Patent Application
20240094106
Rapid and label-free separation of target cells in a large volume of biological samples provides unique opportunities for disease diagnostics and treatment. The ability to separate and enrich populations of scarce target cells, such as rare circulating tumor cells (CTCs), can provide valuable research tools and insights into diseases such as metastatic cancers. Other target cells, such as T lymphocytes, provide valuable candidate therapeutics for treatments such as immunotherapies. The limited availability and difficulty of obtaining useful amounts of rare CTCs is a hurdle in such research. Similarly, the ability to quickly purify sufficient amounts of T lymphocytes hinders the availability of advanced immunotherapy treatments such as adoptive cell transfer (ACT) immunotherapy. However, even with advanced technologies for cell separation, the limiting throughput, high cost and low separation resolution of currently available cell separation platforms still prevents the effectiveness of such technologies in processing large volumes of biological samples.
According to various aspects, the present disclosure provides microfluidic devices, kits, and methods for separating and/or enriching cells and/or micro-particles in samples. The devices, kits and methods combine inertial focusing and ferrohydrodynamic separation to achieve size-based separation of particles in a sample with high resolution and a at thigh throughputs.
Embodiments of multi-stage microfluidic devices provided in the present disclosure for separating cells and/or micro-particles in a sample include a first microfluidic channel, an inertial focusing stage, at least two sheathing fluid channels, a ferrohydrodynamic separation stage, a magnetic source, and three or more outlets. The first microfluidic channel has a first and second end, and there is a first fluid inlet at the first end of the microfluidic channel and configured to receive a fluid sample comprising the sample combined with a ferrofluid. In embodiments, the inertial focusing stage is at the second end of the first microfluidic channel, wherein the first microfluidic channel splits into two or more serpentine focusing channels at a first end of the inertial focusing stage, each serpentine focusing channel having a plurality of alternating micro-curves configured to focus cells/particles within the sample into a narrow stream to produce a focused fluid sample stream and wherein the two or more serpentine focusing channels form a convergence at a second end of the inertial focusing stage. The at least two sheathing fluid channels are fluidly connected to one or more sheathing fluid inlets, the sheathing fluid channels configured such that the convergence of the serpentine focusing channels is between the at least two sheathing fluid channels. The ferrohydrodynamic separation stage is at the second end of the inertial focusing stage, wherein the convergence of the serpentine focusing channels further converges with the at least two sheathing fluid channels at a first end of the ferrohydrodynamic separation stage to form a ferrohydrodynamic separation channel configured such that the focused fluid sample stream exiting the convergence of the focusing channels enters the ferrohydrodynamic separation channel in a central portion of the ferrohydrodynamic separation channel and a sheathing ferrofluid exiting the sheathing fluid channels enters the ferrohydrodynamic separation channel on the periphery of the ferrohydrodynamic separation channel serving to further narrow the focused fluid sample stream and adjust its starting position in the ferrohydrodynamic separation channel. The magnetic source in the ferrohydrodynamic separation stage can be configured produce a substantially symmetric magnetic field having a field maximum along an inner longitudinal axis of the ferrohydrodynamic separation channel sufficient to cause cells/particles flowing in the ferrohydrodynamic separation channel to be deflected away from the center of the ferrohydrodynamic separation channel towards the sides of the ferrohydrodynamic separation channel as a function of the size of the cells/particles. In embodiments, the device also has three or more outlets at a second end of the ferrohydrodynamic separation stage, each outlet positioned to receive cells/particles in fluid flowing along a different portion of the ferrohydrodynamic separation channel from each of the other outlets such that cells/particles in the sample fluid are separated by size.
The present disclosure also provides kits for enriching and/or sorting unlabeled, microparticles in a fluid sample. In embodiments, the kit includes a multi-stage microfluidic device of the present disclosure and a superparamagnetic composition. The superparamagnetic composition can include a plurality of magnetic nanoparticles and a surfactant. The superparamagnetic composition is adapted to be combined with a carrier fluid to make a superparamagnetic fluid, wherein the superparamagnetic fluid can be the ferrofluid, sheathing ferrofluid, or both, for use in the multi-stage microfluidic device of the kit.
Methods of enriching and/or separating unlabeled, microparticles in a sample comprising a plurality of components are also provided in the present disclosure. In embodiments, methods of the present disclosure for enriching/separating unlabeled, microparticles in a sample include first introducing a sample fluid comprising the sample with the unlabeled, microparticles and a first ferrofluid into the first fluid inlet of a multi-stage microfluidic device for the present disclosure at a first flow rate. Next, the method includes, flowing the fluid sample through the inertial focusing stage and focusing the microparticles in the fluid sample into a focused fluid sample stream and then combining the focused fluid sample stream from the inertial focusing stage with a sheathing ferrofluid at the first end of ferrohydrodynamic separation stage such that the sheathing ferrofluid serves to further narrow the focused fluid sample stream of microparticles in the fluid sample. The focused fluid sample stream of microparticles is then flowed in the ferrohydrodynamic separation channel such that the substantially symmetric magnetic field produced by the magnetic force hydrodynamically causes cells/particles flowing in the channel to be focused away from the center of the channel towards the sides of the channel as a function of the size of the particles, such that larger particles move further toward the sides of the channel than smaller particles. The separated particles can then be collected from the at least 3 outlets.
Other systems, methods, features, and advantages of the present disclosure will be or will become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.
Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, material science, cellular biology, microfluidics, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20-25° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications and patents that are incorporated by reference, where noted, are incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. Any terms not specifically defined within the instant application, including terms of art, are interpreted as would be understood by one of ordinary skill in the relevant art; thus, is not intended for any such terms to be defined by a lexicographical definition in any cited art, whether or not incorporated by reference herein, including but not limited to, published patents and patent applications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.
The articles “a” and “an,” as used herein, mean one or more when applied to any feature in embodiments of the present invention described in the specification and claims. The use of “a” and “an” does not limit the meaning to a single feature unless such a limit is specifically stated. The article “the” preceding singular or plural nouns or noun phrases denotes a particular specified feature or particular specified features and may have a singular or plural connotation depending upon the context in which it is used.
