Patent Application
20200171509
This invention relates to the static or continuous magnetic separation of specific entities from mixtures where the separation of target entities is done in a one-step process. It is universally applicable to the harvest or removal of such entities for bioprocessing of biological molecules, cells of all types, virus particles, and the like, and for small- to large-scale separations of the same. By manipulation of the scientific principles that underlie this invention, specific targets can be conveniently captured for analysis, harvested or subjected to further processing on a collection surface free of bystander components. The invention also employs newly discovered properties of a class of magnetic nanoparticles that enable these materials to be used to perform one-step static separations and enable continuous magnetic separations. The principles employed and the methods disclosed completely obviate the need for washing of targeted entities such as cycles of resuspension and magnetic separation for removal of contaminating substances in the case of static separation or complex cycles of sample introduction and harvest to perform continuous processing.
Magnetic separations in industrial applications and in biological systems are well known in the art. In the case of removing ferromagnetic contaminants from dry mixtures or slurries in a variety of manufacturing processes, simple solutions for continuous operations are well known. On the other hand, in biological systems, truly continuous devices and protocols for magnetic separations that yield high purity product at reasonable yields have not, in fact, been realized.
To create a continuous separation process in its simplest form for the above-mentioned biological entities, leveraging an intrinsic property of the entities to be separated has been the most successful route. For example, owing to their different sizes but relatively similar-sized nuclei, mammalian cells have differing densities, with the smaller cells being denser. Thus cells of different sizes are readily separated in density gradients by centrifugation. The continuous introduction of cell mixtures into specialized centrifuge heads containing density gradients and the continuous removal of a density layer containing desired cells is well known in the art. Also well known in the art is free-flow electrophoresis for the separation of proteins and macromolecules. In that case, a stream containing a mixture of proteins or macromolecules is introduced at some position near the top of a suitable vessel containing appropriate flowing electrolyte and with an electric potential placed on its sides. If the sample contains materials of differing electrophoretic mobility, the different species will move apart from each other as they flow downwards and can be harvested from different ports along the bottom of the apparatus. Each of the foregoing examples is enabled by the fact that one or more entities of interest can be moved out of a mixture or away from the other species therein effectively by a single step.
Positive selection magnetic separation for cells and other biological entities are typically done by batch methods. For separations done in vessels (e.g., tests tubes, beakers, bags), several process steps must be performed to obtain pure product. In magnetic separations, such steps are typically done in 2-3 cycles to obtain purified product, and could comprise the following steps: 1) magnetically labeled entities are pulled to the side of the vessel; 2) supernatant containing unlabeled entities is removed and discarded; 3) the vessel is removed from the magnetic gradient; 4) wash buffer is added; 5) entities are re-suspended; and 6) the re-suspended entities are again magnetically separated. In the case where magnetic labeling is such that high-magnetic-field-gradient columns are employed, the process could be as follows: 1) the mixture containing magnetically labeled entities is passed through an appropriate column in a magnetic field; 2) labeled entities magnetically adhere to the column; 3) the column is washed free of sample and unlabeled entities that might have been trapped in the column; 4) the column is removed from the magnetic field; and 5) cells are recovered by passing buffer through the column, sometimes with the augmentation of vibration of the column. For both of these systems, not only are they complicated by their many steps, they do not lend themselves to continuous separation.
Many inventions address attempts to create continuous magnetic separation systems or, in some cases, one-step magnetic separation procedures by employing a variety of means toward those ends. Those works are summarized below:
Ching-Jen, et al.'s patents (U.S. Pat. Nos. 6,129,848A, 6,132,607A and U.S. Pat. No. 6,036,857A) describe methods for the continuous separation of chemicals, cells or components from blood (e.g., WBCs). Ching-Jen, et al.'s methods represent a series of batch-mode separations to effect a continuous separation (i.e., discontinuous or batch processing).
