individual paths (p) controls the characteristics of the streamlines. Any number of paths, e.g., 1, 2, 4, 8, 12, 16, 32, etc., or any range in between, may be employed depending on the width of the chamber. In other embodiments, the flow rate of fluid through the chamber may be 1 mL/hr, 2 mL/hr, 3 mL/hr, 4 mL/hr, 5 mL/hr, 6 mL/hr, 7 mL/hr, 8 mL/hr, 9 mL/hr, 10 mL/hr, 15 mL/hr, 20 mL/hr, 25 mL/hr, 30 mL/hr, 40 mL/hr, 50 mL/hr, and so forth. The chamber is desirably rectangular but may also be circular, semi-circular, V-shaped, or any other appropriate shape.
Angle (θ) is the angle between the horizontal axis with zero slope and a line having a slope along which the posts are distributed. (In this example, the angle provides a measure of the negative slope (tan (θ)=Δy/Δx) of a row of posts.) In this example, it is understood that the posts may have a positive or negative slope, as each row of posts is arranged diagonally. Here, it is understood that diagonal may encompass any orientation that is not parallel or perpendicular to the chamber, e.g., an angle between 1 and 89 degrees, between 91 and 179 degrees, between 181 and 269 degrees, or between 271 and 359 degrees, clockwise or counter clockwise. In general, multiple rows of posts are present in a chamber, and each row has the same slope or substantially the same slope as defined by angle (θ). When cells 100 enter the chamber 105 of the microfluidics device, the cells flow through a plurality of paths (p) between the posts 115 in a lateral manner, towards a side of the chamber 105, until reaching a side of the chamber. For concentrating cells, the cells generally do not cross rows of posts, but rather, the cells typically travel along a particular path (p). The cells then exit the microfluidics chamber via output mechanism 122. In other embodiments, the posts may have a curvilinear component along the length of the chamber 105.
In some embodiments, the posts are arranged at an interval (dx) along a line having a slope, wherein the interval is regularly spaced or fixed. For example, a post may be placed every 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 85 μm, 90 μm, 95 μm, 100 μm, and so forth, including any value in between these ranges depending upon the size of the cell to be concentrated.
In other embodiments, the posts are distributed along a line having a slope, such that the interval is not fixed but instead varies between any two successive posts. Any interval (dx) is permitted, provided that the interval is small enough to keep the cell from escaping path (p). For example, for a variable interval, a first post may be placed at a particular location, the second post may be placed 5 μm from the first post, a third post may be placed 4 um from the second post, and so forth. The interval can be selected based on the size of the cell.
In some embodiments, the interval can be selected to be sufficiently large to allow concentration of a particular type of cell, while allowing smaller cells to exit via output mechanism 120. For example, a blood sample may comprise a plurality of different cell types of relatively small size, including red blood cells, neutrophils (e.g., 12-14 μm in diameter), eosinophils (e.g., 12-17 μm in diameter), basophils (e.g., 14-16 μm in diameter), lymphocytes (e.g., 10-14 μm in diameter), and monocytes (e.g., 20 μm in diameter). Other types of cells in the human body are much larger, e.g., ranging from 40 to 100 μm in diameter or more. In such cases, the posts may be spaced to allow red and white blood cells to pass through, while concentrating larger cells (e.g., normal cells, tumor cells, etc.). The configuration of the posts (spacing) is determined based upon the type of cell being isolated. Different sheath-to-sample flow ratios can affect size-based sorting performance.
Each post has an associated length (l), width (w), also referred to as in-plane dimensions and a height (h), also referred to as an out of plane dimension (see also
In the examples presented herein, the posts have an oval or rectangular shape. In some embodiments, the length of the post is 1.1 to 10 times greater than the width of the post; 1.1 to 5 times greater than the width of the post; 2 to 4 times greater than the width of the post; 3 to 4 times greater than the width of the post; or 2-3 times greater than the width of the post. In still other embodiments, the length and the width of each post in the matrix are the same. In yet other embodiments, if the posts are circular, the radius of each post in the matrix is the same.