As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
In this disclosure, “consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. “Consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
As used herein, “about,” “approximately,” “substantially,” and the like, when used in connection with a numerical variable, can generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within +/−10% of the indicated value, whichever is greater. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
As used herein, the terms “optional” or “optionally” indicates that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
As used herein, “kit” refers to a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
As used herein, “instruction(s)” refers to documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents and are meant to include future updates.
As used herein, the term “biocompatible,” with respect to a substance or fluid described herein, indicates that the substance or fluid does not adversely affect the short-term viability or long-term proliferation of a target biological particle within a particular time range.
“Curved” or “curve,” as described herein, indicates a non-linear shape, where curved can include a single curve, multiple curves, and multi-directional curves, including crescent-shaped, U-shaped, serpentine, sigmoidal, and the like.
As used herein, the term “cells/particles” refers to particles, cells, or a combination of both, in a sample or mixture.
In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, embodiments of the present disclosure, in some aspects, relate to devices, kits, and methods for label-free separation of cells and/or other small particles with high throughput resolution, and the like. Devices, kits, and methods of the present disclosure provide for inertial based focusing of cells/particles in a sample in a focusing stage and then ferrohydrodynamic, size-based separation in a separation stage. Embodiments of such devices, kits and methods facilitate/provide the ability to separate and enrich target cells/particles from a sample with high resolution and efficiency.
High-throughput and high-resolution separation of target cells in a label-free manner from a large volume of biological samples has increasingly found applications in both fundamental biological research and clinical assays.1-3 These target cells could harbor important information about diseases, as in the case of rare circulating tumor cells (CTCs) and metastatic cancers,4-9 or could be candidates for potent therapeutic cells, ss in the case of T lymphocytes in cancer immunotherapies.10-12 For example, CTCs are cancer cells that detached from primary tumors and are carried through the vasculature to potentially seed distant site metastases in vital organs. Increased knowledge about CTCs has significant implications in cancer research and clinical utilities of diagnosing and treating cancers.4-9. However, one major bottleneck of CTC research has been the limited availability of CTCs for investigations, due to their rarity as well as physical and biological heterogeneities in blood circulation, which typically yield only about 1-10 CTCs from one milliliter of human whole blood. The scarcity and heterogeneity of CTCs highlighted a need for new cell separation methods that could quickly enrich CTCs from a large quantity of contaminating blood cells (˜107-108 white blood cells) in a clinically relevant amount of blood (˜10 milliliters).
Another area that could potentially benefit from high-throughput and high-resolution cell separation devices and methods is adoptive cell transfer (ACT) immunotherapy, in which T lymphocytes are purified, genetically modified, and infused into cancer patients to mediate anti-tumor effects.10-12. One major bottleneck and a significant contributor to the high price point of ACT has been the cost associated with the purification of T lymphocytes from concentrated samples of human white blood cells (WBCs), which includes ˜109 WBCs and other blood components including platelets and red blood cells.13 In order to derive potent therapeutic cells in ACT, high-purity T lymphocyte separation from WBCs has become critical, which in turn spurred the development of high-throughput and high-resolution cell separation methods that can enrich a large quantities of T lymphocytes from concentrated WBCs in a low-cost manner.
Current cell separation platforms haven't met the needs for the above-mentioned applications because they faced challenges including high cost, low separation resolution and limited sample processing volume. Traditionally, target cells were separated from contaminating cells using methods including magnetically activated cell sorting (MACS) or fluorescently activated cell sorting (FACS). MACS was a label-based method that relied on the interaction between magnetic beads and surface antigens of target or contaminating cells.14 This method had high costs due to the use of expensive equipment, antibodies and magnetic beads. On the other hand, the throughput of the FACS method was limited to ˜103 cells/s,15 far below the needed throughput (˜105 cells/s) in separation of CTC or lymphocytes (e.g., B or T lymphocytes) from blood samples, often loses as much as 50% or more of cells, and also requires expensive equipment. A majority of existing microfluidic cell separation methods, despite their precision, also suffered from low throughput as they typically were limited to processing only small amounts of biological sample in the range of microliters to a few milliliters (see Example 2, below, for comparison).16-21 Recently developed inertial force based microfluidic systems, resulted in focusing and separation of biological particles with extremely high throughputs (>105 cells/s), but suffered from low separation resolution resulting in low target cell recovery.22-27 These challenges in currently available methods highlighted an urgent need to develop a high-throughput and high resolution method that can separate target cells from a large volume of biological samples in a low-cost manner.
To address this need, the present disclosure provides a label-free, inertial-ferrohydrodynamic cell separation (inertial-FCS) method that integrates inertial focusing and ferrohydrodynamic separation of cells according to their physical size/diameters. Briefly described, first a sample (e.g., lysed blood sample including target cells/particles and non-target cells/particles) is combined with a ferrofluid to produce a fluid sample, which is then introduced into the device of the present disclosure. The fluid sample passes through an inertial focusing stage which functions to focus cells/particles within the sample into a narrow stream. The focused fluid sample is then combined with a sheathing fluid (e.g., additional ferrofluid) as it enters a ferrohydrodynamic separation stage. The addition of the sheathing fluid produces sheath flow which further focuses the cell stream and adjusts the starting position of the stream in the ferrohydrodynamic separation channel, where cells/particles in the focused stream from the focusing stage are then separated based on their volumes under a magnetic field as a function of their size-based magnetic buoyancy.