U.S. Pat. No. 4,910,148 to Sorenson, et al. relates to a method and device for separating magnetized particles from biological fluids, particularly white blood cells using a monoclonal antibody to link the cells to magnetic beads. Sorenson's separation is static (i.e., no flow) and is conducted in a plastic blood bag. The magnetic beads are linked to malignant white blood cells by an agitation process and then a magnetic field is applied to keep the white blood cells bearing magnetic beads in the disposable plastic bag. The Sorenson device also requires space between the magnets, which does not optimize the magnetic force. The back plate of the Sorenson device is a soft magnetized material and the magnets are samarium-cobalt. Sorenson has a volume limitation since it uses a blood bag (150 mL) and there is no decoupling between the beads and the white blood cells. Further, the cells remain in the disposable blood bag after separation.
U.S. Pat. No. 5,514,340 to Lansdorp, et al. relates to a device for separating magnetically labeled cells in a sample using an applied magnetic field. Lansdorp uses magnetized screens to attract the magnetic particles allowing the biological fluid to be caught in the magnetic wires of the screen. The magnets used in Lansdorp must constantly be cleaned since there is contact between the magnets and the blood cells.
U.S. Pat. No. 5,567,326 to Ekenberg, et al. relates to an apparatus and methods for separating magnetically responsive particles from a non-magnetic test medium in which the magnetically responsive particles are suspended. In Ekenberg, small patch amounts of biological fluid are placed in a tube then a magnetic pin is inserted in the fluid for separation.
U.S. Pat. No. 4,988,618 to Li, et al. relates to a magnetic separation device for use in immunoassay or hybridization assay procedures. The Li device comprises a base having a plurality of orifices for receiving non-ferrous containers which hold the sample and the assay components, including ferrous particles. The orifices are surrounded by a plurality of magnets which are spaced about the peripheral of the orifices.
U.S. Pat. No. 4,935,147 to Ullman, et al. relates to a method for separating a substance from a liquid medium, particularly applicable for separation of cells and microorganisms from aqueous suspension, but also for the determination of an analyte. Although Ullman discusses a method with a reversible non-specific coupling, the method is not continuous nor does it utilize a multi-dimensional gradient.
U.S. Pat. No. 5,968,820 to Zborowski et al describes a quadrupole based continuous separator that employs laminar flow of magnetically labeled sample adjacent to flowing buffer to effect continuous separation. No performance data is given. However, the system has been employed for the isolation of clustered pancreatic islet cells by Weegman et al [J. Diabetes Res. 2016, Article ID 6162970, (2016)]. That system can only give high purity at very low concentrations which is a significant limitation.
U.S. Pat. No. 5,541,072 to Wang et al. describes a continuous feed separator that captures target cells in a hydrodynamically designed flow cell placed between arrays of alternating bucking magnets. That system proved very effective for negative selection, whereas positively selected cells were difficult to recover. In the work leading up to that disclosure, attempts were made to employ a two vector system—unidirectional downwards flow and a strong magnetic gradient, similar in concept to '820—to create a continuous separation system. The notion employed was analogous to free flow electrophoresis where a stream of mixed proteins is directed into a downward flowing rectangular column while a strong electric potential is exerted in the horizontal direction. In that system, proteins of high charge/mass ratio rapidly separate from the stream and can be harvested because of their lateral displacement from the original stream.