Additional features include the rotation angle (ϕ) of the post, the height or the out of plane dimension of the post, spacing between the rows (dy), and offset of the rows (xo), which are described in additional detail in
Referring to
Referring to
As indicated previously, the posts are distributed along a line having a negative slope. By orienting the cells along this line, the cells flowing through path (p) are directed laterally, towards a side of the chamber, while the solution that the cells are originally suspended in is able to flow in a manner (e.g., near horizontally or horizontally) to exit the microfluidics chamber via output mechanism 120.
Referring to
In other embodiments, the posts are distributed along a vertical line, such that the interval (dy) is not fixed but instead varies between any two successive rows of posts. For example, a first row of posts may be placed at a particular location, the second row of posts may be placed 5 μm from the first row of posts, a third row of posts may be placed 4 μm from the second row of posts, and so forth, aligned with respect to a vertical line. The interval (dy) can be selected based on the size of the cell to be concentrated. Any interval (dy) is permitted, provided that the interval is sufficiently wide to allow flow of the solution through the chamber without requiring high pressures, which could damage the microfluidics device. In some embodiments, successive rows of posts may have a zero offset (xo).
Referring to
The posts may be made out of any suitable material, including poly(dimethylsiloxane) (PDMS), glass, plastic, elastomers, silicon, etc. PDMS in general can be fabricated to have sub-micron resolution (e.g., below 0.1 μm features). In some embodiments, the chamber 105 and/or posts 115 may be made of PDMS. In other embodiments, the chamber may be fabricated of glass and/or silicon and/or plastic, and the posts may be fabricated using PDMS (e.g., the bottom of the chamber may be glass or silicon and the top of the chamber may be glass or plastic). Lithography, which is well known in the art, may be utilized to fabricate the chambers and posts as described herein. Materials having similar properties to PDMS may also be used to construct the microfluidic devices set forth herein.
Additionally, specific embodiments may comprise a number of additional features, including valves (e.g., between an input mechanism and the chamber, between the chamber and an output mechanism, between an output mechanism and another input mechanism or another chamber, etc.), pumps and mixers. In some embodiments, valves can be placed into PDMS during curation.
A variety of techniques can be employed to fabricate a microfluidics device. The microfluidics device may be formed from one or more of the following materials: poly(methylmethacrylate) (PMMA), polycarbonate, polystyrene, polyethylene, polyolefins, silicones (e.g., poly(dimethylsiloxane) PDMS), silicon, and combinations thereof. Other materials are well known in the art.
Methods for fabricating a chamber with posts and a microfluidics device using the materials referenced herein are also known in the art and include but are not limited to embossing, laser micromachining, milling, molding (e.g., thermoplastic injection molding, or compression molding), photolithography (e.g., stereolithography or x-ray photolithography), silicon micromachining, wet or dry chemical etching, etc.
Silicon fabrication techniques using photolithography followed by wet (KOH) or dry etching (reactive ion etching with fluorine or other reactive gas) can be employed for glass materials. For example, a glass master can be formed by conventional photolithography, which serves as a master template for molding techniques to generate a plastic or PDMS-based device.
A microfluidics device may be fabricated in one or more layers that are joined together, e.g., by adhesives, clamps, heat, solvents, etc. Alternatively, the microfluidics device may be fabricated as a single piece, e.g., using stereolithography or other three-dimensional fabrication techniques.
In some embodiments, due to the deformability of cells, a gap distance may be selected that is about 5 μm or less as compared to the size (e.g., a diameter) of the cell being concentrated. In other embodiments, the gap distance may be selected that is about 5 μm or greater as compared to the size (e.g., a diameter) of the cell being sorted. For rigid cells or microspheres having the same or similar diameter as a deformable cell, the gap distance may be different (e.g., larger for rigid cells) as compared to the gap distance for deformable cells of the same diameter.
In some embodiments, additional simulations may be performed to confirm other types of configurations for different cells.