Embodiments, of multi-stage microfluidic devices of the present disclosure for separating cells/particles in a sample can include an inertial focusing stage, a ferrohydrodynamic separation stage, a magnetic source, and outlets as described below. In embodiments, the inertial focusing stage includes two or more serpentine focusing channels at a first end of the inertial focusing stage, with each channel having a plurality of alternating micro-curves configured to focus cells/particles within the sample into a narrow stream to produce a focused fluid sample stream. In embodiments, the two or more serpentine focusing channels form a convergence at a second end of the inertial focusing stage. The ferrohydrodynamic separation stage is at the second end of the inertial focusing stage, where the convergence of the serpentine focusing channels further converges with at least two sheathing fluid channels to form a ferrohydrodynamic separation channel. In embodiments, this area is configured such that the focused fluid sample stream exiting the convergence of the focusing channels enters the ferrohydrodynamic separation channel in a central portion of the ferrohydrodynamic separation channel and a sheathing ferrofluid exiting the sheathing fluid channels enters the ferrohydrodynamic separation channel on the periphery of the ferrohydrodynamic separation channel. This configuration serves to further narrow the focused fluid sample stream and adjust its starting position in the ferrohydrodynamic separation channel. Devices of the present disclosure also include a magnetic source in the ferrohydrodynamic separation stage configured produce a substantially symmetric magnetic field having a field maximum along an inner longitudinal axis of the ferrohydrodynamic separation channel. This magnetic field causes cells/particles flowing in the ferrohydrodynamic separation channel to be deflected away from the center of the ferrohydrodynamic separation channel towards the sides of the ferrohydrodynamic separation channel as a function of the size of the cells/particles. At the second end of the ferrohydrodynamic separation stage, devices of the present disclosure include outlets (e.g., 3 or more). The outlets are positioned to receive cells/particles in fluid flowing along a different portion of the ferrohydrodynamic separation channel from each of the other outlets such that cells/particles in the sample fluid are separated by size. Additional description of these and other features of devices of the present disclosure are provided below.
In embodiments of devices and methods described in greater detail below and illustrated in
In the inertial focusing stage, the first microfluidic channel splits into two or more serpentine channels having multiple alternating micro-curves which serve to focus cells/particles within the sample into a narrow stream. In embodiments, such as depicted in
In embodiments, such as illustrated in
In embodiments the overall inertial focusing stage (e.g., the combined serpentine inertial focusing channels and any areas between sections of micro-curves) can include 1 or more larger bends/curves (e.g., in an L or U-shaped configuration such as illustrated in
In embodiments of the microfluidic separation device of the present disclosure, as illustrated in
The sheathing fluid channels are fluidly connected to one or more sheathing fluid inlets (identified as “Inlet B-sheath flow” in the embodiment illustrated in
The ferrohydrodynamic separation stage also includes a magnetic source configured to produce a substantially symmetric magnetic field having a field maximum along an inner longitudinal axis of the ferrohydrodynamic separation channel sufficient to cause cells/particles to be focused away from the center of the channel towards the sides of the channel as a function of the size of the particles. In embodiments the device is configured such that the magnetic field causes larger cells/particles to be focused further towards the sides/periphery of the channel, such as illustrated in
In embodiments, the magnetic source is provided by an array of magnets including a top array and bottom array, wherein the ferrohydrodynamic separation stage is sandwiched between and substantially centrally aligned between the top magnet array and the bottom magnet array, wherein the magnets in the top array are oriented to repel the magnets in the bottom array. In embodiments, the magnetic source is an arrangement of magnets such as illustrated in
After the ferrohydrodynamic separation stage, the device includes three or more outlets fluidly connected to an end of the ferrohydrodynamic separation channel (see reference number (6) of
The ferrofluid and sheathing ferrofluid each include a plurality of magnetic microparticles, a surfactant, and a carrier fluid. In embodiments, the ferrofluid and sheathing ferrofluid can have the same makeup, in other words they can be the same fluid but are introduced at different points of the device as described above. In embodiments the ferrofluid/sheathing ferrofluid is a biocompatible superparamagnetic fluid including magnetic nanoparticles, a biocompatible surfactant, and a biocompatible carrier fluid. In embodiments, the surfactant improves the biocompatibility of the magnetic nanoparticles. In embodiments, the ferrofluid/sheathing ferrofluid has concentration of magnetic nanoparticles of about 0.001-1% (v/v), such as about 0.01-1% (v/v) and other intervening ranges.
In embodiments, the devices of the present disclosure can be used to separate circulating tumor cells, lymphocytes or other target cells from white blood cells and other non-target/contaminating cells in a sample, such as a blood sample. As described in greater detail in the discussion and examples below, the devices, kits, and methods of the present disclosure can separate target cells from contaminating cells with a high throughput (e.g., about 104 cells/s to 2×105 cells/s, such as, for example, 105 cells/s), a high sample processing flow rate (e.g., about 200-1400 μL/min such as, for example, 60 mL/h (1000 μL/min) and a high separation resolution (e.g., about 0.5-2 μm, such as, for example, 1-2 μm) in cellular diameter difference. Due to its label-free nature, this method didn't require the use of antibodies and magnetic beads and could lower the cost of cell separations.
The present disclosure also provides kits for enriching and/or sorting unlabeled, microparticles in a fluid sample, where the kits include a multi-stage microfluidic focusing and separation device of the present disclosure along with a superparamagnetic composition for making the ferrofluid/sheathing ferrofluid for use in the device. In embodiments the superparamagnetic composition includes a plurality of magnetic nanoparticles and a surfactant. The superparamagnetic composition is adapted to be combined with a carrier fluid to make a superparamagnetic fluid that can be the ferrofluid, sheathing ferrofluid, or both, for use in the multi-stage microfluidic device. In embodiments, kits of the present disclosure can also include instructions for combining the magnetic nanoparticles, surfactant, and carrier fluid to make the superparamagnetic fluid and instructions for using the superparamagnetic fluid and the multi-stage microfluidic device to separate cells/particles in a fluid sample. Instructions can also include information such as desired flow rates, concentration of ferrofluids/sheathing fluids, and the like.
In embodiments of kits of the present disclosure, the surfactant is biocompatible such that it renders the superparamagnetic composition biocompatible for use with samples that include cells, other biological particles, or both. In such embodiments the kit may also include instructions for combining the superparamagnetic composition with a biocompatible carrier fluid. In embodiments, the ferrofluid and the sheathing ferrofluid have a concentration of magnetic nanoparticles of about 0.001-1% (v/v).
