Patent Application
20220387999
The present invention is directed to devices that allow for the separation of key cellular blood components of the immune system, directly applicable to the development of medicines for the treatment of infectious disease and cancer, as well as the separation of cells of interest in suspension from a heterogeneous population.
The isolation of key cell types in suspension from heterogeneous mixtures is an essential preparative step in medicine and biotechnology. For example, applications such as liquid biopsy, cancer immunotherapy, and cell manufacturing processes require high throughput and fine separation of cell subpopulations of the immune system. While the traditionally used density-based gradient separation methods provide enough cell numbers for many applications, the cell pelleting process often results in undesired ex-vivo immune cell activation at suboptimal isolation sensitivity. Other bulk cell centrifugation methods, such as counterflow centrifugation elutriation, have clinically relevant isolation yields, but rely solely on physical properties such as cell density and therefore lack specificity towards cell surface composition.
Magnetic activated cell sorting methods, on the other hand, solve the specificity issue by tagging a subpopulation of cells with antibodies conjugated to magnetic beads, which are then collected to achieve the separation. However, this technique requires the interaction of beads and cells and requires expensive reagents to operate. Alternative high throughput, label-free solutions to this problem include inertial microfluidics, and acoustofluidic techniques, yet these methods are prone to size-biased discrimination, preventing, for instance, the sensitive separation of equally sized cell subpopulations. Thus, there is a need for the development of label-free, high throughput sorting techniques that simultaneously maximize cell viability and purity, while minimizing size bias and non-specific interactions.
It is an objective of the present invention to provide devices that allow for the separation of key cellular blood components of the immune system, directly applicable to the development of medicines for the treatment of infectious disease and cancer, as well as the separation of cells of interest in suspension from a heterogeneous population, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.
The present invention features a microfluidic device that combines two key technologies to achieve high throughput sorting of cells in suspension. First, by creating flow vortices within the microfluidic chip, select sizes of cells may be trapped. Afterward, an array of electrodes may be used to exert a force to discriminate between cells of equal size, yet with different electrical properties. As an example, this device may be used to purify cell mixtures containing different subpopulations with overlapping sizes. This would be useful for applications where one wishes to extract specific cell types for bioengineering while maximizing cell viability in the process.
The present invention features a system for high-throughput cell sorting. The system may comprise a microfluidic platform that may comprise a main microfluidic channel, and one or more cavity acoustic transducers (CATs). The one or more CATs may be dead-end channels coupled to the main microfluidic channel. The microfluidic platform may be coupled to an external acoustic source. The system may further comprise a fluid disposed through the main microfluidic channel comprising cells having different sizes and different electric properties. The fluid may intersect the CATs to form one or more interfaces. The system may further comprise a set of electrodes disposed underneath the microfluidic platform. The set of electrodes may be configured to apply an alternating current (AC) to the cells. The CATs may be configured to oscillate the interfaces to produce one or more microstreaming vortices, such that each microstreaming vortex is capable of selectively trapping cells based on size. Applying the AC to the cells may cause the cells to move relative to the set of electrodes based on electric properties through dielectric polarization.
The present invention features a high-throughput method for cell sorting. The method may comprise providing a microfluidic platform. The microfluidic platform may comprise a main microfluidic channel and CATs. The one or more CATs may be dead-end channels coupled to the main microfluidic channel. The microfluidic platform may be coupled to an external acoustic source. The method may further comprise providing a set of electrodes disposed underneath the microfluidic platform. The method may further comprise flowing a fluid through the main microfluidic channel comprising cells having different sizes and different electric properties. The fluid may intersect the CATs to form one or more interfaces. The method may further comprise applying acoustic energy to the CATs via the external acoustic source to oscillate the interfaces. Oscillating the interfaces may produce one or more microstreaming vortices such that each microstreaming vortex is capable of selectively trapping cells based on size. The method may further comprise applying, by the set of electrodes, an AC to the cells. Applying the AC may cause the cells to move relative to the set of electrodes based on electric properties through dielectric polarization.
One of the unique and inventive technical features of the present invention is the application of both LCAT-induced microvortices and dielectrophoresis to a plurality of cells in a microfluidic platform. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously provides for the high-throughput sorting of cells based on both size and electric properties. None of the presently known prior references or work has the unique inventive technical feature of the present invention.