In this example, a second input mechanism 112 is present in this system. A second solution (e.g., a buffer, which is different from the first solution entering the chamber through the first input mechanism 110) enters the chamber through the second input mechanism 112. In some embodiments, the first solution and the second solution do not substantially mix, and thus, the upper region of the chamber 155 comprises the first solution and has little if any of the second solution, while the lower region of the chamber 165 comprises the second solution and has little if any of the first solution. Thus, as the cells are deflected laterally and exit the chamber through output mechanism 122, the solution that exits the chamber via output mechanism 122 is in the second solution buffer.
In some aspects, buffer changes may occur sequentially, in a cascade design, as shown in
In general, the input mechanism can be driven by a syringe pump or by manual depression of a syringe. In some aspects, for multiple inputs, one or more syringe pumps can be used to drive each input in an automated manner. In other aspects, for multiple inputs, manual depression of one or more syringes can be used to drive each input manually.
In this example, a second input mechanism 114 is present in this system. A second solution (e.g., which may or may not be different from the first solution and comprises molecules capable of binding to the cell surface (binding moieties) of the cells in the first solution, enters the chamber at input mechanism 114. In some embodiments, the binding moieties mix with the cells, and bind to the cells having suitable markers on their cell surface.
Additionally, a third input mechanism 112 is present in this system. A third solution (e.g., a buffer, which may or may not be different from the first solution entering the chamber through the first input mechanism 110 and may or may not be different from the second solution entering the chamber through the second input mechanism 114) enters the chamber through the third input mechanism 112. In some embodiments, the first/second solutions and the third solution do not substantially mix, and thus, the upper region of the chamber 155 comprises the first/second solutions and has little if any of the third solution, while the lower region of the chamber 165 comprises the third solution and has little if any of the first/second solutions. Thus, as the cells exit the chamber through output mechanism 122, the solution that exits the chamber via output mechanism 122 comprises the third buffer, enriched in cells bound to the binding molecule.
In general, the input mechanism can be driven by a syringe pump or by manual depression of a syringe. In some aspects, for multiple inputs, one or more syringe pumps can be used to drive each input in an automated manner. In other aspects, for multiple inputs, manual depression of one or more syringes can be used to drive each input manually.
A second solution (e.g., a buffer), which is different from the first solution entering the chamber through the first input mechanism 110(1), enters the chamber through the second input mechanism (112). In some embodiments, the first solution and the second solution do not substantially mix, and thus, the upper region of the chamber (155) comprises the first solution and has little if any of the second solution, while the lower region of the chamber (165) comprises the second solution and has little if any of the first solution. The cells (enriched and in the buffer from input mechanism 112) exit chamber 105(2) through output mechanism 122(2).
Once in the second chamber 105(2), the cells are concentrated by the matrix of posts 115 as described herein. Upon exiting chamber 105(2), the cells are directed towards a region that comes into contact with laser 200, where cells bound to the marker are killed upon illumination by the laser (e.g., by 6 mJ, 45 Hz, 6 ml/hr). Any suitable tag that is activated by a laser (e.g., a tag activated into a form capable of killing a cell, such as heat generation, energy dissipation, cleavage into a toxic form, etc.) can be used to kill cells to which the tag is bound. In one embodiment, gold (Au) nanorods, nanocages, or other nanoparticles are adsorbed onto IgG antibodies that are specific to a type of cell in the solution. Once the cells are illuminated by the laser, the Au particles heat up, and kill the cells to which the gold labeled antibodies are attached. The dead cells are separated from living cells, by utilizing dielectrophoresis (see, Pohl H. A. et al., Science (1966), 152(3722): 647). Dead cells exit at output mechanism (126), and the live cells exit at output mechanism (128).
The techniques presented herein can be combined with other protocols to isolate particular cells from a biological sample. For example, in one embodiment, a blood sample is obtained, mixed with one or more gold labeled antibodies that recognize particular cell surface markers (e.g., CD2, CD14, CD19, CD45, CD61 CD66b, Glycoprotein A, etc.) The mixture may be injected into the system of
The techniques presented herein may also be used with cells that are stimulated by an antigen. Antigens include but are not limited to CMV pp65, OKT3, etc. The techniques presented herein may also be used with any cell type that underdoes an increase in size upon stimulation, e.g., by an antigen, by an overexpressed protein, or other growth/size enhancing factor.