The present disclosure also provides methods for enriching and/or separating microparticles in a sample that includes plurality of components of different sizes. For instance, methods of the present disclosure can be used to separate particles (e.g., cells and other particles) from each other based on the size of the target cells/particles. In embodiments, the cells/particles (both target and/or non-target cells/particles can be unlabeled, since labeling is not needed for separation with the devices and methods of the present disclosure. In some of the examples below, the cells/particles may be labeled for purposes of visualization of particle streams for demonstration and/or for identification of cell/particle type after testing; however, such labeling is for verification/demonstration and is not needed for cell separation as in other methods.
According to some embodiments, methods of the present disclosure include providing a sample fluid including a sample with unlabeled microparticles (e.g., cells or other microparticles) and a first ferrofluid. Then, the method includes using inertial focusing forces to focus the microparticles in the fluid sample into a focused fluid sample stream. Next the focused fluid sample stream is combined with a sheathing ferrofluid such that the sheathing ferrofluid serves to further narrow the focused fluid sample stream of microparticles in the fluid sample (and to also adjust the location of the focused fluid sample stream, e.g., in a device). Then methods of the present disclosure include flowing the focused fluid sample stream of microparticles through a substantially symmetric magnetic field such that the substantially symmetric magnetic field hydrodynamically causes microparticles flowing in the focused fluid sample stream to move relative to a center of the stream as a function of the size of the microparticles, such that larger microparticles are deflected further away from the center of the stream than smaller microparticles. Then the separated microparticles can be collected (e.g., in outlets) in groups based on location of the microparticles relative to the center of the stream. The groups of separated microparticles will have different sizes depending on the location since the location at collection was based on deflection as a function of the size of the microparticles.
In embodiments of methods of the present disclosure, a sample fluid (e.g., whole blood, lysed blood, other biological fluids, etc.) and a first ferrofluid is introduced into a multi-stage microfluidic separation device of the present disclosure. The sample fluid includes microparticles (e.g., unlabeled microparticles), and the sample fluid can be combined with the first ferrofluid prior to or at the time of introduction to the device, this mixed fluid sample/ferrofluid is also referred to as simply the “fluid sample”. The fluids are introduced at the first fluid inlet of the device at a first flow rate. Then the fluid sample is flowed through the inertial focusing stage where the microparticles in the fluid sample are focused into a focused fluid sample stream. The focused fluid sample stream from the inertial focusing stage is then combined with a sheathing ferrofluid at the first end of ferrohydrodynamic separation stage such that the sheathing ferrofluid serves to further narrow the focused fluid sample stream of microparticles in the fluid sample and adjust the starting positions of the focused fluid sample stream in the channel. This focused fluid sample stream of microparticles is then flowed through the ferrohydrodynamic separation channel such that the substantially symmetric magnetic field produced by the magnetic force hydrodynamically causes cells/particles flowing in the channel to be focused away from the center of the channel towards the sides of the channel as a function of the size of the particles, such that larger particles move further toward the sides of the channel than smaller particles. These separated particles form size-based fluid particle streams that flow toward the outlets, such that streams with larger particles move toward outlets located near the periphery/sides of the channel, and streams with smaller particles flow toward outlets more centrally-aligned with the center portion of the ferrohydrodynamic separation channel. The method then includes collecting separated particles from the outlets. The devices of the present disclosure include 3 or more outlets, as described above. Some embodiments have about 3-10 outlets.
In some methods of the present disclosure, the sample is a lysed blood sample, the microparticles are cells the target cells are selected from circulating tumor cells and lymphocytes, and the cells include white blood cells and target cells. According to some methods the target cells are selected from circulating tumor cells and lymphocytes. The microparticles in some embodiments, have varying physical diameters (e.g., different types of particles have different diameters), such as particle diameters from about 4-40 μm. In some embodiments, after exiting the inertial focusing stage and/or when first entering the first end of the ferrohydrodynamic separation stage, the focused fluid sample stream has a width of about 4-100 μm. In embodiments, the fluid sample is processed in the device at a flow rate of about 200-1400 μL/min.
Having described the devices, kids, and methods of the present disclosure generally, some various aspects are set forth below, followed by some representative examples.
The present disclosure further includes the following aspects and embodiments.
Aspect 1: A multi-stage microfluidic device for separating cells and/or micro-particles in a sample, the device comprising:
Aspect 2: The multi-stage microfluidic device of aspect 1, wherein the magnetic source comprises an array of magnets comprising a top array and bottom array, wherein the ferrohydrodynamic separation stage is sandwiched between and substantially centrally aligned between the top magnet array and the bottom magnet array, wherein the magnets in the top array are oriented to repel the magnets in the bottom array.
Aspect 3: The multi-stage microfluidic device of aspect 2, wherein the array of magnets comprises six magnets arranged in a sextupole configuration.
Aspect 4: The multi-stage microfluidic device of any of aspects 1-3, wherein the first microfluidic channel comprises one or more filters between the inlet and the second end of the first microfluidic channel, the filters configured to separate debris from the fluid sample.
Aspect 5: The multi-stage microfluidic device of any of aspects 1-4, wherein the first microfluidic channel comprises one or more bends at the second end of the first microfluidic channel before the inertial focusing stage.
Aspect 6: The multi-stage microfluidic device of any of aspects 1-5, wherein the two or more serpentine focusing channels each comprise about 30-50 alternating micro-curves.
Aspect 7: The multi-stage microfluidic device of any of aspects 1-6, wherein the serpentine focusing channels comprise alternating small and large micro-curves.
Aspect 8: The multi-stage microfluidic device of aspect 7, wherein an interior channel width of each serpentine focusing channel varies along the length of said channel, wherein the interior channel width at a crest portion of each smaller micro-curve is about 50-200 μm and wherein the interior channel width at a crest portion of each larger micro-curve is about 100-400 μm.
