Patent Application
20240026267
Rapid separation of rare 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), from biological samples can provide valuable research tools and insights into diseases such as metastatic cancers. The limited availability and difficulty of obtaining useful amounts of rare CTCs is a hurdle in such research. However, even with advanced technologies for cell separation, the limiting throughput, high cost and low separation resolution of currently available cell separation platforms still prevent the effectiveness of such technologies in processing large volumes of biological samples.
Profiling circulating tumor cells (CTCs) in cancer patients' blood samples is critical to understanding the complex and dynamic nature of metastasis. This task is further complicated by the fact that CTCs are not only extremely rare in circulation but also highly heterogenous in their molecular programs and cellular functions, even within a single sample. Label-based CTC separation technologies were developed to selectively enrich a subset of CTCs from blood, primarily through the use of specific biological markers including epithelial cell adhesion molecule (EpCAM). These antigen-based labels were a rate-limiting factor in effective CTC separation, as the inherent heterogeneity of CTCs render these technologies ineffective for general use. The vast array of various biomarkers that might or might not be expressed, and which cannot be predicted to remain expressed in CTCs undergoing Epithelial-to-Mesenchymal Transitions (EMT) are cumbersome and confounding in these label-based methods. Furthermore, most label-based technologies do not conveniently enable comprehensive molecular analysis of separated CTCs because they are either dead or immobilized to a surface. Thus, a variety of label-free methods have been developed to exploit specific physical markers in order to deplete non-CTCs in blood and thereby enrich cancer cells. While such methods may be used to separate CTCs based upon, for example, size, the existence of large white blood cells, such as monocytes, that may have overlapping sizes with CTCs complicate these label-free methods and reduce the purity of the sample obtained. Other devices have attempted to incorporate two or more of these methods, but still suffer from the time-consuming and laborious sample preparation due to the complications discussed above for labeling CTCs.
Furthermore, due to the heterogeneity of CTCs in the blood and the different health threat posed by different types of CTC's there is a great interest in further characterizing the types of CTCs circulating in a patient's bloodstream. For instance, only some CTC's transition to an invasive phenotype with the ability to actively invade distant organs. Identification of these invasive phenotype CTCs that pose the greatest threat of metastasis could help predict patient outcomes, identify those in need of more aggressive therapies, and identify new treatments. Thus, in addition to separation of CTC's from other components of blood, there is additional interest in further characterization of isolated CTC's with respect to their phenotype and to differentiate and identify passive and invasive CTC phenotypes.
There remains a need for improved devices and methods for separating circulating tumor cells that overcome the aforementioned deficiencies.
Embodiments of the present disclosure provides for microfluidic devices, systems, kits, and methods of using multi-stage microfluidic devices are provided for high throughput sorting, separation/enrichment of target rare cells from a sample, and can additionally provide for characterization/phenotyping of circulating tumor cells (CTCs) and other unlabeled rare cells in a biological sample such as blood, where the rare cells do not need to be labeled.
In an aspect the present disclosure provides for a method of enriching target rare cells in a biological sample, the method comprising: combining the biological sample with a plurality of magnetic microbeads adapted to specifically conjugate with white blood cells (WBCs) such that a majority of WBCs in the sample are conjugated to one or more magnetic microbeads to produce a magnetically labeled biological sample; combining the magnetically labeled biological sample with a colloidally stable ferrofluid to produce a mixed ferrofluid biological sample; flowing the mixed ferrofluid biological sample through an inertial focusing stage comprising two or more sigmoidal microchannels with a plurality of alternating curvatures, such that rare cells and WBCs in the mixed ferrofluid biological sample are focused into one or more or two or more narrow focused fluid sample streams; flowing the one or more or two or more focused fluid sample streams through a ferrohydrodynamic separation stage comprising a ferrohydrodynamic separation channel and 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 the white blood cells conjugated to the magnetic beads flowing into the ferrohydrodynamic separation channel to be focused toward a central longitudinal axis of the ferrohydrodynamic separation channel and to cause target rare cells in the sample to be deflected towards an outer portion of the ferrohydrodynamic separation channel; separating the magnetic-bead-conjugated WBCs flowing along a central longitudinal axis of the ferrohydrodynamic separation channel from target rare cells flowing along an outer portion of the ferrohydrodynamic separation channel to produce an enriched biological sample. The rare cells can be circulating tumor cells (CTCs) and the biological sample is a red blood cell-lysed blood sample from a patient. The flow rate of the mixed ferrofluid biological sample can be about 500-2000 μl/min. The concentration of the ferrofluid can be about 0.005-0.05%. Over 95% of WBC's cab be separated from the biological sample to produce the enriched biological sample. In an aspect, over 99% of WBC's can be separated from the biological sample to produce the enriched biological sample. The magnetic source can comprise an array of magnets comprising a first array and second array, wherein the ferrohydrodynamic separation stage is sandwiched between and substantially centrally aligned between the first magnet array and the second magnet array, wherein the magnets in the first array are oriented to repel the magnets in the second array. The array of magnets comprises six magnets that can be arranged in a sextuple configuration. The method further comprises introducing the enriched biological sample into a microfluidic chemotactic cell migration unit and sorting the target rare cells into different migratory phenotypes based on at least one of a speed and distance of migration of target rare cells with respect to one or more chemo-modulatory compounds flowing in a portion of the microfluidic chemotactic cell migration unit. The target rare cells can be circulating tumor cells (CTCs) and the one or more chemo-modulatory compounds comprises at least one compound that is a chemoattractant for CTCs having an invasive phenotype. The chemoattractant for CTCs can be selected from the group consisting of Fetal Bovine Serum (FBS), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). The rare cells can be target circulating tumor cells (CTCs) and the one or more chemo-modulatory compounds comprises at least one compound that is a migration inhibitor for WBCs. The at least one migration inhibitor compound for WBCs can be Slit2. The enriched biological sample can be incubated in microfluidic chemotactic cell migration unit with the chemo-modulatory compounds for about 3 to 48 hours. A gradient of chemo-modulatory compounds can be maintained by continuous perfusion during the incubation time. Conjugating the WBCs in the sample to magnetic microbeads comprises: combining the WBC with one or more WBC biomarker antibodies and then conjugating the biomarker antibodies to the magnetic microbeads. The biomarker antibodies can be biotinylated and the magnetic microbeads are coated with streptavidin, and wherein the one or more or two or more WBC biomarker antibodies are selected from the group consisting of: CD45, CD45RA, CD66b, CD16, and CD3.
An aspect of the present disclosure provides for an integrated inertial ferrohydrodynamic cell separation (i2FCS) microfluidic unit comprising: a sample inlet configured to receive a mixed ferrofluid biological sample comprising a biocompatible ferrofluid combined with a biological sample comprising target rare cells and magnetically labeled white blood cells (WBCs); a filter section fluidly connected to the sample inlet, the filter section comprising a first microfluidic channel and one or more filters configured to remove a first plurality of waste particles from the mixed ferrofluid biological sample; an inertial focusing stage fluidly connected to the filter section, wherein the first microfluidic channel splits into two or more sigmoidal microchannels with a plurality of alternating micro-curves, each sigmoidal microchannel configured to focus cells within the sample into a narrow stream to produce a focused fluid sample stream; a ferrohydrodynamic separation stage fluidly connected to the inertial focusing stage such that the focused fluid sample streams from the two or more sigmoidal microchannels flow into a ferrohydrodynamic separation channel; a waste outlet fluidly connected to and axially aligned with the ferrohydrodynamic separation channel to receive materials flowing through a central portion of the channel, one or more target cell outlets each fluidly connected to the ferrohydrodynamic separation channel and offset from the center of the channel and configured to receive material flowing near the outer portion of the channel; and one or more magnetic sources adjacent to ferrohydrodynamic separation stage and configured to produce a substantially symmetric magnetic field having a field maximum along a length of the ferrohydrodynamic separation channel sufficient to cause the white blood cells conjugated to the magnetic beads in the ferrohydrodynamic separation channel to be focused toward a center of the channel and to exit the channel via the waste outlet and to cause target rare cells to be deflected towards an outer portion of the ferrohydrodynamic separation channel and to exit the channel via the one or more target cell outlets. The magnetic source comprises an array of magnets comprising a first array and second array, wherein the ferrohydrodynamic separation stage is sandwiched between and substantially centrally aligned between the first magnet array and the second magnet array, wherein the magnets in the first array are oriented to repel the magnets in the second array. The array of magnets comprises six magnets arranged in a sextuple configuration. A distance between a center of the ferrohydrodynamic separation channel and a magnet junction of the array of magnets is from about 0-50 μm. The array of magnets produces a magnetic flux density of about 1.0-3.0 T within the ferrohydrodynamic separation channel and a magnetic flux gradient of about 500-5000 T m−1. The number of alternating micro-curves in each of the sigmoidal microchannels comprises about 30-40 micro-curves. The alternating micro-curves comprise alternating small and large micro-curves. The diameter under each large micro-curve is about 600-2400 μm and the diameter under each small micro-curve is about 300-800 μm. 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 150-400 μm and wherein the interior channel width at a crest portion of each larger micro-curve is about 330-850 μm. The internal channel width of the ferrohydrodynamic separation channel is about 800-1600 μm. The ferrohydrodynamic separation channel can have a u-shaped curve, where the length of the channel before the curve is about 51000-59500 μm and the length after the curve is about 51500-60000 μm. The channel height in the inertial focusing stage and the ferrohydrodynamic separation phase is about 30-300 μm.