When that simple notion was explored for a ferrofluid-based system (a system employing highly magnetic colloidal nanoparticles such as those described by Liberti et al. in U.S. Pat. Nos. 5,597,531 and 5,698,271), it was found that when a stream of cells labeled with magnetic nanoparticles (90-140 nm) was introduced into a rectangular volume of downwardly flowing buffer, instead of magnetically labeled cells being pulled out of the stream towards the higher gradient region and becoming separated from non-target cells, the entire stream moved as a phase towards the magnetic gradient that was applied to the system. To further understand this behavior, '072 discloses the results of an in-depth series of experiments to explore our discovery of this phenomenon, which was referred to as Ferro-phasing. Just like the streaming experiments previously mentioned, when a droplet of magnetic nanoparticles mixed with food coloring was introduced into a magnetically inert fluid such as water in a microtiter well which was positioned in a magnetic quadrupole device, the colored liquid and the magnetic nanoparticles (i.e., ferrofluid) immediately formed an annular cylinder distributed around the periphery of the microtiter well while the water formed a clear cylinder within the annulus. In other words, the ferrofluid/food dye mixture moved as a unitary phase to the regions of highest magnetic field gradient. Over time, the ferrofluid separated to the wall of the vessel, leaving behind a diffuse ring of food coloring. Importantly, the initial phenomenon clearly demonstrates that the food coloring acted like it was incorporated into a phase. If instead the food coloring was first mixed with the water and ferrofluid within the microtiter well and subsequently placed in a quadrupole separator, ferrofluid separated to the wall, leaving the food coloring behind.
One hypothesis offered to explain those experiments could be a consequence of the fact that ferrofluids and other magnetic nanoparticles are known to form long chains under the influence of magnetic field gradients (Liberti, unpublished observations, Ugelstad also). It can be shown that 8 μg (based on Fe mass) of 130 nm particles that are about 80% magnetite placed in a 1 cm3 chamber could form about 30,000 linear chains. Presuming that such strands would likely align parallel to each other, one could imagine that because of their highly hydrophilic nature, they would strongly interact with neighboring water molecules, resulting in a gel-like structure. Evidence of this gel-like structure, or Ferro-phase as it is referred to, is provided in U.S. provisional application No. 62/546,700 where it is disclosed how this phenomenon can be used to move or position non-magnetic entities such as small molecules, macromolecules, and cells contained therein.
Based on the observations that a two-phase system, comprising an inert fluid and a Ferro-phase, can be formed and maintained, it was concluded in '072 that a simple approach to magnetic separation analogous to free-flow electrophoresis using colloidal nanoparticles is not feasible. As noted, '072 does disclose an invention for large-scale continuous separation, but the need to further process collected target cells because of entrained non-target cells limits the invention.
The discoveries made and disclosed herein, as well as those previously disclosed in U.S. provisional application 62/480,397, tend to show that the conclusion made in '072 as regards the use of colloidal magnetic nanoparticles for doing one-step static or continuous magnetic separations was incorrect. Our recent discoveries on how Ferro-phasing can be overcome by density adjustments make such separations possible.
The present invention and that of provisional application U.S. 62/480,397 overcome the aforementioned problems, regarding the inability to create a magnetic separation system analogous to free-flow electrophoresis employing colloidally stable magnetic nanoparticles, by an effective and simple means for overcoming counteracting Ferro-phasing. It was discovered that Ferro-phasing can be counteracted by adjusting the density of the ferrofluid-containing phase, the non-ferrofluid-containing phase, or both. For example, if in a microtiter well, a ferrofluid-containing solution (about 3-10 μg Fe/mL) is layered over a buffer containing 1% sucrose, those layers will be stable over long periods of time. For the purposes of this invention, the term “layer” refers to a layer of medium or the like. On the other hand, if the microtiter well is placed over a downward-pulling magnetic device, the upper ferrofluid layer (more easily visualized by the inclusion of small amounts of food dye) will immediately move downward as a phase towards the magnet and become layered under the sucrose-containing buffer. If the well is subsequently moved off the magnet, the phases will revert to their original positions. It is notable that these phenomena can be repeated several times. On the other hand, if the sucrose level of the lower buffer layer is increased to 5% and the well is placed on the magnet, the phases will not move, though the ferrofluid will begin to move through the sucrose-containing lower layer toward the magnet. Thus Ferro-phasing can be overcome by adjusting the density of the solutions in accordance with the direction of the magnetic gradient. If target cells are being pulled upwards through a non-magnetic phase, the density of the lower ferrofluid-containing phase needs to be increased, with the degree of increase being related to the ferrofluid concentration. Thus a bottom layer of ferrofluid-containing solution with 0.5% sucrose overlaid with buffer when placed under an upward-pulling magnetic device will Ferro-phase such that the lower layer will move as a phase and replace the top layer.