Aspect 9: The multi-stage microfluidic device of any of aspects 1-8, wherein the ferrohydrodynamic separation channel comprises a first section and a second section connected by a substantially u-shaped curve, such that the ferrohydrodynamic separation channel passes through the magnetic field twice to increase separation of the particles.
Aspect 10: The multi-stage microfluidic device of any of aspects 1-9, wherein the sample is a lysed blood sample, and the cells/particles comprise white blood cells and target cells.
Aspect 11: The multi-stage microfluidic device of aspect 10, wherein target cells are selected from circulating tumor cells and lymphocytes.
Aspect 12: The multi-stage microfluidic device of any of aspects 1-11, wherein the ferrofluid and the sheathing ferrofluid each comprise a plurality of magnetic nanoparticles, a surfactant, and a carrier fluid.
Aspect 13: A kit for enriching and/or sorting unlabeled, microparticles in a fluid sample, the kit comprising:
Aspect 14: The kit of aspect 13, further comprising:
Aspect 15: The kit of any of aspects 13-14, wherein the surfactant is biocompatible.
Aspect 16: The kit of any of aspects 13-15, wherein the ferrofluid and the sheathing ferrofluid have a concentration of magnetic nanoparticles of about 0.001-1% (v/v).
Aspect 17: A method of enriching and/or separating unlabeled, microparticles in a sample comprising a plurality of components, the method comprising:
Aspect 18: The method of aspect 17, wherein the sample is a lysed blood sample, the microparticles are cells, and the cells include white blood cells and target cells.
Aspect 19: The method of any of aspects 17-18, wherein the target cells are selected from circulating tumor cells and lymphocytes.
Aspect 20: The method of any of aspects 17-19, wherein the microparticles have varying physical diameters in a range of about 4-40 μm.
Aspect 21: The method of any of aspects 17-20, wherein the focused fluid sample stream has a width of about 4-100 μm when it enters the first end of ferrohydrodynamic separation stage.
Aspect 22: The method of any of aspects 17-20, wherein the fluid sample is processed in the device at a flow rate of about 200-1400 μL/min.
Aspect 23: A multi-stage microfluidic device for separating cells/particles in a sample, the device comprising:
The features of the device of aspect 23 can also be combined with any of the features of the devices of aspects 1-12 above, with the kits of aspects 12-16, and with the methods of aspects 17-22.
Aspect 24: A method of enriching and/or separating unlabeled microparticles in a sample comprising a plurality of components, the method comprising:
The features of the method of aspect 24 can be implemented with any of the devices of aspects 1-12 and/or 23 above, and with any of the kits of aspects 12-16.
Additional details regarding the devices, kits, and methods, of the present disclosure are provided in the Examples below. The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent.
It should be emphasized that the embodiments of the present disclosure, particularly, any “preferred” embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and protected by the following claims.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y′, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y′, and ‘greater than z’. In some embodiments, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
Now having described the embodiments of the disclosure, in general, the included examples and figures describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
The present example describes the design and testing of an embodiment of an ultrahigh-throughput microfluidic technology of the present disclosure, referred to in this example as inertial-ferrohydrodynamic cell separation (inertial-FCS), that rapidly sorted through over 60 milliliters of samples at a 100,000 cells/second throughput in a label-free manner, differentiating the cells based on their physical diameter difference with ˜1-2 μm separation resolution. Through the integration of inertial focusing and ferrohydrodynamic separation, it was demonstrated that the resulting inertial-FCS devices could separate viable and expandable circulating tumor cells from cancer patients' blood with high recovery rate and high purity. This example also shows that the devices could enrich lymphocytes directly from white blood cells based on their physical morphology without any labeling steps. This label-free method could address needs of high throughput and high-resolution cell separation in circulating tumor cell research and adoptive cell transfer immunotherapy.
These above-mentioned challenges in currently available methods highlighted a need for a high-throughput and high resolution method that can separate target cells from a large volume of biological samples in a low-cost manner. To address this need, devices and methods of the present disclosure were developed, and example embodiments described here provide a label-free inertial-ferrohydrodynamic cell separation (inertial-FCS) method that was based on the integration of inertial focusing and ferrohydrodynamic separation of cells according to their physical diameters. This demonstrated separation of target cells from contaminating cells with a high throughput of ˜105 cells/s, a high sample processing flow rate (˜60 mL/h) and a high separation resolution of ˜1-2 μm in cellular diameter difference. Due to its label-free nature, this method didn't require the use of antibodies and magnetic beads and could lower the cost of cell separations. This example illustrates the working principle of the inertial-ferrohydrodynamic cell separation (inertial-FCS) approach, the design and optimization of an example device for high-throughput and high-resolution cell separation, and the validation of the device using spiked cancer cells, blood samples from cancer patients, and human white blood cells.