An aspect of the present disclosure provides for a combined integrated inertial ferrohydrodynamic cell separation (i2FCS) and cell migration device comprising: the i2FCS unit as described above and herein and a chemotactic cell migration unit comprising: an enriched sample inlet to receive an enriched biological sample from the i2FCS unit; a chemo-modulatory fluid inlet to receive a chemo-modulatory fluid comprising one or more chemo-modulatory compounds; one or more sample flow channels fluidly connected to the enriched sample inlet and configured to flow the enriched biological sample; one or more chemo-modulatory channels fluidly connected to the chemo-modulatory fluid inlet, configured to flow the chemo-modulatory fluid, and oriented substantially parallel to the one or more sample flow channels; a plurality of migration microchannels connecting at least one sample flow channel to at least one chemo-modulatory channels, each of the plurality of migration microchannels oriented substantially perpendicular to the sample flow channel and the chemo-modulatory channel and in fluidic communication with both the sample flow channel and the chemo-modulatory channel, wherein the height of each migration microchannel is smaller than the height of each of the sample flow channel and the chemo-modulatory channels; and at least one migratory target cell outlet at and end of a chemo-modulatory channel opposite the chemo-modulatory fluid inlet configured to collect migratory target cells that have migrated from the enriched sample inlet. Each migration microchannel can have dimensions to allow migration of a single cell at a time. Each migration microchannel has a width of about 7-50 μm, a height of about 3-8 μm, and a length of about 200-2000 μm.
An aspect of the present disclosure provides for a cell separation system for enriching target rare cells in a biological sample and characterizing the target rare cells, the system comprising: a plurality of magnetic microbeads adapted for specific conjugation to white blood cells in the biological sample to provide a magnetically labeled biological sample having a majority of the white blood cells in the biological sample conjugated to two or more magnetic microbeads; a biocompatible ferrofluid comprising a plurality of magnetic nanoparticles and a biocompatible surfactant, the biocompatible ferrofluid adapted to be combined with the magnetically labeled biological sample and an optional biocompatible carrier fluid to make a mixed ferrofluid biological sample; the integrated i2FCS microfluidic unit as described above and herein; one or more chemo-modulatory compounds that affect the migratory behavior of one or more different phenotypes of target cells; a chemotactic cell migration unit comprising: an enriched sample inlet to receive an enriched biological sample from the i2FCS unit; a chemo-modulatory fluid inlet to receive a chemo-modulatory fluid comprising the one or more chemo-modulatory compounds; one or more sample flow channels fluidly connected to the enriched sample inlet and configured to flow the enriched biological sample; one or more chemo-modulatory channels fluidly connected to the chemo-modulatory fluid inlet, configured to flow the chemo-modulatory fluid, and oriented substantially parallel to the one or more sample flow channels; a plurality of microchannels connecting at least one sample flow channel to at least one chemo-modulatory channels, each of the plurality of microchannels oriented substantially perpendicular to the sample flow channel and the chemo-modulatory channel and in fluidic communication with both the sample flow channel and the chemo-modulatory channel, wherein the height of each microchannel is smaller than the height (or width) of each of the sample flow channel and the chemo-modulatory channels; and at least one migratory target cell outlet at and end of a chemo-modulatory channel opposite the chemo-modulatory fluid inlet configured to collect migratory target cells that have migrated from the enriched sample inlet.
Further aspects of the present disclosure will be readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.
In various aspects, microfluidic systems, devices, and methods of using microfluidic devices are provided for separating/enriching rare cells, such as circulating tumor cells (CTCs), in a biological sample such as whole blood. The methods do not involve labeling of the rare cells and are capable of high throughputs with high levels of retention and separation of the rare cells. In embodiments, the methods, systems, and devices also provide for phenotypically characterizing the separated/enriched rare cells according to certain migration phenotypes.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and 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. The skilled artisan will recognize many variants and adaptations of the embodiments described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of biochemistry, molecular 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° C. and 1 atmosphere.
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. Functions or constructions well-known in the art may not be described in detail for brevity and/or clarity.
It should be noted that ratios, concentrations, amounts, and other numerical data can 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 numerical range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (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’”.
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.
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, trouble-shooting, 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, “attached” can refer to covalent or non-covalent interaction between two or more molecules. Non-covalent interactions can include ionic bonds, electrostatic interactions, van der Walls forces, dipole-dipole interactions, dipole-induced-dipole interactions, London dispersion forces, hydrogen bonding, halogen bonding, electromagnetic interactions, 7-7 interactions, cation-7 interactions, anion-7 interactions, polar 7-interactions, and hydrophobic effects.
A “biocompatible” substance or fluid, as described herein, indicates that the substance or fluid does not adversely affect the short-term viability or long-term proliferation of a target cell 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.
As used herein the term “chemogradient” refers to a fluid chemical gradient created in some embodiments of the present disclosure characterized by a varying % concentration of a chemical compound (e.g., a chemo-modulatory compound) in one area of the device of the present disclosure to another. For instance, near where a chemo-modulatory fluid is introduced to the device the concentration of the chemo-modulatory fluid would be higher than in an area of the device further away from where the chemo-modulatory fluid was introduced.
The term “chemotactic” and “chemo-modulatory” as used herein refer to a chemical compound (e.g., a chemical compound in a fluid) that has the ability to induce chemotaxis, e.g., modulate the movement of a cell within a fluid, for instance, either toward (attraction) or away from (repellant) the chemical compound. For instance, a chemo-attractant has the effect of attracting a certain cell or cell-type, while a chemo-repellent has the effect of repelling a certain cell or cell-type. In embodiments, a chemo-modulatory fluid can include one or more fluids, e.g., a compound that may be a chemo-attractant for a first type of cell and a different compound that may be a chemo-repellent for a different type of cell. A chemo-modulatory compound or substance can induce chemotactic movement, chemotaxis, of a cell or entity in response to the chemical stimulus produced by the compound or substance.
As used herein, channels that are “substantially parallel” refers to channels of a device of the present disclosure that, with respect to a reference channel, while not absolutely parallel throughout the entire device, are parallel for a majority of the length of the channel, particularly in a portion of the channel in which cell migration is being observed.
Microfluidic Devices & Systems and Methods of Use Thereof
In various aspects, microfluidic devices, systems, kits, and methods of using multi-stage microfluidic devices are provided for high throughput sorting, separation/enrichment of target rare cells from a sample, and can additionally provide for characterization/phenotyping of circulating tumor cells (CTCs) and other unlabeled rare cells in a biological sample such as blood, where the rare cells do not need to be labeled.
Although microfluidics-based methods have been explored as a new avenue to enrich and study CTCs for the past decade, previous approaches were based on the use of specific tumor antigens (marker-dependent) or cell size threshold (cell size-dependent) for enrichment, and suffered disadvantages due to limitations of these approaches. For example, marker-dependent methods that relied on EpCAM or other combination of tumor cell surface antigens were rendered ineffective due to inherent heterogeneity of tumor subtypes. CTCs are highly heterogeneous in their biological and biophysical characteristics with multiple phenotypes co-existing, which can evolve dynamically over the course of metastasis. The significant difference among various markers and their expression levels in CTCs undergoing EMT was difficult to predict, resulting in incomplete recovery of CTCs from clinical samples.
Cell size-dependent methods, on the other hand, based on a presumed size difference between blood and cancer cells, proved largely ineffective because a significant percentage of CTCs in circulation were comparable or smaller than blood cells. Measurements of white blood cells (WBCs) and cultured cancer cells revealed that there was a significant size overlap between the two, and an appreciable percentage (e.g., ˜35% for DMS 79 and H69 small cell lung cancer cell lines) of cancer cells were smaller than ˜10 μm. In addition, clinically isolated CTCs were reported to be as small as ˜6 μm. As a result, few cell size-dependent methods could achieve complete recovery and low WBC contamination simultaneously. Furthermore, due to their fragile nature, CTCs need to be processed with gentle enrichment conditions to keep their viability and tumorigenic capability for downstream studies. As a result, current microfluidic methods for invasiveness phenotyping of tumor cells were mostly confined to cultured cancer cells rather than patient-derived CTCs. Thus, new devices and methods are needed that can enrich viable CTCs from patient samples, regardless of their surface antigens and size profiles, and maintain their functionality so that the properties of the invasive cells can be identified.