By making such density adjustments, it has been discovered that it is possible to magnetically separate target cells out of a ferrofluid-containing phase, such that the target cells leave the phase that originally contained the ferrofluid solution and enter the non-ferrofluid-containing phase. Most importantly, it has been discovered that the non-ferrofluid-containing phase serves to effectively “wash” target cells free of non-target cells, giving exceptional purities in one step. This “washing” effect is confirmed by experiments which show that the height of the column of the non-ferrofluid-containing phase through which target cells traverse is proportional to the purity of the product.
Static One-Step Magnetic Separations
To evaluate the parameters of the herein disclosed two-phase separation system (i.e., a magnetic phase and a non-magnetic phase), extensive experiments were performed in microtiter wells wherein a solution containing ferrofluid-labeled cells was used as the bottom layer. A volume of buffer solution was layered on top of the bottom layer such that the well volume was slightly exceeded, forming a convex meniscus; in this way, when a slide was placed on top of the well, a small amount of buffer spilled out of the well, but no air gap was present between the buffer solution in the well and the slide. This arrangement is illustrated in
To determine the extent of “washing” by the buffer layer that is required to obtain high purities, further experiments were performed in microtiter wells wherein the height of the buffer column was varied (total column height was fixed at 11 mm). HPB cells (human-derived T cell line; CD3+) were labeled with biotinylated anti-CD3, subsequently labeled with streptavidin-functionalized ferrofluid, and spiked into buffer containing 5% sucrose. Cell mixtures were placed into microtiter wells with column heights of 2, 3, 4 and 5 mm, and appropriate volumes of sucrose-free wash buffer were subsequently layered on top of each cell mixture to fill each microtiter well to its maximum capacity. Polylysine-coated slides were placed on top of each filled vessel, and a bucking magnet device was used to provide a magnetic field gradient for 10 min. In each case, it was determined that target cells could be pulled through the various heights of wash buffer and that they were recovered essentially quantitatively (estimated by counting cells adhered to the slides). We then tested mixtures of magnetically labeled HPB cells (pre-stained with a red intracellular stain for identification) and unlabeled U937 cells (human-derived CD3− monocyte cell line; pre-stained with a green intracellular stain for contrast). It was determined that only the magnetically labeled HPB cells were pulled through the buffer layer and that no U937 cells were captured. Following these experiments, human red blood cells were spiked into the system at levels as high as 15% hematocrit, and again, only HPB cells were captured on the polylysine-coated slides.
Based on the ability to pull magnetically labeled target cells up from the ferrofluid layer, through the buffer layer in a highly purified state, and immobilize them on a polylysine-coated surface, attempts were made to recover cells from the collection surface. This was accomplished by placing cell mixtures of appropriate density containing ferrofluid-labeled cells into microtiter wells and layering less dense buffer on top. A Parafilm-wrapped, upward-pulling magnetic device was then placed atop the well in direct contact with the buffer layer. After 10 min of magnetic separation, the Parafilm-wrapped to magnetic device was lifted off the well, and the droplet adhering to the Parafilm was recovered and examined microscopically. With the demonstration that target cells could be recovered in a highly purified state, a more sophisticated system was created that would allow qualitative and quantitative analysis of product.