Overview of Inertial-Ferrohydrodynamic Cell Separation
The underlying working principle of inertial-ferrohydrodynamic cell separation (inertial-FCS) was the integration of the inertial focusing and ferrohydrodynamic separation of cells of interests based on their physical diameters (
Optimization of Inertial-Ferrohydrodynamic Cell Separation
First, the inertial-ferrohydrodynamic cell separation (inertial-FCS) device was optimized for a high cell-processing throughput separation of target cells. The throughput performance targets of the inertial-FCS device in cell separation included: (1) a cell-processing throughput of ˜105 cells per second, and a sample processing flow rate of >60 mL per hour, and (2) a high recovery and purity of separated cells. These performance metrics were chosen after considering separation requirement on the samples that contained cancer cells or lymphocytes, as well as the challenges facing existing cell separation methods (see supplementary information).16-27
Systematic optimization of high-throughput inertial-FCS devices focused on the effects of device geometry, magnetic field and its gradient, sample flow rates, as well as ferrofluid concentration on device performance, including cell-processing throughput, recovery rate and purity of separated cells. This optimization was conducted using a previously developed physical model that took into consideration of the balanced magnetic buoyance force and hydrodynamic viscous force on cells in laminar low conditions.37,38 Firstly, the microchannel dimensions were determined for both inertial focusing and ferrohydrodynamic separation stages by balancing a goal of processing at least 60 milliliters of samples within one hour, and a goal to achieve inertial focusing of cells in the sigmoidal microchannels with alternating curvatures. Inertial focusing channel dimensions were optimized so that the channel Reynold's number (Rc) was 63.8 and the particle Reynold's number (Rp) was 1.3 when the flow rate was 1000 μL min−1 or 60 mL h−1, ensuring that the cells would experience inertial focusing and self-organize into narrow streams prior to the separation.28 The schematic and prototype microchannel of an inertial-FCS device are shown in
Secondly, the generation of external magnetic field gradient was optimized because the amplitude of magnetic force on cells was proportional to the amplitude of magnetic field gradient.31-36 In order to maximize the field gradient, a sextupole magnet configuration was adopted in the inertial-FCS device that could generate a magnetic flux density in the range of 0-3.2 T (1.1-1.3 T within the separation microchannel), and a magnetic flux density gradient up to 670 T m−1 (
The remaining optimization focused on the effect of ferrofluid concentration (volumetric fraction of magnetic materials in the ferrofluid) and cell-processing sample flow rate on the performance of the ferrohydrodynamic separation stage. For this part of optimization, an output was calculated—a separation distance in the y-direction between cells with different diameters, denoted as ΔY. ΔY was optimized using parameters including ferrofluid concentration (0-0.3% v/v) and sample flow rate (100-1200 μL min−1, i.e., 6-72 mL h−1). The goal was to maximize the separation distance while achieving the highest sample flow rate simultaneously. We first optimized the flow ratio between the sample flow and the sheath flow to be 2 when the sample flow rate was 600-1200 μL min−1 through simulation (
Optimization of Inertial-Ferrohydrodynamic Cell Separation for High Separation Resolution
The inertial-ferrohydrodynamic cell separation (inertial-FCS) device was next optimized for a high-resolution separation of target cells. The aim was to use the inertial-FCS device to separate cells that had ˜1-2 μm difference in their physical diameters. Being able to differentiate cells with such a small diameter difference allowed the device to selectively enrich target cells from contaminating cells that had similar physical morphologies. For this purpose, it was first investigated the theoretical separation resolution of the inertial-FCS device through simulations, by considering realistic biological samples containing cells with polydisperse physical diameters. The separation distance between particles that had just 1 μm in their diameter difference was obtained, denoted as ΔY1 μm, and its dependence on the particle diameter and sample flow rates through simulation in
A major observation from this simulation study (
For particles of 10-20 μm in diameter, the separation resolution was relaxed to 2 μm in their diameter difference in order to achieve sufficient separation distance and high sample flow rates.
To search for parameters that worked efficiently for particles of >20 μm in diameter, the separation distance (ΔY2 μm)'s dependence on the ferrofluid concentration was simulated at a constant sample flow rate (1,500 μL min−1).
Validation of Inertial-Ferrohydrodynamic Cell Separation with Biological Samples
After the completion of the inertial-FCS optimizations, the devices were verified in two cell separation applications. The first application was the use of the device in recovering circulating tumor cells from lysed whole blood. The second application was the use of the device in separating lymphocytes from human white blood cells. Due to the large volume of samples and the large quantity of cells, as well as the physical diameter distribution of cells in these samples, we chose sample flow rates of 1,000-1,200 μL min−1 (60-72 mL h−1) and a ferrofluid concentration of 0.05% (v/v) in the inertial-FCS device to achieve high throughput and high separation resolution.
Initially, the spiked cancer cell validation was conducted with the aim of using inertial-FCS devices to recover CTCs from cancer patients' blood. Most CTCs of epithelial origin had a diameter range of 15 μm-25 μm, and were on average larger than other blood components such as red blood cells (RBCs: 6-9 μm in diameter), and the majority of white blood cells (WBCs: 8-14 μm in diameter).1 However, measurements on the cell diameter of cancer cell lines and WBCs showed that there was a significant diameter overlap between WBCs and cancer cells.39. There was also a significant percentage of patient-derived CTCs that were smaller than 10 μm in diameter.39 Because of the polydispersity in the cancer cells' diameter, label-free cancer cell separation methods for CTC applications needed to have a high separation resolution, preferably ˜1-2 μm so that target cells could be precisely separated based on their physical diameter while the contamination was minimized. The spiked cancer cell validation demonstrated that the inertial-FCS device could separate cancer cells with a resolution of ˜2 μm, which led to a high recovery rate and purity of isolated cells.
The inertial-FCS device was then used to separate a non-small lung cancer cell line (H1299) based on their physical diameters. A typical separation process can be visualized in
A typical separation process can be visualized in
As shown in
The inertial-FCS device was then validate using a blood sample from four stage IIIB/IV lung cancer patients. The patients were recruited and consented from the University Cancer and Blood Center (Athens, Georgia) under an approved IRB protocol (University of Georgia, VERSION00000869). Blood was drawn from the patients prior to any cancer related treatment and processed by inertial-FCS devices for CTC separation. After separation, isolated cells were divided for cell identification and cell culture. Cells in the identification portion were stained with the epithelial marker (EpCAM), mesenchymal markers (vimentin and N-cadherin), leukocyte marker (CD45) and nucleus staining DAPI for their identification. CTCs were identified as epithelial positive (EpCAM+/CD45−/DAPI+), mesenchymal positive (Vim+/CD45−/DAPI+, N-cad+/CD45−/DAPI+ or Vim+/N-cad+/CD45−/DAPI+), or both epithelial and mesenchymal positive (EpCAM+/Vim+/N-cad+/CD45−/DAPI+), while WBCs were identified as CK−/Vim−/N-cad−/CD45+/DAPI+. The numbers of identified CTCs for all patients are listed in Example 2 below.