Broadly described, the present disclosure provides methods for separating/enriching rare cells from a sample without labeling the rare cells in the sample prior to separation and for further characterizing the phenotype (such as the migratory phenotype) of the separated/enriched rare cells as well as systems and devices for carrying out the methods of the present disclosure.
In general, methods of the present disclosure include providing a sample containing unlabeled rare cells (such as CTCs), magnetically labeling white blood cells in the sample, passing the sample through an inertial focusing device to produce a focused sample in a narrow stream, passing the focused sample through a ferrohydrodynamic separation device to separate magnetically labeled white blood cells in the sample from rare cells in the sample to produce an enriched sample having a higher concentration of target rare cells and a lower concentration of white blood cells than the original sample, and then passing the enriched sample through a chemotactic cell migration unit to further identify and characterize the target rare cells in the sample according to migratory phenotype in response to one or more chemo-modulatory/chemotactic agents. The present disclosure further provides variations of this method as well as various embodiments of systems and devices designed to carry out the methods of the present disclosure.
In embodiments, methods of enriching target rare cells in a biological sample include combining the biological sample with a plurality of magnetic microbeads adapted to specifically conjugate with white blood cells (WBCs) such that a majority of the WBCs are conjugated to one or more magnetic microbeads to produce a magnetically labeled biological sample. In embodiments, nearly all WBCs are conjugated to at least one magnetic microbead and a majority of WBCs in the sample are conjugated to two or more magnetic microbeads. In embodiments, conjugating the WBCs in the sample to magnetic microbeads includes combining the WBC with two or more WBC biomarker antibodies and then conjugating the biomarker antibodies to the magnetic microbeads. In embodiments, the biomarker antibodies are biotinylated and the magnetic microbeads are coated with streptavidin and multiple biomarker antibodies are used to increase the percent of magnetic microbeads that are conjugated to WBC and reduce the amount of unconjugated magnetic microbeads which can clog the channels of the microfluidic devices. In embodiments, the one or more or two or more WBC biomarker antibodies are selected from the group including but not limited to: CD45, CD45RA, CD66b, CD16, and CD3.
Then the magnetically labeled biological sample is combined with a colloidally stable ferrofluid to produce a mixed ferrofluid biological sample. The method then includes flowing the mixed ferrofluid biological sample through an inertial focusing stage. The inertial focusing stage includes two or more sigmoidal microchannels with a plurality of alternating curvatures (described further below, and an embodiment of which is depicted in
In embodiments of the methods of the present disclosure the rare cells are circulating tumor cells (CTCs) and the biological sample is a red blood cell-lysed blood sample from a patient. The flow rate of the sample helps to optimize the inertial focusing as well as the ferrohydrodynamic separation and to achieve high throughput. In embodiments the flow rate of the mixed ferrofluid biological sample is about 500-2000 μl/min, such as about 500-1200 μl/min. The concentration of the ferrofluid also has an effect on the performance of the device, and affect the maximal separation distance between particles. In embodiments, the concentration of the ferrofluid is about 0.005-0.05%, such as about 0.015-0.03% (% v concentration of ferrofluid after mixing with the sample). As demonstrated in the examples below, methods of the present disclosure achieve separation of over 95% of WBC's from the biological sample to produce the enriched biological sample. In embodiments of these methods of the present disclosure, over 99% of WBC's are separated from the biological sample to produce the enriched biological sample, such as up to 99.992%.
In the ferrohydrodynamic separation stage, in embodiments, the magnetic source is an array of magnets comprising a first array and second array. In embodiments, the ferrohydrodynamic separation stage can be arranged between and substantially centrally aligned between the first magnet array and the second magnet array, such that the magnets in the first array are oriented to repel the magnets in the second array. In some embodiments, described in greater detail below, the array of magnets comprises six magnets arranged in a sextuple configuration. Greater details about the magnetic field properties are provided in the description below of the systems and devices of the present disclosure.
Methods of the present disclosure can also include further processing the enriched sample to further characterize the rare target cells enriched after the integrated inertial and ferrohydrodynamic separation. In embodiments, methods of characterizing the enriched target cells (e.g., CTCs) include observing certain migratory phenotypes of the target rare cells to differentiate cells with various migratory phenotypes. Embodiments of such methods include introducing the enriched biological sample into a microfluidic chemotactic cell migration unit and sorting the target rare cells into different migratory phenotypes based on at least one of a speed and distance of migration of target rare cells with respect to one or more chemo-modulatory compounds flowing in a portion of the microfluidic chemotactic cell migration unit. In some embodiments of the methods, the target rare cells are circulating tumor cells (CTCs) and the one or more chemo-modulatory compounds includes at least one compound that is a chemoattractant for CTCs having an invasive phenotype. In some embodiments the chemoattractant for CTCs is selected from, but not limited to factors such as, Fetal Bovine Serum (FBS), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). In some embodiments, the rare cells are target circulating tumor cells (CTCs) and the one or more chemo-modulatory compounds comprises at least one compound that is a migration inhibitor for WBCs, such as but not limited to Slit2. In yet other embodiments, the chemo-modulatory compounds can include both a chemoattractant for CTCs or certain subtypes of CTCs as well as a compound that repels WBCs, such that CTCs are attracted to certain areas of the migratory separation stage that include the chemoattractant(s) and WBCs are repelled from certain regions of the device including the WBC repellant compound. In embodiments, the enriched biological sample is incubated in microfluidic chemotactic cell migration unit with the chemo-modulatory compounds for a sufficient period of time to allow for migration, such as from about 3 to 48 hours. For instance, the enriched biological sample can be incubated with the chemo-modulatory compounds for about 24 hours. In embodiments, a gradient of chemo-modulatory compounds is maintained by continuous perfusion during the incubation time.
Many other variations to the methods of the present disclosure can be envisioned by a skilled artisan, and some such variations are described in greater detail in the examples below.
In some aspects, multi-unit, multi-stage microfluidic systems are provided for enriching and characterizing CTCs in a biological sample. In embodiments, the systems use a combination of a separation unit to separate target cells from non-target cells in a sample to produce an enriched sample and a migration unit to further characterize the target cells with respect to their migratory phenotype in response to different stimuli. In embodiments, systems of the present disclosure include an integrated inertial ferrohydrodynamics cell separation (i2FCS) approach to produce an enriched sample and a chemotactic cell migration approach for phenotypic characterization of the enriched cells. An illustration of a combined system of the present disclosure is shown in
Embodiments of the cell separation systems and kits of the present disclosure include a cell separation system and/or kit for enriching and/or characterizing CTCs in a biological sample. In addition to the microfluidic devices described herein, the cell separation/characterization systems of the present disclosure also include various compositions and compounds employed in the use of the systems and methods of the present disclosure. In embodiments, the cell separation systems can include a plurality of magnetic micro beads adapted for specific conjugation to white blood cells in the biological sample (e.g., the micro beads will not conjugate target cells, such as CTCs), a biocompatible ferrofluid including a plurality of magnetic nanoparticles and a biocompatible surfactant. The biocompatible ferrofluid can also be combined with the magnetically labeled biological sample and an optional biocompatible carrier fluid to make a mixed ferrofluid biological sample for processing in the systems/devices of the present disclosure. The systems/devices of the present disclosure allow enrichment of CTCs are size independent and do not require labeling the CTCs.
In embodiments the plurality of magnetic microbeads can include various biocompatible magnetic materials, such as, but not limited to iron oxide based magnetic material (e.g., magnetite (Fe3O4), maghemite (Fe2O3), or combinations of these. The magnetic materials for the microbeads should be non-toxic to cells. The magnetic microbeads of the present disclosure are adapted for conjugation with white blood cells (WBCs) such that the microbeads bind/conjugate with WBCs in the sample and do not substantially bind/associate with the CTCs in the sample (e.g., they do not specifically bind/conjugate/associate with the CTCs or other components present in the sample other than WBC's, and any non-specific association with CTCs, if present, is insignificant/negligible). In embodiments the magnetic microbeads are functionalized for specific binding to WBCs, such as by surface-functionalization with one or more binding agent(s) for specific binding to WBCs but that will not substantially bind the CTCs (e.g., streptavidin/avidin conjugated to biotinylated white blood cell-specific antibodies). In embodiments the magnetic microbeads comprise streptavidin coated magnetic Dynabeads (Life Technologies, Carlsbad, CA). In embodiments, the streptavidin coated magnetic beads are further conjugated to white-blood cell specific antibodies for specific binding to white blood cells. Examples of white blood cell-specific antibodies include, but are not limited to antibodies to CD45, CD45RA, CD66b, CD16, and CD3, and combinations thereof (while other antibodies can be used, a combination of the above antibodies will target substantially all WBC's present in a blood sample). Preferably one or more or two or more WBC biomarker antibodies are used, and in some embodiments all 5 of ate above are used.