A chart providing approximate ranges of gap distances and flow rates for separating cells of various sizes is presented. It will be appreciated that cell diameters typically vary within a population of cells. Accordingly, gap distances similar to the values provided below may also be used to separate cells of a given size.
In another aspect, the cell sorting and concentrating device can be additionally configured to have chambers in the floor of the device, positioned between the sorting mechanism and the chamber exit. This configuration traps cells in the floor of the device. In other embodiments, the flow rate is adjusted to trap cells in the gap between posts, allowing smaller cells to pass through. For cells that become trapped, the flow rate may be increased to dislodge the trapped cells.
In still other aspects of the techniques presented herein, the chamber may be used to concentrate cells appearing in urine. For example, bladder cancer cells frequently are shed, and therefore, may be present in urine. The chambers described herein may concentrate such cells.
In other aspects, the techniques presented herein may utilize capture antibodies bound to the posts to remove specific cells (e.g., CTCs) from the sample, or alternatively, may be used to remove populations of cells that are to be excluded from processing.
It is understood that the device may perform any of the functions described herein, including cell concentration, cell sorting, buffer changes, etc.
In another aspect, the device can be used as a preprocessing step to select cells for transfection, e.g., to improve transfection yields. In this example, larger active cells, e.g., undergoing cell division, are selected based on larger size, and the collected larger cells are used for transfection.
In another aspect the device can be used to identify metastatic cells. Some metastatic cells become softer than their non-cancerous counterparts, and therefore, are able to deform through smaller gap distances. By adjusting the gap distance and the flow rate, conditions can be established to allow metastatic cells to become separated from normal cells. Normal cells that are more rigid are not able to pass through smaller gap distances are not collected.
It is noted that other types of cancers, such as breast cancer, may lead to an increase in cell stiffness or viscoelasticity. In this case, the flow rate may be established to allow normal cells to deform through a smaller gap distance, while more rigid cells would not be able to pass through.
In yet another aspect, the present techniques may be used to collect cells while harvesting supernatant. For instance, during large scale protein production processes, cells may be cultivated to express a therapeutic monoclonal antibody or other biologic. The techniques disclosed herein provide a way in which to quickly collect these cells, while collecting supernatant containing the expressed antibody, without a centrifugation or filtration step.
In other aspect, one or more microfluidics chambers may be configured to isolate cells having a range of sizes. For example, by configuring two chambers in a serial orientation, such that the first chamber has a cut off target of 8 μm, and the second chamber has a cutoff target of 10 μm, cells having sizes in between these ranges can be targeted for collection.
In another aspect, the optimal cutoff size for a particular type of cell can be determined by generating a probability cutoff curve, as shown in
In another aspect, PDMS or any material sufficiently similar to PDMS may be used to construct part or all of the chamber as described herein.
The methods described herein may employ only one of the above steps or any combination of the above steps, in any order, to concentrate and/or separate cells. The methods described herein may recover at least 75%, 80%, 90%, 95%, 98%, or 99% of the desired cells in the sample.
In another aspect, the microfluidics chambers described herein may be used to isolate, perform buffer changes, and/or concentrate cancer cells that are larger than other types of cells within a sample. In other embodiments, the microfluidics chambers described herein may be used to concentrate or perform buffer changes of cancer cells within a population of noncancerous cells of about the same size. The cancer cells may be isolated in a subsequent (secondary) sorting step, e.g., using staining specific to cancer cells, antibodies specific to cancer cells, etc.
It should be apparent to those skilled in the art that many more modifications besides those already described are possible. The systems, methods and devices disclosed herein are not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
Cells may be obtained from a biopsy, surgical resection, or from a blood draw. Cells may be collected and stored according to standard techniques known in the art.