To perform a one-step magnetic separation using the device of
To perform a separation with direct immuno-magnetic labeling, a cell-containing solution 3 is incubated with ferrofluid (in the vessel 1, if desired), to which appropriate targeting molecules are coupled (e.g., monoclonal antibodies or other recognition molecules). In the case where an indirect immuno-magnetic labeling method is used, appropriate targeting molecules are incubated with the cell-containing solution 3 for an appropriate interval (5-15 min) and unbound targeting molecules are removed by various methods well known in the art. In many cases when employing ferrofluids, this removal step is unnecessary and the ferrofluid can be directly added, initially mixed, and allowed to bind. Since ferrofluid binding to targets is not affected by continual mixing (diffusion-controlled reaction), samples can be placed directed into the vessel 1 and positioned on the base 6. While incubation is taking place, the layering process can be performed. As no wash or re-suspension steps are required by this invention, target cells can be separated in as little as 20 min when employing direct labeling. For the indirect method, an additional 10-15 min would be required.
It should be understood that a system similar to that which is depicted in
It should also be noted that the system depicted in
To determine if larger magnetic particles which do not exhibit Ferro-phasing could be used in the single-step separation process disclosed herein, we examined 2.8 μm streptavidin-coated Dynabeads® (Dynabeads® M280 Streptavidin, Thermo Fisher Scientific) in a static separation. Cells that had been labeled with biotinylated antibody, subsequently labeled with streptavidin-coated Dynabeads®, and densified with sucrose were placed into a vessel 1. This dense cell-containing solution 3 was overlaid with a buffer layer 2, a collection plate 4 was placed atop the vessel 1, and an upward-pulling magnetic device 5 was placed atop the collection plate 4 as described above for the ferrofluid-based system. We determined that a one-step separation can be achieved, yielding high purity target cells. For the device used, yields were about 20% less than with ferrofluid, but this could likely be improved.
Comparing these two classes of magnetic particles, ferrofluids have some significant advantages over Dynabeads®. For example, ferrofluids are colloidal and their reactions are diffusion controlled, which allows the magnetic nanoparticles to remain suspended indefinitely and eliminates the need for mixing. In contrast, optimal reactions with Dynabeads® require mixing, and labeled cells must be processed in a timely manner to prevent settling. Nonetheless, the ability to incorporate density layering, where one layer contains a densified magnetically labeled mixture in contact with a less dense “washing” buffer which permits one-step separations without the need for additional cycles of re-suspension and re-separation, has wide utility.
Continuous Magnetic Separation
Based on the ability to 1) magnetically pull cells out of a dense phase and upward through a less dense phase—or alternatively, magnetically pull cells out of a less dense phase and downward through a dense phase—and 2) pull target cells through a sufficiently large column of wash buffer, which is very effective at preventing non-target cells from reaching the collection surface, there is clearly the potential to use this finding to create a novel system for continuous magnetic separation providing the phases can be introduced into, flowed through, and collected from an appropriate vessel without significant mixing. Hence, by eliminating Ferro-phasing, two useful systems are created.
To perform a continuous separation, the TDMS device can be conveniently loaded with the two liquids of different densities such that a distinct boundary 16 between them is established and maintained. This can be accomplished by pumping the denser of the two liquids (not containing the cell mixture) at a controlled rate into the chamber via inlet port 12 to fill the chamber 11 to a defined level (indicated by the dashed line in
As a cell-containing solution 3 flows to the right, magnetically labeled cells will move upwards in the chamber 11 tracing out an arc, the curvature of which will be a function of the speed with which the cells traverse the chamber 11, the densities and viscosities of the cell-containing solution 3 and wash buffer 2, the gradient of the magnetic field strength produced by the magnetic device 5, and each cell's magnetic load. It should also be clear that while the length of the chamber 11 will not affect the speed with which cells move upwards, the longer the chamber 11, the more rapidly the solutions can be flowed through it. By appropriately controlling flow, solution densities and viscosities, magnetic gradient, degree of magnetic labeling, and length of the chamber 6, targeted cells will move into the upper wash buffer layer 2 and exit through outlet port 15, where they can be harvested. It should be understood that there is essentially no limitation on the volume of cell suspension that can be processed through the TDMS device.