Furthermore, the inventive technical feature of the present invention is counterintuitive. The reason that it is counterintuitive is because it contributed to a surprising result. One skilled in the art would expect that the high hydrodynamic forces on the cells from the microvortices would be too strong to counterbalance with DEP forces to control the position and sort them out. Surprisingly, the present invention uses DEP forces not to completely halt particles but rather to change their location within the microfluidic chip so that they experience different hydrodynamic forces. The process results in the entrapment of larger cells while allowing the flow of cells of finite smaller sizes, which are then sorted by electric properties only. Thus, the inventive technical feature of the present invention contributed to a surprising result and is counterintuitive.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:
Following is a list of elements corresponding to a particular element referred to herein:
As used herein, the term “subset of the plurality of cells” refers to any selection of cells from the plurality of cells with a number of cells less than or equal to the number of cells in the plurality of cells. Thus, the subset may comprise every cell in the plurality of cells, zero cells, or any number of cells in between.
As used herein, Cavity Acoustic Transducers (CATs) are simple on-chip actuators that are easily fabricated and can be actuated using a battery-operated portable electronics platform. CATs are dead-end channels that are in the same plane laterally with respect to the microchannels. In some embodiments, the CATs require no additional fabrication steps other than those needed to produce a single layer or multilayer device. When the device is filled with liquid, CATs trap bubbles creating an interface that can be excited using an external acoustic source such as a piezoelectric transducer. The interface generated by an LCAT may be selected from a group comprising a gas-liquid interface, a liquid-liquid interface, a lipid membrane, a polymer membrane, a nano-particle membrane, or a combination thereof. In some embodiments, the liquid-liquid interface may comprise a plurality of immiscible liquids. As used herein, the term “immiscible liquids” refers to a set of liquids that are incapable of mixing (e.g. water and a hydrophobic liquid such as oil). In other embodiments, the liquid-liquid interface may comprise a thin physical barrier between the liquids, in which case the liquids may be immiscible or miscible. As used herein, the term “thin” refers to a membrane with a width of 2 to 100 nm. In some embodiments, the lipid membrane may comprise a lipid bilayer. In some embodiments, the polymer membrane may comprise a synthetically created membrane capable of enacting a driving force (e.g. pressure or concentration gradients) on particles on either side of the polymer membrane.
As used herein, “air” may refer to a gas or mixture of gasses, such as atmospheric air, oxygen, nitrogen, helium, neon, argon, an inert gas, or a reactive gas.
Near the air-liquid interfaces of the CAT, there may be a narrow gap where streamlines are directed forward in the direction of the bulk of the flow (see
Referring now to
In some embodiments, the plurality of cells may comprise one or more cell subtypes. The general idea is that CAT vortices allow size-based separation, while application of the AC-signal (either intermittently or continuously) can move cell subtypes relative to the overall device flow depending on their electric properties. The net result is that a cell subtype can be extracted at the outlet of the device, even when the different cell subtypes could have an overlapping size distribution.
In some embodiments, the oscillation may be controlled by a piezoelectric transducer (PZT) voltage. In some embodiments, the CATs (130) may be configured to induce pumping of the fluid (150), thereby eliminating the need for external pumping. In some embodiments, the interfaces (180) may comprise a gas-liquid interface, a liquid-liquid interface, a lipid membrane, a polymer membrane, a nano-particle membrane, or a combination thereof. In some embodiments, the system (100) may be used to purify cell mixtures that may comprise different subpopulations with overlapping sizes. For example, the system (100) may be used to separate lymphocyte subtypes in a high throughput manner for biomedical applications. In some embodiments, the electrodes (200) may be disposed parallel or perpendicular to a flow direction of the main microfluidic channel (120).
Referring now to
In some embodiments, the oscillation may be controlled by a piezoelectric transducer (PZT) voltage. In some embodiments, the CATs (130) may be configured to induce pumping of the fluid (150), thereby eliminating the need for external pumping. In some embodiments, the interfaces (180) may comprise a gas-liquid interface, a liquid-liquid interface, a lipid membrane, a polymer membrane, a nano-particle membrane, or a combination thereof. In some embodiments, the method may be used to purify cell mixtures that may comprise different subpopulations with overlapping sizes. For example, the method may be used to separate lymphocyte subtypes in a high throughput manner for biomedical applications. In some embodiments, the electrodes (200) may be disposed parallel or perpendicular to a flow direction of the main microfluidic channel (120).