In embodiments, the biological sample is whole blood. In embodiments, the sample is whole blood that has been treated with a lysis buffer to lyse/remove red blood cells. In embodiments, the biological sample is combined with the magnetic microbeads to label the WBCs in the sample prior to introduction to the device of the present disclosure to produce a magnetically labeled biological sample, such that the WBCs in the sample are substantially conjugated to magnetic microbeads prior to introduction. In embodiments, about 95% to 99.9%, or more of the white blood cells are conjugated to one or more magnetic microbeads. In embodiments, a majority of white blood cells in the magnetically labeled sample are conjugated to about 2 or more magnetic microbeads. In embodiments, a majority of white blood cells in the magnetically labeled sample are conjugated to about 1 to about 60 magnetic microbeads. In embodiments, the white blood cells in the magnetically labeled sample are conjugated to an average of about 20-50 magnetic microbeads. As used herein, the terms “magnetically labeled biological sample” and “magnetically labeled sample” refer to embodiments described above where a biological sample is combined with magnetic microbeads adapted for specific conjugation with WBC's and not to CTC's or other components of the sample, which are not substantially associated with the magnetic microbeads or otherwise magnetically labeled. Thus, the terms “magnetically labeled biological sample” and “magnetically labeled sample” should not be interpreted to indicate any magnetic labeling of any other components of the biological sample other than the WBCs.
In embodiments, the biocompatible ferrofluid of the present disclosure is a colloidal suspension of magnetic nanoparticles, coated by a biocompatible surfactant and suspended in a carrier fluid. The ferrofluid of the present disclosure is biocompatible and non-toxic to CTCs. In embodiments, the magnetic nanoparticles are a non-toxic magnetic material, such as, but not limited to iron oxide materials (e.g., magnetite (Fe3O4), maghemite (Fe2O3), and combinations of these. In embodiments, the magnetic nanoparticles are iron oxide particles (e.g., maghemite (Fe2O3)). Materials such as iron, cobalt, cobalt ferrite, and FePt are potentially toxic to cells, but could potentially be used if first rendered biocompatible/nontoxic by biocompatible coatings, etc. The magnetic nanoparticles can have a diameter of about 1-20 nm. In embodiments they have an average diameter of about 8-12 nm (e.g., about 11 nm). In embodiments, the magnetic nanoparticles are coated in a biocompatible surfactant to reduce agglomeration and to increase biocompatibility. In embodiments, the biocompatible surfactant can include electric double layer surfactant, polymer surfactant, inorganic surfactant, or a combination thereof. In embodiments, the surfactant is polymethyl methacrylate-polyethylene glycol (PMMA-PEG). In embodiments, the carrier medium can include biocompatible carrier fluids, such as, but not limited to, water, salt solution, or a combination. In embodiments, the carrier medium is a balanced salt solution, such as Hank's balanced salt solution (HBSS). In an embodiment, the biocompatible ferrofluid includes maghemite nanoparticles (Fe2O3) coated with polymethyl methacrylate-polyethylene glycol (PMMA-PEG) and 10% (v/v) 10× Hank's balanced salt solution (HBSS). In embodiments, the pH is about 7, and the osmotic pressure is close to that of a biological (e.g., human) cell. In embodiments, the ferrofluid concentration (volume fraction of magnetic particles) is about 0.005-0.05% and intervening ranges. For instance, in embodiments the ferrofluid concentration can be about 0.015-0.03%. (v/v). In embodiments the concentration is about 0.028%. The viscosity of the biocompatible ferrofluid varies based on the concentration of magnetic particles, the surfactant chosen, as well as the carrier fluid. In embodiments, the viscosity of the ferrofluid is about 0.95 mPa·s to 1.8 mPa·s, such as for instance, 1.7 mPa s−1 at room temperature. In embodiments the saturation magnetization was about 1,107 A m−1 and volume fraction of the ferrofluid was about 0.298% v/v.
In embodiments of systems or kits of the present disclosure, the system may include the fully prepared biocompatible ferrofluid as described above, and/or a prepared ferrofluid along with instructions for diluting the fluid with additional carrier fluid to adjust the concentration/volume fraction of magnetic nanoparticles in the fluid. In embodiments, systems or kits of the present disclosure may include a biocompatible ferrofluid composition that includes the plurality of magnetic nanoparticles and instructions for combining the magnetic nanoparticles/biocompatible superparamagnetic sheathing composition with a biocompatible surfactant and biocompatible carrier fluid to make a biocompatible ferrofluid of the present disclosure. In embodiments, systems/kits of the present disclosure can include the plurality of magnetic nanoparticles and the biocompatible surfactant (separately or mixed (e.g., such that the surfactant coats the magnetic nanoparticles) and instructions for combining the biocompatible superparamagnetic sheathing composition with a biocompatible carrier fluid and/or a patient sample to make a biocompatible superparamagnetic sheathing fluid of the present disclosure.
Integrated Inertial Ferrohydrodynamic Cell Separation (i2FCS) Unit
In embodiments, systems and devices of the present disclosure include a cell separation unit that has an integrated inertial focusing stage and a ferrohydrodynamic separation stage, as well as an inlet, filter stage/region, and at least a waste outlet for collection of WBCs and a target outlet for collection of an enriched sample having a greater concentration of rare cells and a lower concentration of WBCs compared to an initial sample.
The device/unit can include at least two stages, although there may be more in some embodiments. Therefore, the terms first, second, third and so-on, when used to describe the stages, should not be considered limiting on the total number of stages but may be used for simplicity to describe the relative ordering of the stages. Additional stages, not explicitly described, may in some aspects appear before the first stage.
The i2FCS units of the present disclosure comprise a sample inlet configured to receive a mixed ferrofluid biological sample comprising a biocompatible ferrofluid combined with a biological sample comprising target rare cells and magnetically labeled WBCs. After the sample inlet, the i2FCS unit includes a filter section fluidly connected to the sample inlet. The filter section includes a first microfluidic channel and one or more filters configured to remove a first plurality of waste particles from the mixed ferrofluid biological sample. The filter section is fluidly connected to the sample inlet and has a first microfluidic channel and one or more filters configured to remove a first plurality of waste particles from the mixed ferrofluid biological sample. In embodiments, the one or more filters are configured to remove a first plurality of waste particles from the biological sample (e.g., large particulate/agglomerated matter in the sample). After the first filter section, the filtered biological sample proceeds to the first separation stage.
After the filter section the units/devices of the present disclosure include an inertial focusing stage. The inertial focusing stage of the units, devices, systems, and methods of the present disclosure differs from the first generation inertial focusing cell separation (iFCS) devices and methods described in PCT/US22/70512, incorporated herein by reference. First, the first generation iFCS device was for separation of completely unlabeled samples based on particle size, which missed some target CTCs closer in size to some WBCs, vs the use of magnetic labeling and separation of WBCs in the present disclosure. The difference in principles of separation are illustrated in
The inertial focusing stage of the i2FCS microfluidic unit of the present disclosure is fluidly connected to the filter section. The first microfluidic channel of the filter section splits into two or more sigmoidal microchannels (as shown in
After the inertial focusing stage, the i2FCS microfluidic unit of the present disclosure includes a ferrohydrodynamic separation stage fluidly connected to the inertial focusing stage such that the focused fluid sample streams from the two or more sigmoidal microchannels flow into a ferrohydrodynamic separation channel, as illustrated in
The i2FCS microfluidic unit of the present disclosure also includes a waste outlet fluidly connected to and axially aligned with the ferrohydrodynamic separation channel to receive materials flowing through a central portion of the channel. This device/unit also includes one or more target cell outlets each fluidly connected to the ferrohydrodynamic separation channel and offset from the center of the channel and configured to receive material flowing near the outer portion of the channel. An illustration of an embodiment of the geometry of the unit at the end of the ferrohydrodynamic separation channel and the fluid connection to the waste outlet and the one or more target cell outlets is provided in
The i2FCS microfluidic unit of the present disclosure also includes one or more magnetic sources adjacent to the ferrohydrodynamic separation stage and configured to produce a substantially symmetric magnetic field having a field maximum along a length of the ferrohydrodynamic separation channel sufficient to cause the white blood cells conjugated to the magnetic beads in the ferrohydrodynamic separation channel to be focused toward a center of the channel and to exit the channel via the waste outlet. The substantially symmetric magnetic field is also configured to cause target rare cells to be deflected towards an outer portion of the ferrohydrodynamic separation channel and to exit the channel via the one or more target cell outlets. In embodiments, the magnetic source comprises an array of magnets comprising a first array and second array, wherein the ferrohydrodynamic separation stage is sandwiched between and substantially centrally aligned between the first magnet array and the second magnet array, wherein the magnets in the first array are oriented to repel the magnets in the second array. An embodiment of the arrangement of the magnets with respect to the ferrohydrodynamic separation stage is illustrated in
In embodiments of the microfluidic device of the present disclosure, the rare cells (e.g., CTCs) that exit via the one or more target cell outlets comprise about 95% or more, 97%, or more, or 99% or more of the total number of rare cells present in the biological sample inserted into the sample inlet when in operation.