The primary purpose of the TDMS device depicted in
Since the arc that a magnetic entity makes as it moves toward the collection plate 4 where it binds is a function of the flow rate, solution density and viscosity, gradient of the magnetic field strength, and magnetic load of an entity, there are several manipulations that could be applied. For example, assuming a system of labeled entities which all have similar magnetic loads and are similar in size and shape, if the collection plate 4 is sufficiently long, those entities would be collected in a band along the length of the collection plate 4. If the direction of flow is from the left to the right, then increasing the flow rate should move the collected band to the right. Therefore if one desires to collect such entities along some particular length of the collection plate 4, the flow rate of the solutions would be increased (or decreased) as the separation proceeds. Thus the user can control the location and spread of magnetically captured entities.
On the other hand, should the magnetically labeled entities be heterogeneous as regards magnetic labeling, density, size, or shape, this will manifest in how they are distributed on the collection plate 4. Assuming a population of cells has a distribution of receptors, then regardless of the method of magnetic labeling (i.e., indirect or direct), their magnetic load would have an analogous distribution to the receptor distribution. As such, with appropriate control of the flow, solution properties, and magnetic field gradient, the collection pattern of such a population on the collection plate 4 would reflect its receptor distribution. That is, high-density-receptor cells with high magnetic loading would collect closer to the inlet than low-density-receptor cells with less magnetic loading. Hence, the TDMS device so described has analytical capabilities that are a direct result of the physics that the system imposes on magnetically labeled entities.
For a system where magnetic entities are to be captured on the collection plate 4, it may be desirable to confine them to a narrower band (i.e., in the dimension orthogonal to the direction of flow) as opposed to being spread across the entire width of the collection plate 4. To date, TDMS devices we have tested have had rectangular cross sections. Hence, with magnetic entities being pulled from one phase to the other phase, collected entities will be spread over the entire width of the collection plate 4. There are at least two means for narrowing the width of the collection band. One is to use a magnetic device whose gradient across the width of the collection plate 4 is non-uniform such that magnetic entities can effectively be collected in a narrow band. This can be achieved to simply by employing a bucking magnet arrangement where opposing magnets abut with no spacer (unlike the arrangement described above). In that case, the gradient of the magnetic field strength at the contact plane formed by the opposing magnets is extraordinarily high and non-uniform; in fact, this type of magnetic arrangement is well known to collect such magnetically labeled entities in a relatively narrow band. Alternatively, the cross section of the chamber 17 can be designed so as to force magnetically labeled entities to form a narrow band on the collection plate 4. It should be clear that both of these strategies can be employed in tandem to narrow the width of the collection band.
The following examples demonstrate the basic principles of this invention and various means for employing this invention for magnetic separations.
We have demonstrated that the greater the column height of the non-magnetic phase (i.e., buffer), the greater the purity without a significant change in yield. This effect was demonstrated using ferrofluid-labeled HPB cells (CD3+ cell line) spiked into RBC (15% hematocrit) and placed into microtiter wells with different column heights, over which buffer was layered of reciprocal column heights such that the total column heights of the two-phase systems were the same. As shown in the data below, the greater the column height of the non-magnetic phase (i.e., buffer), the more pure the product.
Leukapheresis product was labeled with anti-CD3 and subsequently labeled with ferrofluid. The suspension of magnetically labeled target cells and non-magnetically labeled non-target cells was diluted two-fold and placed in a rectilinear open-top vessel with interior dimensions of 1.0 cm wide×4.0 cm long×1.5 cm tall. In one case, 3 mL of labeled cell suspension was added to the vessel, followed by 3 mL of buffer layered on top (i.e., sample constituted 50% of the column height). In another case, 1.5 mL of labeled cell suspension was added, followed by 4.5 mL of buffer layered on top (i.e., sample constituted 25% of the column height). A cover slip was placed on top of each vessel, above which a magnetic device with a strong upward-pulling magnetic gradient was positioned. After a 10 min separation, the cover slip and the magnetic device were lifted off the vessel in tandem and rotated 180°. The cover slip was then removed from the magnetic device to retrieve the captured cells. By performing flow cytometry on the recovered cells, it was determined that the purity of the product (i.e., the % CD3+) was 97.8% for the 50% column height sample and 99.0% for the 25% column height sample.