For patient 1, a total of 1452 CTCs were separated from 10 mL of blood from this patient (145 CTCs per mL of blood). Examples of intact CTCs from device outputs are shown in
Next, lymphocyte validation was conducted with the aim of using inertial-FCS devices to purify lymphocytes from white blood cells. Lymphocytes consisted of B and T cells, which were 6-8 μm in diameter, and larger natural killer cells, which were 12-15 μm in diameter.40 These lymphocytes co-exited with a large quantity of granulocytes that were 10-15 μm in diameter, and monocytes that were 15-30 μm in diameter.40 The inertial-FCS device was used to separate the lymphocytes from other white blood cells. In this validation, 10 mL lysed human blood with ˜60 millions of WBCs was suspended in a 0.05% (v/v) ferrofluid and processed in the device with a flow rate of 1,000 μL min−1 (60 mL h−1) and a corresponding throughput of ˜100,000 cells/s. After separation, the diameter distributions of cells collected from the device inlet and outlets #1-5 were examined.
It was also observed that the platelets with the diameter of 2-3 μm were present in outlets #2-5, due to the fact that the inertial focusing stage was less effective in focusing small cells such as platelets (see supplementary information). The cells from the outlet #3, which had a diameter distribution of 6.39±2.12 μm, were collected and analyzed because this diameter distribution coincided with the lymphocyte diameter (6-8 μm) reported in the literature.23,40. The analyses of these cells included flow cytometry for the cell composition, immunofluorescent and hematologic stains for cell differentiation (
The data and results of this study demonstrate a label-free inertial-ferrohydrodynamic cell separation (inertial-FCS) method and device that integrated both inertial focusing and ferrohydrodynamic separation for cell separation based on their physical diameter difference. This method leveraged both the high throughput of the inertial focusing and high resolution of the ferrohydrodynamic separation to enable rapid and precise cell separations that were urgently needed in fundamental biological research and clinical assays. Systematic optimization of the inertial-FCS method was performed and operating parameters were determined that enabled it to process more than 60 mL of biological samples within one hour at an extremely high 100,000 cells/s throughput, a feature that was desired in a variety of biological applications that involve processing a large volume of biological samples to search for target cells.
The high resolution nature of this method allowed it to differentiate cells with ˜1-2 μm in their physical diameter difference, a feature that was sought for label-free and low-cost cell separation applications, which often had polydispersed cells with overlapping physical sizes. The inertial-FCS devices could separate spiked cancer cells in a biocompatible manner from white blood cells with high throughput and high resolution. Isolated cancer cells showed a high recovery rate (94.8%) and a high purity (11%), which implied that this method could be used in enriching circulating tumor cells from cancer patients. This was confirmed by using inertial-FCS devices to process blood samples from stage IIIB/IV lung cancer patients. The inertial-FCS devices could also purify lymphocytes directly from white blood cells based on their physical diameters at an extremely high throughput, which could potentially lower the cost associated with adoptive cell transfer therapy because lymphocytes were precursors for potent therapeutic cells. Future optimization of the inertial-FCS method could potentially lead to devices that can process a single blood sample to simultaneously purify both circulating tumor cells and lymphocytes with non-overlapping size profiles.
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Ferrofluids Synthesis and Characterization
Maghemite nanoparticles (diameter: 10.91±4.86 nm) were synthesized by a chemical co-precipitation method as previously descried (see reference 33, Zaho, et al., Lab Chip, 2017, 17, 2243-2255, incorporated by reference herein), hereby incorporated by reference herein for preparation of maghemite nanoparticles). Size and morphologies of nanoparticles were characterized using transmission electron microscopy (TEM; FEI, Eindhoven, the Netherlands). The viscosity of ferrofluid was measured with a compact rheometer (Anton Paar, Ashland, VA) at room temperature. Volume fraction of magnetic materials and saturation magnetization of the ferrofluid were characterized with a vibrating sample magnetometer (VSM: MicroSense, Lowell, MA). In order to achieve biocompatibility, pH of the ferrofluid was adjusted to 7 and the osmotic pressure was balanced with Hank's Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA). The concentration of undiluted ferrofluid was measured to be 0.3% (v/v) and corresponding viscosity of the ferrofluid was 1.68 mPa-s at 23° C.
Cell culture and sample preparation Cancer cell lines (ATCC, Manassas, VA) including two human breast cancer cell lines (MCF7 and MDA-MB-231), and two human lung cancer cell lines (H1299 and H3122) were used in this study. MCF7 and MDA-MB-231 were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA), and H1299 and H3122 cell were cultured in RPMI medium. DMEM and RPMI medium were supplemented with 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 1% (v/v) penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA), and 0.1 mM non-essential amino acids solution (Thermo Fisher Scientific, Waltham, MA). Cells were released through incubation with 0.05% trypsin-EDTA solution (Thermo Fisher Scientific, Waltham, MA) at 37° C. for 5 minutes. The concentration of harvested cells was measured with automated cell counter (Countess™, Thermo Fisher Scientific, Waltham, MA). After dilution with PBS, the exact number of cells were counted with a Nageotte counting chamber (Hausser Scientific, Horsham, PA). Desired cancer cells were spiked into 1 mL ferrofluid.
Inertial-FCS Device Fabrication and Assembly
The mold of inertial-FCS device was fabricated by SU-8 2025 photoresist (Kayaku Advance Materials, Westborough, MA) with a channel height of 60 μm, which was measured by a profilometer (Veeco Instruments, Chadds Ford, PA). Polydimethylsiloxane (PDMS) devices were prepared with a Sylgard 184 silicone elastomer kit (Ellsworth Adhesives, Germantown, WI) using a 1:7 ratio of cross linker and base, followed by a curing at 70° C. for 3 hours. The fabricated microchannel was placed in the sextupole permanent magnet array (N52, K&J Magnetics, Pipersville, PA) and held in a custom-made aluminum manifold. Each magnet was 50.8 mm in length, 6.35 mm in both width and thickness, with a residual magnetic flux density of 1.48 T.