Systems and devices of the present disclosure include a chemotactic cell migration unit to further characterize the enriched rare cells after exiting the i2FCS unit described above. In embodiments chemo-modulatory compounds are used to help further characterize the migratory phenotypes of the enriched rare cells to determine various phenotypes such as migratory phenotypes that might be associated with a more invasive cell type posing a greater health risk to a patient, as described in greater detail in the Example below. An embodiment of a design of a chemotactic cell migration unit is illustrated in
In embodiments, the chemotactic cell migration units of the present disclosure is designed to be used with one or more chemo-modulatory compounds that affect the migratory behavior of one or more different phenotypes of target cells. For instance, the one or more chemo-modulatory compounds can include at least one compound that is a chemoattractant for CTCs having an invasive phenotype. In other embodiments, the chemoattractant for CTCs is selected from the group consisting of Fetal Bovine Serum (FBS), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF). In some other embodiments, the one or more chemo-modulatory compounds include at least one compound that is a migration inhibitor for WBCs, such as, but not limited to Slit2.
The chemotactic cell migration unit/device includes various features. It includes an enriched sample inlet to receive an enriched biological sample from the i2FCS unit. It also includes a chemo-modulatory fluid inlet to receive a chemo-modulatory fluid comprising one or more chemo-modulatory compounds. In embodiments, the sample inlet and chemo-modulatory fluid inlet are located on opposite sides of the unit. In embodiments, the chemotactic cell migration unit also includes one or more sample flow channels fluidly connected to the enriched sample inlet and configured to flow the enriched biological sample. The unit/device also includes one or more chemo-modulatory channels fluidly connected to the chemo-modulatory fluid inlet, configured to flow the chemo-modulatory fluid. The flow of the chemo-modulatory fluid from the chemo-modulatory fluid inlet through the chemo-modulatory channels of the device can create a chemogradient within the device or areas of the device. In embodiments, the chemo-modulatory channel(s) are oriented substantially parallel to the one or more sample flow channels. Due to the orientation of the inlets and the flow of the fluids, the enriched sample fluid may, at least initially, flow in an opposite direction of the flow of chemo-modulatory compounds from the chemo-modulatory fluid inlet.
In addition to the sample flow channel and the chemo-modulatory channels, the cell migration unit also includes a plurality of migration microchannels connecting at least one sample flow channel to at least one chemo-modulatory channels. Each of the plurality of migration microchannels oriented substantially perpendicular to the sample flow channel and the chemo-modulatory channel and in fluidic communication with both the sample flow channel and the chemo-modulatory channel. The migration microchannels are small so that fluid flows between the chemo-modulatory channels toward the sample flow channel to create a chemo-gradient, and only about 1 cell can pass through the migration microchannel at a time. In embodiments, the height (or width) of each migration microchannel is smaller than the height (or width) of each of the sample flow channel and the chemo-modulatory channels. In embodiments, each migration microchannel has dimensions to allow migration of about a single cell at a time, for instance in embodiments, each migration microchannel has a width of about 7-50 μm (e.g., about 30 μm), a height of about 3-8 μm (e.g., about 5 μm), and a length of about 200-2000 μm.
The cell migration unit also includes at least one migratory target cell outlet at and end of a chemo-modulatory channel opposite the chemo-modulatory fluid inlet configured to collect migratory target cells that have migrated from the enriched sample inlet. As described above, the distance and speed with which a target rare cell has migrated with respect to the chemo-modulatory fluid within the device can differentiate the cells into different migratory phenotypes. For CTCs, more invasive phenotypes have also been shown to have more invasive characteristics and thus this can provide additional diagnostic approaches as well as novel ways to investigate phenomena such as epithelial to mesenchymal transition that appears to accompany a transition to a more high-risk invasive phenotype.
Embodiments of the present disclosure include combined integrated inertial ferrohydrodynamic cell separation (i2FCS) and cell migration devices that incorporate the i2FCS unit of the present disclosure as well as the chemotactic cell migration units of the present disclosure. The i2FCS unit can be used according to methods of the present disclosure to produce an enriched biological sample that has an enriched population of rare cells, such as CTCs. Then the combined device also includes an integrated chemotactic cell migration unit for further characterizing the target cells in the enriched sample as described above. While the units can function independently, the combined device includes the two units in an integrated or conjoined device. In embodiments, the two units can be included on a same microfluidic platform or on separate substrates where the enriched sample is transferred from one unit to the next
Cell separation systems of the present disclosure include the i2FCS unit as well as the chemotactic cell migration unit of the present disclosure along with other components useful for operation of the system. One other such component is a plurality the magnetic microbeads form producing the magnetically labeled biological sample (with magnetically labeled WBCs) as well as the biocompatible ferrofluid (or ingredients to make it). Another feature of some systems of the present disclosure is one or more chemo-modulatory compounds for use in the cell migration unit. The chemo-modulatory compounds are as described above, compounds that affect the migratory behavior of one or different phenotypes of target cells.
The present disclosure also includes methods described above using the microfluidic devices of the present disclosure and the systems of the present disclosure (e.g., microfluidic device plus biocompatible superparamagnetic sheathing fluid and magnetic microbeads) to enrich CTCs in a biological sample. In some aspects, the methods are capable of isolating a majority of the unlabeled rare cells. In some aspects, the unlabeled rare cells are circulating tumor cells in a whole blood sample, and the majority of the circulating tumor cells comprises about 90%, about 95%, about 97%, about 99%, or more of the circulating tumor cells as compared to a total number of circulating tumor cells present in the biological sample inserted into the first fluid inlet when in operation.
In some aspects, the biological sample includes whole blood, wherein the whole blood includes a plurality of components. In some aspects, the plurality of components comprises magnetically labelled white blood cells, and wherein at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are not collected in the one or more circulating tumor cell outlets as compared to a total number of white blood cells present in the whole blood inserted into the first fluid inlet when in operation. This can mean, for instance, that at least 95%, at least 98%, at least 99%, at least 99.9%, or more of the white blood cells are collected in one or more of the filters and the waste outlet as compared to a total number of white blood cells present in the whole blood inserted into the sample fluid inlet when in operation. This can result in, for example, that at least 90%, 92%, 95%, or more of the unlabeled rare cells are collected in the one or more circulating tumor cell outlets as compared to a total number of unlabeled rare cells present in the whole blood inserted into the sample inlet when in operation.
Methods are provided for enriching, separating, or isolating unlabeled rare cells such as circulating tumor cells from a sample, e.g., a biological sample such as whole blood. In some aspects, the biological sample is or includes whole blood. In some embodiments the biological sample includes whole blood treated with a lysis buffer to lyse red blood cells as described above. In some aspects, the biological sample includes about 1 to 1000 circulating tumor cells per milliliter of the biological sample, including both natural samples and samples spiked with CTCs for research purposes. In embodiments, the biological sample comprises about 1-10 circulating tumor cells per milliliter of the biological sample. Examples of the circulating tumor cells can include those selected from the group consisting of a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
In embodiments, the biological sample is a sample from an animal host (e.g., a mammal, human, etc.). In embodiments, the biological sample is a fluid having a plurality of components (e.g., cells, fluids, etc.) (e.g., blood, urine, saliva, exudate, homogenized tissue in a biocompatible fluid, etc.). In embodiments, the biological sample is from a human. In embodiments, the biological sample is whole blood or whole blood that has been treated with lysis buffer to lyse red blood cells. In embodiments, the biological sample is whole blood, or RBC lysed blood from a human host having cancer or suspected of having cancer.