In some embodiments, fine needle aspiration is performed using a sharp 25-gauge long needle (e.g., from Becton Dickinson). The length of the needle is selected based upon the location of the tumor within the patient. The needle is attached to flexible plastic tubing, which is connected to an aspirating syringe. The tip of the needle is advanced into the tumor, and aspiration is used to collect a sample of tumor cells. The procedure is monitored using imaging, e.g., sonography, or any other suitable technology.
Each sample is placed into a tube or flask with suitable cell culture media for mammalian cells (e.g., 5% fetal bovine serum (FBS); a fungicide and/or bacteriocide/antibiotic). Cells are cultured as a monolayer, e.g., in 6 well plates, grown to confluence, e.g., for 2 weeks or until reaching a suitable cell density. The cultured cells are then collected, optionally undergo a wash, and are centrifuged into a pellet. The cells are resuspended and stored in liquid nitrogen for future use.
In other embodiments, a blood sample may be obtained from a patient and mixed with an isotonic saline solution, according to known techniques in the art.
Unless indicated otherwise, devices were fabricated using standard soft lithography techniques known in the art.
Soft lithography may be used to generate polydimethylsiloxane (PDMS) structures described herein. In general, a mold (master) is produced by photolithography, wherein the mold represents the empty spaces of the microfluidic chamber. In this example, the master is solid for regions in which fluid will flow, and is empty in regions where posts are present. For example, photolithography techniques may be found in, e.g., U.S. Pat. No. 8,372,579.
PDMS is formed by mixing a component containing silicon hydride with another component containing a vinyl group. This mixture is poured into the master mold, and is allowed to cure, e.g., for an hour or two at 70° C. Once solidified, the PDMS structure can be peeled from the master mold (while remaining intact), and the master mold can be used again for making additional PDMS molds.
After removing the PDMS structure from the master, the PDMS structure is sealed to another surface, e.g., glass, silicon, or PDMS, in a reversible or irreversible manner. In some embodiments, adhesive silicon and/or cellophane tape can be used in conjunction with PDMS to create reversible seals, suitable for lower pressures. Higher pressures may necessitate an irreversible seal, formed by exposing PDMS to oxygen plasma. Alternatively, PDMS can be sealed with the aid of polar solvents, wherein a thin film of a polar solvent is placed between PDMS layers and then heated, evaporating the solvent and sealing the layers.
In other embodiments, more complex structures can be created by “sandwiching” or stacking multiple layers of PDMS. In such embodiments, micro-stages are utilized to ensure perfect alignment after oxidation. These techniques are known in the art.
Alternatively, 3D printing techniques can also be used to generate a master mold. This technology prints small amounts of a material (e.g., silicon, etc.), as multiple layers in an additive manner (as represented by a CAD file) to generate a three dimensional structure.
3D printing permits rapid prototyping, and can achieve a resolution suitable for some microfluidic devices. In this example, the master would then be used in the same way as in the previous example to create PDMS chambers.
Alternatively, a CAD file could be utilized to directly print the chamber structure (instead of the inverse structure). The structure could then be coated with PDMS to have desired surface properties. (Waheed, et al., Lab Chip (2016) 16:1993-2013.)
These examples are purely intended to be exemplary and are not intended to be limiting, as numerous different embodiments are understood to fall within the scope of the systems, methods, and devices disclosed herein.
In one embodiment, a biological sample comprising cells was obtained. The sample was loaded into a syringe, which was connected by tubing to an input mechanism 110 of the microfluidics device (see, e.g.,
In another embodiment, a biological sample was obtained. The sample was loaded into a syringe, which was connected by tubing to an input mechanism of the microfluidics device (e.g., an input port in fluid communication with a chamber) (see, e.g.,
In general, the input mechanism can be driven by a syringe pump or by manual depression of a syringe. In some aspects, for multiple inputs, one or more syringe pumps can be used to drive each input. In other aspects, for multiple inputs, manual depression of one or more syringes can be used to drive each input.