Microfluidic Experiment Setup and Procedure
The inertial-FCS microchannel was first treated with plasma for 3 minutes, followed by an ethanol (70%) flushing for 10 minutes. The microchannel was then primed with PBS supplemented with 0.5% (w/v) bovine serum albumin (BSA) and 2 mM EDTA (Thermo Fisher Scientific, Waltham, MA). Sample fluids and sheath fluids were individually controlled with syringe pumps (Chemyx, Stafford, TX) at variable flow rates. Images and videos of cells were obtained with an inverted microscope equipped with a CCD camera (Carl Zeiss, Germany).
CTC Processing
Cancer patient samples collected at the University Cancer and Blood Center (Athens, Georgia) were approved by the University of Georgia Institutional Review Board (IRB) (VERSION00000869) before study initiation and informed consent was obtained from the participants. Blood was first lysed with RBC lysis buffer (eBioscience, San Diego, CA) for 10 minutes at room temperature, centrifuged at 500×g for 5 minutes, and resuspended in 0.05% ferrofluid. After processing with the inertial-FCS device, collected cells were centrifuged at 500×g for 5 minutes at room temperature and resuspend in DMEM/F12 medium supplemented with B27 supplement (1×; Thermo Fisher Scientific, Waltham, MA), epidermal growth factor (20 ng/mL; Millipore Sigma, Burlington, MA), basic fibroblast growth factor (10 ng/mL; Thermo Fisher Scientific, Waltham, MA), L-Glutamine (2 mM; Thermo Fisher Scientific, Waltham, MA), and Penicillin-Streptomycin (1×; Thermo Fisher Scientific, Waltham, MA). Cells were cultured in a vented T25 flask at 37° C. with 5% CO2. Cultures were supplemented with fresh medium every 3 days and washed every 5 days with 1×PBS.
Human White Blood Cells
Human whole blood cells from donors was purchased from a commercial source (Zen-Bio, Research Triangle, NC) and lysed with RBC lysis buffer (eBioscience, San Diego, CA) for 5 minutes to remove red blood cells at room temperature. Remaining blood cells were then suspended in the same volume of ferrofluid (0.05% v/v) containing 0.1% (v/v) Pluronic F-68 non-ionic surfactant (Thermo Fisher Scientific, Waltham, MA) before device processing.
Flow Cytometry of WBCs
Types of separated WBCs were confirmed using flow cytometry (Agilent Quanteon, Agilent, Santa Clara, CA). WBC size and granularity were used to distinguish the granulocytes, lymphocytes and monocytes. Size information were collected from the forward scatter (FSC) while the granularity of the cells was predicted with the side scatter. Fluorescent signals were used to further identify the WBC types: CD45+/CD3+ was classified as T lymphocytes; CD45+/CD19+ was classified as B lymphocytes.
Immunofluorescence Staining
After inertial-FCS device processing, all the outlet sample were collected and resuspend with PBS. The collected cells were fixed with 4% (w/v) paraformaldehyde solution (Santa Cruz Biotechnology, Inc., Dallas, TX) for 10 minutes and subsequently permeabilized with 0.1% (v/v) Triton X-100 (Alfa Aesar, Haverhill, MA) in PBS for 10 minutes. The cells were blocked with a blocking reagent (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes to reduce non-specific binding, and immunostained with primary antibodies. In the CTC experiments, the primary antibodies used were anti-EpCAM, anti-CD45, anti-vimentin, anti-N-cadherin (Santa Cruz Biotechnology, Inc, Dallas, TX). Anti-CD45, anti-CD3, and anti-CD19 (Santa Cruz Biotechnology, Inc, Dallas, TX) were used in the lymphocytes experiment. After immunofluorescence staining, the cells were washed and resuspended with PBS. A small portion of cells was cover slipped with mounting medium supplied with DAPI (Electron Microscopy Sciences, Hatfield, PA) for imaging.
Hematologic Staining of WBCs
Separated WBCs was differentiated using Wright's stain (Millipore Sigma, Burlington, MA) following manufacture's protocol. Briefly, isolated cell was placed on poly-L-lysine coated glass slides for 1 hour at 4° C. The slides were flooded with 1 mL Wright Stain. After 30 seconds, 1 mL deionized water was added and mixed thoroughly for 1 minute. The slides were thoroughly rinsed with deionized water and air-dried before inspection and imaging.
Cancer Cell Recovery Rate and Purity Calculation
Collected cells from device outlets were stained with 2 μM DAPI (Thermo Fisher Scientific, Waltham, MA) to identify the nucleated cell. The number of cells was counted with a Nageotte counting chamber. Cells with CellTracker signal was identified as cancer cells, while other cells with DAPI signal was classified as white blood cells. The recovery rate was calculated by the number of collected cancer cells/number of spiked cancer cells. The purity was calculated by the number of collected cancer cells/number of collected nucleated cells.
Cell Diameter Measurement
Cells were deposited onto microscope slides and imaged with a microscope in bright field mode. Images of cells were analyzed by the ImageJ software. The effective diameter of the cells was calculated using their surface areas with the assumption that cells were spherical.
This Example provides data and information supplemental to Example 1, above.
Where ρ is density of the fluid, v is the maximum channel velocity, L is characteristic channel dimension, μ is dynamic viscosity of the fluid, W is the channel width, and H is the channel height.
R
p
=R
c×(D/L)2 (2)
Where D is the particle diameter.
The sample flow rate was 1000 μL min−1 and the sheath flow rate was 500 μL min−1.
Purity is defined as the ratio of the number of CTCs and the number of total cells found at the collection outlet of the inertial-FCS device.
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This application claims the benefit of U.S. Provisional Application Ser. No. 63/145,391, titled “Devices, Kits, and Methods for Label-Free Inertial Ferrohydrodynamic Cell Separation with High Throughput and Resolution,” filed Feb. 3, 2021, which is incorporated herein by reference in its entirety.
This invention was made with Government support under 1150042, 1659525, and 1648035, each awarded by the National Science Foundation, and under TR002378 and EB028191, each awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/070512 | 2/3/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63145391 | Feb 2021 | US |