Untreated biological samples have various amounts of circulating tumor cells, dependent on the type of cancer, the stage of cancer, other cancer treatments, etc. In general, un-spiked samples from a host have about 1-10 circulating tumor cells/ml of the biological sample. However, for research purposes, sometimes samples are spiked with additional CTCs. Also the level of CTCs can be higher depending on the tumor grade of a host. Thus, in embodiments, the biological sample can have from about 1 to about 1000 circulating tumor cells/ml of biological sample. It will be understood that for research purposes even higher amounts of circulating tumor cells may be present in a sample and still be within the scope of the present disclosure. However, unlike some previous methods, the present methods/devices/systems are able to detect/enrich CTCs from a sample having 10 or fewer CTCs/ml of biological sample.
In embodiments, the CTCs can include, but are not limited to, a primary cancer cell, a lung cancer cell, a prostate cancer cell, a breast cancer cell, a pancreatic cancer cell, and a combination thereof.
Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. 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.
Profiling circulating tumor cells (CTCs) in cancer patients' blood samples will lead to greater understanding of the complex and dynamic nature of metastasis. This task is challenged by the fact that CTCs are not only extremely rare in circulation but also highly heterogenous in their molecular programs and cellular functions. The present example describes an embodiment of a combinational approach for the simultaneous biochemical and functional phenotyping of patient-derived CTCs, using an integrated inertial ferrohydrodynamic cell separation (i2FCS) method and a single-cell microfluidic migration assay. This combinatorial approach offers unique capability to profile CTCs on the basis of their surface expression and migratory characteristics. We achieve this using the i2FCS method that successfully processes whole blood samples in a tumor-cell-marker and size-agnostic manner. This i2FCS method enables an ultrahigh blood sample processing throughput of up to 2×105 cells s−1 with a blood sample flow rate of 60 mL h−1. Its short processing time (10 minutes for a 10 mL sample), together with a close-to-complete CTC recovery (99.70% recovery rate) and a low WBC contamination (4.07-log depletion of leukocytes), result in adequate and functional CTC for subsequent studies in the single-cell migration device. For the first time, this example describes this novel approach to query CTCs with single-cell resolution in accordance with their expression of phenotypic surface markers and migration properties, revealing the dynamic phenotypes and the existence of a high-motility subpopulation of CTCs in blood samples from metastatic lung cancer patients. This method can be applied to study the biological and clinical values of invasive CTC phenotypes.
Circulating tumor cells (CTCs) are implicated in the formation of metastatic tumors, which is responsible for as much as 90% of cancer-related mortality.1-6 While the number of tumor cells in blood circulation has been correlated to clinical outcomes,7-9 it has become clear that enumeration of CTCs alone is not sufficient in understanding their multifaceted role in metastasis, in which CTCs participate in nearly all aspects of the process.3 ⋅ 10⋅ 11 Cancer patients have CTCs of varying phenotypes in their blood circulation; 1⋅ 4 ⋅ 10⋅ 12-15 while some cells passively detach themselves from the primary tumor,16 a fraction of them gain the ability to actively invade distance organs through modifying their cellular programs, morphology and surrounding tissues.17 Cells of this invasive phenotype often exhibit a high-motility trait that facilitates hematological spread, giving them the greatest threat of metastasis.3⋅ 10⋅ 11 ⋅ 19 Despite rapid advances in the understanding of the molecular mechanisms of CTCs,4⋅ 13⋅ 15 functional properties of invasive CTC phenotype(s) remain poorly understood due to the limitations of existing CTC isolation and phenotyping methods.20-22
CTC's extreme scarcity in blood circulation (<10 CTCs per one milliliter of whole blood) and a lack of methods for the isolation of adequate and functional cells represent a significant bottleneck in studying the invasive phenotypes of CTCs.20⋅ 22 CTCs are highly heterogeneous in their biological and biophysical characteristics with multiple phenotypes co-existing, which can evolve dynamically over the course of metastasis.3⋅ 10⋅ 11 Existing isolation techniques relying on the expression of tumor cell surface epitopes bias the sampling population and reduce the heterogeneity of captured cells.20 These techniques also lead to immobilized and non-functional CTCs and limit possibility of conducting functional studies.20 Physical property separation methods relying on size-based selection can separate larger CTCs from smaller leukocytes without limiting to molecular markers for selection. However, the isolated cells are contaminated with a large number of leukocytes and may also miss CTCs that were morphologically similar to the leukocytes. As such, current microfluidic methods for invasiveness phenotyping of tumor cells were mostly confined to cultured cancer cells rather than patient-derived CTCs.23-27 New methods are needed to isolate adequate and functional CTCs from patient samples so that the properties of invasive cells can be identified and characterized.
Here we report a novel combinational approach, which first uses an integrated inertial ferrohydrodynamic cell separation (i2FCS) method to recover virtually all CTCs from blood samples with minimal contamination using an approach that is not reliant on tumor cell markers and cell size for separation. Adequate and functional CTCs isolated from this method enable quantitative profiling of their biochemical and functional properties using a microfluidic assay that can track single tumor cell's chemotactic migration over time. In isolating CTCs when they are present at extremely low levels in the whole blood, we find that i2FCS method enables an ultrahigh blood sample processing throughput of up to 2×105 cells s−1 with a sample flow rate of about 60 mL h−1, resulting in an approximate 10 minute device processing time for a standard 10 mL of blood sample. The short processing time, together with a close-to-complete CTC recovery rate of 99.70% and a low WBC contamination of ˜507 WBCs carryover per milliliter blood processed, preserve isolated CTCs' viability and biological functions, allowing simultaneous biochemical and functional phenotyping of single tumor cells isolated from cancer patient's blood. Using this approach, we reveal a great diversity of biochemical and functional phenotypes of CTCs with single-cell resolution. CTCs with different levels of epithelial and mesenchymal marker expression exhibit varying chemotactic migration profiles, and there exists a high-motility subpopulation of CTCs in the patient's sample.
Overview of the i2FCS Approach
The integrated inertial ferrohydrodynamic cell separation (i2FCS) approach leverages the integration of cell size-based inertial focusing and cell magnetization-based ferrohydrodynamic separation (
In the second stage of the approach, inertially-focused cell streams are ferrohydrodynamically separated into different spatial locations according to their magnetization difference. Its physical principle, illustrated in
Design Principles of the i2FCS Approach
In the present example, the i2FCS approach was optimized to realize an isolation of functional CTCs in a tumor cell marker and size agnostic manner. Optimized i2FCS devices have the following characteristics: (1) a nearly complete isolation of CTCs from blood samples with 99.70% recovery rate; (2) an ultrahigh throughput of >600 millions of nucleated cells per hour (up to 200,000 cells s−1) and a ultrahigh sample flow rate of 60 mL h−1; (3) an extremely low carryover of ˜507 WBCs for every 1 mL of blood processed; (4) isolated CTCs preserving their initial viability and functions and enabling their biochemical and functional analysis. These performance characteristics were realized through optimizing i2FCS devices' geometry, magnetic field pattern, WBC functionalization, sample flow rate and ferrofluid concentration. A physical model that could predict the dynamics of cells in the i2FCS devices was developed for the optimization process.32⋅ 33
Firstly, the channel dimensions of both inertial focusing and ferrohydrodynamic separation stages in i2FCS were designed to accommodate a high blood sample flow of 60 mL h−1, which greatly reduced the device processing time of blood samples (10 minutes for a standard 10 mL blood sample). For the inertial focusing stage, we designed it so that both tumor and blood cells with diameters larger than 4 μm could be efficiently focused at a flow rate of 60 mL h−1. The geometry of the inertial focusing stage was fine-tuned so that the particle Reynolds number (Rp) was about 5.4, and the channel Reynolds number (Rc) was about 51.5 when the flow rate was about 60 mL h−1, ensuring a well-focused cell stream (˜100 μm in width) before the ferrohydrodynamic separation stage. For the ferrohydrodynamic separation stage, the channel dimension (about 54.8×about 1.2×about 0.06 mm, length×width×height) was optimized so that the channel Reynold's number was about 21.3 when the sample flow rate was about 60 mL h−1, ensuring unperturbed laminar flow conditions during CTC isolation.
Secondly, we designed the generation of magnetic fields in i2FCS with a sextuple magnet configuration (
Throughput, Recovery, Purity and Biocompatibility of the i2FCS Approach
Using the optimized i2FCS device and operating parameters, we validated it with spiked cancer cells from a total of 11 cultured cancer cell lines, including 4 breast cancer cell lines (MCF7, MDA-MB-231, HCC1806, HCC70), 4 non-small cell lung cancer cell lines (A549, H1299, H3122, H520), 2 small cell lung cancer cell lines (DMS79, H69), and 1 prostate cancer cell line (PC-3). We evaluated the performance of i2FCS in the cancer cell isolation, including sample flow rate and cell-processing throughput, cell recovery rate, WBC contamination, viability and proliferation of isolated cells.