Elongated separation chambers, similar in concept to that depicted in
In order to observe whether mixing of the phases was occurring during their passage through the chamber, sufficient food dye was added to make the liquids distinguishable from one another and make the boundary clearly visible. Using the aforementioned arrangement, it was determined that each solution could be pumped through the chamber at at least 1.5 mL/min with no signs of mixing or boundary disturbance for considerable lengths of time (tested up to 20 min). Since the obtainable flow rates were in excess of that required for a magnetically labeled entity to move from one phase to the extreme side of the other phase, it was apparent that flowing two solutions of different densities through such a chamber was achievable.
The chamber and peristaltic pump arrangement described in Example 3 was used for these experiments. The magnetic nanoparticles employed were proprietary ferrofluids prepared by a modification of Liberti et al. (U.S. Pat. No. 6,120,856). These materials have a mean diameter of 130 nm and are composed of quasi-spherical cores of magnetite (ca. 115 nm) coated with layers of either human or bovine serum albumin. They are highly magnetic, comprising greater than 80% magnetic mass.
Ferrofluid concentrations of 1.0, 2.5, 5.0 and 10 μg/mL were prepared in an isotonic cell buffer with added protein (1% w/v BSA). For experiments wherein the magnetic gradient pulled magnetic entities downwards, the above solutions were layered on top of a similar buffer containing 10% w/v sucrose. Chambers were loaded with layered solutions as described previously. When distinct and unperturbed flowing layers were observed, samples were introduced into the top flowing layer. Initial pumping rates for both solutions were 800 μL/min; hence, the dwell time of a nanoparticle in the chamber was about 5.5 min. At that rate, in all cases, ferrofluid was collected on the bottom of the chamber after traversing approximately 25% of the chamber distance. As expected, by increasing the flow rate, ferrofluid was collected after traversing slightly more than half the chamber distance. At a flow rate of 4 mL/min, the design of the inlet port created turbulence; however, with appropriate design modifications, rates of at least that high are feasible as the barrier between the two phases remained mostly intact.
In the invention disclosed herein, there are several parameters that can be controlled to pull magnetically labeled entities from a more dense solution to a less dense solution, or vice versa. Furthermore, those parameters can be tuned such that magnetically labeled entities that are pulled into the “clean” solution (i.e., the phase which is initially devoid of cells) exit the chamber rather than collecting within the chamber. In the case of an entity such as a mammalian cell, those adjustable parameters include dwell time of the magnetically labeled entities in the chamber (determined by solution flow rates and length of the chamber), solution properties (density and viscosity of the solutions), gradient of the magnetic field strength, and magnetic loading of the labeled entities.
For a particular cell type (e.g., a CD34+ human stem cell), this would be accomplished by labeling a cell suspension (obtained by bone marrow aspiration or from mobilized apheresis product) with anti-CD34 by appropriate incubation, washing to remove unbound antibody, and magnetically labeling with an appropriate magnetic nanoparticle (e.g., a ferrofluid coated with rat anti-mouse IgG or, alternatively, a ferrofluid coated with streptavidin if the anti-CD34 is biotinylated). Employing a chamber similar to that described here—4.35 cm in length—and flow rates through the chamber of between 0.2 and 3.0 mL/min, the degree of magnetic labeling that would prevent the target cells from being collected within the chamber would be determined. This might require decreasing or increasing the length of the chamber. Nonetheless, by controlling simple physical parameters, the appropriate conditions will be determined to permit collection of CD34+ cells with the “clean” solution exiting the chamber.