In still another embodiment, a biological sample (e.g., a blood sample) is obtained from a patient. The sample is diluted with sodium chloride isotonic saline and loaded into a syringe, which is connected by tubing to an input mechanism of the microfluidics device (e.g., an input port in fluid communication with a chamber) (see,
In general, the input mechanism can be driven by a syringe pump or by manual depression of a syringe. In some aspects, for multiple inputs, one or more syringe pumps can be used to drive each input. In other aspects, for multiple inputs, manual depression of one or more syringes can be used to drive each input.
In another embodiment, a biological sample (e.g., a blood sample) is obtained from a patient. The sample is diluted with sodium chloride isotonic saline and loaded into a syringe, which is connected by tubing to an input mechanism of the microfluidics device (e.g., an input port in fluid communication with a chamber) (
In another embodiment, a biological sample (e.g., a blood sample) is obtained from a patient. The sample is diluted with sodium chloride isotonic saline and loaded into a syringe, which is connected by tubing to an input mechanism of the microfluidics device (e.g., an input port in fluid communication with a chamber) (
Once in the second chamber 105(2), the cells are concentrated by the matrix of posts 115 as described herein. Upon exiting chamber 105(2), the cells are directed towards a region that comes into contact with laser 200, where cells bound to the marker are killed upon illumination by the laser (e.g., by 6 mJ, 45 Hz, 6 ml/hr). Any suitable tag that is activated by a laser (e.g., a tag activated into a form capable of killing a cell, such as heat generation, energy dissipation, cleavage into a toxic form, etc.) can be used to kill cells to which the tag is bound. In one embodiment, gold (Au) nanorods, nanocages, or other nanoparticles are adsorbed onto IgG antibodies that are specific to a type of cell in the solution. Once the cells are illuminated by the laser, the Au particles heat up, and kill the cells to which the gold labeled antibodies are attached. The dead cells are separated from living cells, by utilizing dielectrophoresis (see, Pohl H. A. et al., Science (1966), 152(3722): 647). Dead cells exit at output mechanism (126), and the live cells exit at output mechanism (128).
For the following examples and figures involving cell sorting by FACS, bounding boxes (e.g., black polygons) may be determined empirically.
According to aspects presented herein, the microfluidics device can also be used for cell sorting, e.g., to enrich T-cells that have been activated from binding to a specific antigen or from addition of a substance that causes activation of the T cells.
In present studies, as shown in
In some aspects, gap distances can be selected based upon the size and the viscoelastic nature of the cell. In general, cells are deformable, and can deform about 2-3 μm on each side. Thus, to select for an 8 μm cell, which deforms about 2 μm on each side, a 4 μm gap distance may be used. This same trend may hold for cells of larger sizes, e.g., a 6 μm cut off for a 10 μm cell, and a 9 μm cutoff for a 13 μm cell. In some instances, larger cells can deform through smaller gaps, and in this case, the flow rate can be adjusted upwards to reduce this occurrence.
In some aspects, the posts may be coated with reagents that cause the cells to bind to the posts, e.g., antibodies, antigens, etc. In this example, the antigen or antibody inhibits cell movement through the chamber, and the cells can be separated based on size and speed of migration through the chamber.
For the following examples and figures involving cell sorting by FACS, bounding boxes (e.g., black polygons shown in
Referring to
In this example, a solution comprising CD25+ cells (activated T cells) was fed into the chamber after being spiked with between 1 and 10% CD25+ activated T cells. The largest gap, the 9 μm gap, provided the highest enrichment percentage after sorting. For the 9 μm gap, the results showed about 90% after 1 round of sorting and 94% after 2 rounds of sorting. The enrichment factor was determined by quantifying the % of cells expressing the activation marker CD25+ divided by initial mix of CD25+.
Referring to
Referring to
In this series of examples, T cells were stimulated in an antigen-specific manner prior to cell sorting. OKT3, which broadly stimulates T cells, was used as a positive control. Antigen-specific stimulation was performed using pp65. In some embodiments, a pp65 peptide pool, e.g., comprising a plurality of overlapping peptides spanning the full length pp65 protein sequence, was used to stimulate T cells. In other embodiments, a specific antigen corresponding to residues 495-503 of the pp65 protein, pp65495-503, was used to stimulate T cells.