We further challenged the device with 10 additional cancer cell lines with distinct size profiles (
Lastly, we investigated the effect of the device processing on the cells' viability and proliferation. The combination of low ferrofluid concentration (0.015% of magnetic content by volume) and laminar flow conditions in the i2FCS device showed little impact on the viability, intactness and proliferation of the isolated cancer cells.
To evaluate the performance of i2FCS in isolating heterogeneous CTCs in clinical samples, we conducted a study of samples collected from 2 patients exhibiting stage IV metastatic non-small cell lung cancer. Immunofluorescent staining was used to distinguish CTCs and WBCs, and CTCs of different phenotypes. We used the i2FCS devices to process blood samples from the patients, who were recruited and consented at the University Cancer and Blood Center (Athens, Georgia) under an approved IRB protocol (University of Georgia, VERSION00000869). Surface markers corresponding to epithelial and mesenchymal phenotypes were chosen because CTCs are reported to go through EMT, epithelial to mesenchymal transition, in which original epithelial tumor cells transition into stem-like mesenchymal cells.10 ⋅ 11 ⋅ 37 The loss of epithelial characteristics and the acquisition of mesenchymal characteristics are closely linked to the tumor cells' high motility and invasiveness to create a new tumor site.10 ⋅ 37-39 CTCs of this functional phenotype are therefore the focus of this study. 20 mL of blood sample from each patient was processed by the i2FCS devices. A quarter of the isolated cells were used for biochemical phenotyping through immunofluorescent staining with an epithelial marker (EpCAM) that is downregulated in EMT,20⋅ 37 ⋅ 38 two mesenchymal markers (vimentin and N-cadherin) that are upregulated in EMT, 18⋅ 37⋅ 38 a leukocyte marker (CD45), and a nucleus marker (DAPI) for their identification. WBCs were identified as CD45 positive and DAPI positive (EpCAM−/Vim−/N-cad−/CD45+/DAPI+). CD45 negative and DAPI positive CTCs were classified into three different phenotypes including epithelial phenotype (EpCAM+/Vim−/N-cad−), mesenchymal phenotype (EpCAM−/Vim+/N-cad−, EpCAM−/Vim−/N-cad+, or EpCAM−/Vim+/N-cad+), and mixed epithelial and mesenchymal phenotype (EpCAM+/Vim+/N-cad− or EpCAM+/Vim+/N-cad+).
Examples of isolated CTCs are shown in
Isolated CTCs from both patients were intact, indicating a minimal impact of the device processing on the cells' morphology. Consistent with previous reports,32⋅ 41-46 the effective cell diameter of isolated CTCs, defined as the maximum Feret diameter of the cells under bright-field imaging, showed a high level of variation for both patients. The effective diameters of randomly selected (n=75) CTCs from patient A's sample were 13.29±6.13 μm (mean±s.d.), with the smallest diameter being 5.88 μm and the largest being 33.74 μm (
We further characterized the biochemical phenotypes of the isolated CTCs through their surface antigen expression using the above-mentioned epithelial and mesenchymal markers. The proportion of each phenotypic subtypes of CTCs are summarized in
Adequate and functional CTCs isolated from the i2FCS approach enable their simultaneous biochemical and functional phenotyping. In this study, we accessed how CTC subpopulations with different levels of epithelial and mesenchymal marker expression affect their chemotactic migration. We chose cell migration to access CTCs' functions because high motility of these cells are implicated in the metastatic spread, including local invasion into surrounding stroma and intravasation into blood circulation, extravasation into parenchyma of foreign tissue, colonization and formation of metastatic lesions.3 ⋅ 10 ⋅ 11 ⋅ 18 ⋅ 19 The identification of high-motility CTCs would facilitate the prediction of a patient's risk of developing metastasis and the design of personalized therapeutics. i2FCS's ultrahigh recovery rate allows us to isolate virtually all CTCs from the patient samples, which potentially contain a subpopulation of these highly motile CTCs. In order to identify this subpopulation, we developed a new microfluidic assay that tracked cells' chemotactic migration with single cell resolution over a 24-hour period in confined microchannels.
CTC isolation and migration characterization process is shown in
In constructing the microfluidic device and assay for CTCs chemotactic migration phenotyping, we applied the following design principles. Firstly, we chose to use chemotactic migration to guide CTC's migratory direction in the microfluidic assay because CTCs are most efficient when the cell is involved in directed migration.47 ⋅48 We used a spatial gradient of growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and fetal bovine serum (FBS), to guide CTCs' migration in the microchannels,49 and a spatial gradient of Slit2 to inhibit the migration of carryover WBCs.50 ⋅ 51 The gradient of growth factors were maintained via a continuous perfusion for a 24-hour period in the microchannel to enable chemotactic migration of CTCs (
This assay was first validated using H1299 lung cancer cells to show that it could differentiate migratory versus non-migratory subtypes (
We estimated that a total of approximately 1260 CTCs isolated from a 10 mL blood sample were seeded in the microfluidic migration device at the start of the migration assay. The number of CTCs was calculated from the 252 CTCs/mL concentration obtained through immunochemistry for Patient B′ sample. At the end of the 24-hour migration assay, we identified again through immunochemistry that a small percentage of initial CTCs (16.4%, 207 out of the 1260 seeded cells) remained in the migration device and exhibited chemotactic migration towards the growth factors' gradient. The other 83.6% of CTCs were likely apoptotic and washed away by the perfusion within the assay timeframe.
Comparison of i2FCS to Existing CTC Enrichment Methods
We objectively evaluated i2FCS's performance in CTC separation to existing methods, using four commonly used metrics in calibrating CTC isolation methods, including the cell-processing throughput, CTC recovery rate, WBC contamination or carryover at device output and integrity of enriched cells. i2FCS method reported an ultrahigh blood sample processing throughput of up to 2×105 cells s−1 with a blood sample flow rate of 60 mL h−1. It resulted in a close-to-complete recovery of spiked cancer cells (99.70% recovery rate) and an ultralow WBC contamination (4.07-log depletion of leukocytes, removing 99.992% of the leukocytes from the blood samples, with approximately 507 WBC carryover per 1 mL of processed blood). The short processing time of i2FCS (10 minutes for 10 mL of blood) and complete recovery of CTCs produced adequate, viable and functional cells for subsequent cell-migration studies. We compared iFCS's performance to a total of 49 recently published CTC separation methods (see supplementary information) and found that i2FCS had better overall performance in the above-mentioned four metrics than existing methods.
We also compared the performance of i2FCS to two generations of CTC-iChip in Table 1 (
The present example demonstrates an integrated method that allowed for the first time simultaneous biochemical and functional phenotyping of patient-derived single circulating tumor cells. The method leveraged an integrated inertial ferrohydrodynamic cell separation (i2FCS) approach for a tumor cell marker and size agnostic isolation of CTCs from patient samples. This approach yielded remarkable CTC isolation performance including a complete isolation of CTCs from blood samples with a 99.70% recovery rate, an ultrahigh throughput of >600 millions of nucleated cells per hour, a ultrahigh blood processing flow rate of 60 mL h−1, and an extremely low carryover of ˜507 WBCs for every one milliliter of blood processed. Furthermore, isolated CTCs from i2FCS preserved their functional properties and enabled their biochemical and functional phenotypes to be quantitively queried via a single cell migration assay.
In samples collected from two metastatic lung cancer patients, i2FCS and the migration assay enabled the sensitive profiling of CTCs' heterogeneity according to their surface antigen levels and migration phenotypes. CTCs profiled in samples collected from the patients revealed that there was a great level of diversity in the phenotypes of CTCs. CTCs exhibited variable levels of epithelial and mesenchymal antigen expressions and morphologies, confirming the marker and size agnostic isolation of the approach. Isolated cells were accessed for their motility towards a gradient of growth factors in a migration assay with single-cell resolution, revealing the existence of a high-motility subpopulation of CTCs in one of the patients' sample.
The i2FCS and migration assay approach could be potentially adapted to a variety of applications in cancer research. CTCs isolated from the i2FCS can readily be recovered with intactness and preserved biological functions, therefore facilitating further downstream analysis and culture. This approach allows multiplexed queries of functional CTCs, which makes it possible to analyze CTCs for their complex roles in metastasis. Experiments using this approach can be implemented using a standard syringe pump with microfluidic devices that are straightforward to fabricate and operate, making it relatively easy for laboratory adoptions.