The mode of operation of this invention provides the potential to perform in-depth analysis of magnetic materials or materials that are magnetically labeled. Most magnetic separations are binary in nature; that is, in a quadrupole separation, entities either collect on the inner walls of the container or they do not. On the other hand, in TDMS devices, the distance a magnetic entity travels before being captured on the collection surface of the chamber provides information about its magnetic character. For example, if there is a distribution of magnetic labeling due to a distribution of receptor density for a given cell type, that would manifest in the way cells are magnetically collected; that is, a more narrow band on the collection surface would indicate a more uniform distribution of receptors, providing that the magnetic labeling is at saturation.
That would also be applicable for examining the heterogeneity of magnetic nanoparticles. A tight distribution of particle size (i.e., magnetic moment) would be indicated by a narrow band on the collection surface in a device and system so described by this invention. In the case of ferrofluid prepared in our laboratories, our manufacturing process typically yields nanoparticles with a mean size of 130 nm. However, we are aware that small particles (50-90 nm) are also produced in the process, which could be detected using a TDMS device.
Using the device and system described in this invention, two ferrofluid solutions—one having a distribution where 97% of particles are 135 nm and 3% are 80 nm, and a second having a distribution where 65% of particles are 135 nm and 35% range from 50-90 nm—could be tested. The experiments would be carried out using flow rates of 1.5 mL/min with the test ferrofluids in the upper, less dense solution. The distribution of ferrofluid collected on the bottom surface of the chamber would be expected to show a region of narrower deposition for the former sample versus a broader deposition for the more polydisperse sample (i.e., mirroring the size distribution obtained by particle size analysis). Hence, this invention could be used as an analytical tool.
There is considerable utility in this invention. It can be used to capture targets on a surface such that they can be recovered from that surface if that is desired, or they can be maintained on the surface and subjected to various treatments to permit a variety of subsequent analyses. Alternatively, this invention can be used to separate target entities from a complex mixture without the need to capture the target entities by adjusting either flow rate and/or magnetic gradient such that magnetically diverted target entities flow out of the chamber rather than being retained therein. It is noteworthy that samples that might contain rare events, such as circulating tumor cells (CTC), would benefit from this invention either by collecting cells outside the chamber or on a collection surface within the chamber because there is essentially no limit to how much sample can be processed using a TDMS device. This could be critically important in applications that use the presence and/or frequency of CTC as a diagnostic or prognostic indicator, wherein a significant quantity of blood must be processed in order to capture a reasonable number of CTC.
In the systems described in this disclosure, target entities are magnetically labeled, separated, and recovered (i.e., positive selection). It should be understood that non-target entities can be magnetically labeled, separated from the non-magnetically labeled target entities, followed by recovery of the latter population (i.e., negative selection). This might be desirable if recovery of “untouched” target cells is of benefit (e.g., if target cells might be activated upon magnetic labeling).
In the systems described in this disclosure, there are two layers of differing densities wherein, depending on the apparatus, the mixture from which an entity is magnetically separated can be in either layer. It should be understood that more than two layers can be used when practicing this invention. This might be desirable if specific reactions or other processing steps on magnetically labeled entities passing through one or more layers are of benefit. Such possibilities add to the utility of this invention.
While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims.
A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these publications is incorporated by reference herein.
Furthermore, the transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step, or material. The term “consisting of” excludes any element, step, or material other than those specified in the claim and, in the latter instance, impurities ordinarily associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps, or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All devices, device components, and methods described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Nos. 62/480,397 filed on Apr. 1, 2017; 62/529,574 filed on Jul. 7, 2017; 62/546,700 filed on Aug. 17, 2017; and 62/591,833 filed on Nov. 29, 2017 which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/025669 | 4/2/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62480397 | Apr 2017 | US | |
| 62529574 | Jul 2017 | US | |
| 62546700 | Aug 2017 | US | |
| 62591833 | Nov 2017 | US |