In general, the pp65 peptide pool resulted in a higher frequency of stimulated T cells as compared to the pp65495-503 antigen, as the pp65 peptide pool comprises a plurality of different types of antigens capable of stimulating a range of T cells. In contrast, a single type of antigen, pp65495-503, stimulates T cells specific to this antigen, leading to a lower frequency of simulated T cells.
PBMC cells, a mixture of T cells, B cells, and monocytes, were obtained from a donor reactive to the pp65 antigen. In particular, PBMC cells, e.g., from a HLA-A2, CMV-seropositive donor LP381 (a normal response), were thawed. Cells were divided into aliquots containing about 8×106 cells. The aliquots were cultured under various conditions including: (1) in the presence of media only (no stimulation); (2) in the presence of OKT3 (stimulation); (3) in the presence of pp65 peptide pool (stimulation); and (4) in the presence of pp65495-503 (stimulation). On the third day after stimulation, cells from each condition were sorted based on size using a 9 μm gap distance and a Qtotal=10 ml/hr and a sample:sheath ratio of about 1:1. On the fourth day after stimulation, the third and fourth conditions were sorted based on size using a 9 μm gap distance and a Qtotal=10 ml/hr with a sample:sheath ratio of 1:1 or Qtotal=20 ml/hr with a sample:sheath ratio of 4:1. After sorting, the samples were analyzed by flow cytometry (FACS) to record cell counts including FSC, SSC, CD3, CD25, and CMV dextramer (pp65495-503) for the following three time points: before sorting, after sorting (collection), and after sorting (waste).
Monocytes and activated T cells (CD3+CD25+ cells) were frequently isolated together, as monocytes are typically larger than lymphocytes (B cells and T cells). When activated T cells (based on CD25+) were gated (sorted) on all live cells, the percentage of collected activated T cells (purity) was lower due to the presence of monocytes. When activated T cells were gated on CD3+ cells, the percentage of collected activated T cells (purity) was higher, in some cases reaching greater than 90% for peptide pool stimulation.
The percentage of CD25+ activated T cells among live cells (composed of all live PBMC cells including T cells (majority), B cells, monocytes, CD3− lymphocytes, etc.) was calculated. The percentage of CD25+ cells among T cells composed of CD3+ cells (e.g., CD3+CD25− and CD3+CD25+ cells) was also calculated. The percentage of CD25+ cells when gated only on CD3+ T cells was higher (as other cell types were excluded) as compared to gating on live cells.
The enrichment factor is the fold increase in CD25+ percentage (%) after sorting, and was calculated by dividing the percentage of collected CD25+ cells by unsorted cells. For example, and with reference to
The cell sorting assays provided herein may be combined with one or more other assays to isolate cells of interest, e.g., using staining, antibodies, etc. After sorting, the antigen stimulated T cells (CD25+CD3+) were shown to be enriched as compared to their non-sorted counterparts.
Monocytes, which are about the same size as activated T cells, may also appear in the collected fraction, while smaller cells, e.g., CD3+CD25− cells and CD3− lymphocytes are typically excluded.
Referring to
In general, any suitable material may be used to form the posts within the microchannel. Preferably, the material will be gas permeable, e.g., PDMS, to allow trapped gas to escape from the microfluidics channel, and to prevent trapped air bubbles from affecting the flow dynamics within the microfluidics channel. The microfluidics channel and associated tubing may be filled with a suitable buffer and flushed with excess buffer, to allow trapped air bubbles to become dislodged and diffuse through the PDMS out of the microfluidics channel or to exit via tubing connected to the microfluidics channel. In some embodiments, surfactants may be added to the buffer to reduce the occurrence of gas bubbles.
This application claims priority under 35 U.S.C. § 119(e) from U.S. Provisional Patent Application Ser. No. 62/616,126, filed Jan. 11, 2018, the entire contents of which are hereby incorporated by reference
| Number | Date | Country | |
|---|---|---|---|
| 62616126 | Jan 2018 | US |