Magnetic field and particle separation performance was simulated and optimized in MATLAB (MathWorks, Natick, MA) using a physical model, which predicted trajectories of cancer cells and labeled WBCs in the microfluidic channel coupled with a sextuple configuration of magnets.32 ⋅ 33 (incorporated herein by reference)
The master mold containing the microfluidic structures was fabricated using standing photolithography methods with SU-8 2025 photoresist (Kayaku Advanced materials, Westborough, MA). The height of the structures was measured to be 60 μm. The 1 mm thick PDMS layer was prepared with Sylgard 184 silicone elastomer kit (Ellsworth Adhesives, Germantown, WI) in a 1:7 ratio of cross-linker and base, and cured at 60° C. for 4 hours. After bonding with the inlet and outlet layer (5 mm thick PDMS), the devices were oven baked at 80° C. for 20 minutes following by a hotplate at 150° C. for 1 hour. The device was placed within a custom aluminum manifold that held six N52 NdFeB permanent magnets (K&J Magnetics, Pipersville, PA) in a sextuple configuration. The magnets had a geometry of 50.8 mm×6.35 mm×6.35 mm (L×W×H) and had a remanent magnetization of 1.48 T each. Before each use, the devices were sterilized with 70% ethanol and then primed with 1×PBS supplemented with 0.5% (w/v) BSA and 2 mM EDTA (Thermo Fisher Scientific, Waltham, MA).
The water-based ferrofluid was a colloidal suspension of maghemite nanoparticles, synthesized by a chemical co-precipitation method following developed protocol.58⋅ 59 (incorporated herein by reference). The saturation magnetization (1,107 A m−1) and volume fraction of the ferrofluid (0.298%, v/v) were measured by a vibrating sample magnetometer (VSM, MicroSense, Lowell, MA). The viscosity of the ferrofluid (1.7 mPa s−1) was characterized via a compact rheometer (Anton Paar, Ashland, VA) at room temperature. The diameter and morphology of maghemite nanoparticles were determined to be 10.91±4.87 nm (mean±s.d.) with a transmission electron microscopy (TEM; FEI, Eindhoven, the Netherlands).
11 human cancer cell lines including four breast cancer cell lines (MCF7, MDA-MB-231, HCC1806, and HCC70), four non-small cell lung cancer (NSCLC) cell lines (A549, H1299, H3122, and H520), two small cell lung cancer (SCLC) cell lines (DMS79 and H59) and one prostate cancer cell line (PC-3) were purchased from ATCC (Manassas, VA). Cell cultures followed the manufacturing instructions. Breast cancer cell lines MCF7 and MDA-MB-231 were cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA) and the other cell lines were cultured in RPM 1640 medium (Thermo Fisher Scientific, Waltham, MA). DMEM and RPMI medium were supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA), 1% (v/v) penicillin/streptomycin solution (Thermo Fisher Scientific, Waltham, MA), and 0.1 mM non-essential amino acid (NEAA, Thermo Fisher Scientific, Waltham, MA). All the cell lines were cultured at 37° C. with 5% CO2. When the cells grown into 80% confluence, cells were washed twice with PBS by gently shaking the cell culture flask. This step was required to remove dead cells and debris. Cells were released with 0.05% trypsin-EDTA solution (Thermo Fisher Scientific, Waltham, MA), centrifugated (5 min, 500 g) to remove the supernatant, and resuspend in 1× Dulbecco's Phosphate Buffered Saline (DPBS, Thermo Fisher Scientific, Waltham, MA). To track the cell trajectories in the i2FCS device, cells were either stained with 3 μM CellTracker Green or 3 μM CellTracker Orange (Thermo Fisher Scientific, Waltham, MA) for 30 minutes at 37° C. and then washed and resuspended with culture medium. Cells were counted with Countess 2 (Thermo Fisher Scientific, Waltham, MA) and diluted to 104 cells per mL with culture medium. After dilution, the exact number of cells was confirmed with Nageotte counting chamber (Hausser Scientific, Horsham, PA). Variable number (10, 50, 100, and 200) of cancer cells were spiked into 0.015% (v/v) ferrofluid for spiking experiments.
Recovery Rate and Purity Calculation of i2FCS
Cells collected from the CTC outlet and WBC outlet were stained with 2 μM DAPI (Thermo Fisher Scientific, Waltham, MA) to stain cell nucleus, and counted with a Nageotte counting chamber. Cells with CellTracker (Green/Orange) signal were identified as cancer cells, while other cells only expressing DAPI signal were identified as WBCs. The recovery rate of i2FCS was calculated by (Ncancer_cell@CTC_outlet/(Ncancer_cell@CTC_outlet+Ncancer_cell@WBC_outlet))×100%. The purity was characterized by the WBC carryover NWBC@CTC_outlet, the depletion rate (1−N(NWBC@CTC_outlet/NTotal_WBC)×100%, and the log depletion rate log(NTotal_WBC/NWBC@CTC_outlet).
Cells suspended in PBS were deposited on a microscope slide and imaged with an inverted microscope (Axio Observer, Carl Zeiss, Germany) in bright field mode. Cell morphologies were analyzed with ImageJ software. Effective cell diameter was measured as the maximum Feret diameter of the cells under bright-field imaging.
Short-term cell viability of lung cancer cell line H1299 after i2FCS processing was characterized with a Live/Dead assay (Thermo Fisher Scientific, Waltham, MA) following the manufacturer's protocol. All cells are alive at the start of the viability characterization. Dead cells and cell debris were removed by PBS wash after cell culture. For long-term proliferation, the isolated H1299 cells from i2FCS device were washed three times with cell culture medium to remove the ferrofluid, and then the cells were re-suspended with culture medium and transferred into a T25 flask. (Corning, Corning, NY). The cells were then cultured at 37° C. (5% CO2) under a humidified atmosphere. Cellular morphology was inspected every 24 hours.
Complete blood count (CBC) reports of cancer patients' blood samples were used to determine the number of WBCs to optimize WBC labeling. Whole blood was firstly labeled with biotinylated antibodies including anti-CD45 (eBioscience, San Diego, CA), anti-CD45RA (eBioscience, San Diego, CA), anti-16 (eBioscience, San Diego, CA), anti-66b (Biolegend, San Diego, CA), and anti-CD3 (Biolegend, San Diego, CA) for 30 minutes at room temperature. The antibody-conjugated blood was lysed with RBC lysis buffer (eBioscience, San Diego, CA) for 5 minutes following by centrifugation (500 g, 5 minutes) at room temperature. After removing the supernatant, the cells were resuspended with 1×PBS and incubated with washed Dynabeads (Thermo Fisher Scientific, Waltham, MA) for 30 minutes on a rocker. All the labeling and washing procedures were performed following the manufacturer's protocol. Blood cells were suspended in the same volume of 0.015% (v/v) ferrofluid supplemented with 0.1% (v/v) Pluronic F-68 surfactant (Thermo Fisher Scientific, Waltham, MA) before processing using the device.
After device processing, isolated cells were concentrated through centrifugation (600 g, 5 minutes) and immobilized onto poly-L-lysine (Sigma-Aldrich, St. Louis, Mo) coated glass slides. Isolated cells were fixed with 4% (w/v) paraformaldehyde (Santa Cruz Biotechnology, 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 at room temperature. Cells were then blocked with Ultracruz blocking reagent (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes at room temperature to block nonspecific binding sites. Cells were then immunostained overnight at 4° C. with primary antibodies including EpCAM-Alexa Fluor 488, N-cadherin-Alex Fluor 594, Vimentin-Alex Fluor 647 (Santa Cruz Biotechnology, Dallas, TX), CD45-PE (BD Bioscience, San Jose, CA). Cells were stored in mounting medium supplemented with DAPI (Fluoroshield™ with DAPI, Sigma-Aldrich, St. Louis, Mo).
Isolated CTCs were loaded into a microfluidic migration device for single cell migration assay.10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, Waltham, MA), 20 ng mL−1 epidermal growth factor (EGF, Thermo Fisher Scientific, Waltham, MA), and 20 ng mL−1 basic fibroblast growth factor (bFGF, Thermo Fisher Scientific, Waltham, MA) were used as the chemoattractants for the CTCs, while 5 μg mL−1 Slit2 (Thermo Fisher Scientific, Waltham, MA) was used to inhibit the migration of WBCs. After cell loading, migration assay was performed in an incubator (37° C., 5% CO2) for 24 hours. Cells was immunofluorescently stained in the device to identify their cell types.
It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments 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.
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 63/391,984, having the title “METHODS, SYSTEMS, AND DEVICES FOR SEPARATING AND CHARACTERIZING CIRCULATING RARE CELLS FROM BIOLOGICAL SAMPLES,” filed on Jul. 25, 2022, the disclosure of which is incorporated herein in by reference in their entireties.
This invention was made with government support under Grant Nos. 1150042, 1659525 and 1648035 awarded by the National Science Foundation, by No. UL1TR002378 and 1R41EB028191-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63391984 | Jul 2022 | US |