The present invention features a microfluidic platform that incorporates two techniques to improve cell separation sensitivity without compromising particle sorting throughput. The device may implement Cavity Acoustic Transducers (CATs) and Dielectrophoresis (DEP). The device may comprise a microfluidic channel that has lateral air-liquid interfaces that may be actuated by a piezoelectric transducer placed below the chip (see
Using the above-described technologies, the separation works by first switching on the LCAT, which induces the vortex formation and cell trapping by size. After trapping cells of a specific size range in the vortices by this method, the LCAT is switched off and the DEP electrodes on, so that only cells with specific electric properties are attracted towards the electrodes—a mode known as positive DEP (
The remaining cells can also be subsequently collected by switching the field off. The present invention features interplay between acoustic streaming sorting and DEP separation. In some embodiments, the present invention features turning off the acoustic transducer so that the cells settle on the electrodes and are exposed to greater DEP forces. Otherwise, cells that are trapped and swirling in the microfluidic vortices cannot be pulled with sufficient DEP force to the electrodes. A default operation would be to pump cells in and enrich for certain cell size subpopulation via the LCAT, turning off the LCAT so cells settle and DEP is turned on to trap cells with certain dielectric properties, while keeping the DEP force on then turning on the LCAT (or external pump) to remove cells not attracted by DEP electrodes. Likely there would be an on and off switching algorithm that optimizes the interplay between DEP force and acoustic streaming shear force.
The present invention may allow for the separation of lymphocyte subtypes in a high throughput manner. Lymphocytes represent an important component of the adaptive immune system and can be classified into two major classes: B lymphocytes (B-cells) and T lymphocytes (T-cells). T-cells are of particular interest, as they help control the body's immune response to foreign substances, and they destroy cells that have been infected by viruses or become cancerous. Isolating T-cells is critical to studying the body's immune response and is directly applicable to the development of medicines for the treatment of infectious diseases and cancer in the pharmaceutical industry. In some embodiments, the plurality of cells comprise peripheral blood mononuclear cells (which includes monocytes and lymphocytes). Thus the technology could be configured for monocytes with size based separation (CATs), while allowing for refined sorting of B vs. T cells (which have overlapping sizes).
Lymphocyte subpopulations possess different intrinsic electrokinetic properties that have been leveraged to separate and detect them in a label-free manner. For instance, T and B-cells have different electrophoretic mobilities and membrane capacitance. Despite the existence of label-free lymphocyte subtype characterization techniques, there is still a great need for gentle, efficient, and scalable lymphocyte isolation methods from whole blood for further downstream analysis, and processing. The proposed invention implements LCAT devices, which have been proven to be an effective tool for gentle, size-based separation of leukocytes from whole blood. This technology is then combined with the electrophysiological specificity of DEP methods, which have been proven to be effective in the separation of cells with identical sizes, yet different electric signatures.
When compared with other sorting methods such as inertial microfluidics and conventional acoustofluidic devices that rely mostly on size differences, the present invention provides more sensitive and specific isolation of the cell subtypes of interest. Thus, the combination of LCAT and DEP improves the separation capabilities of each of these techniques alone. This is because DEP provides a more sensitive separation that does not rely on size alone to achieve the separation (i.e., it relies on the intrinsic electrophysiological phenotype of cells). Furthermore, the addition of LCAT to a DEP-based method increases the throughput that DEP methods can usually reach alone. These benefits may have the added advantages provided by LCAT: 1) A label-free trapping of cells; 2) Optimized cell viability; 3) Ease of operation, as no external pumping systems are needed; and 4) competitive throughput for the handling of relevant whole blood cell concentrations.
In some embodiments, the CATs (130) may intersect the main channel (120) at an angle. As a non-limiting example, the angle may be between about 40-50 degrees. In other embodiments, the angle may be about 1-10, 10-20, 20-30, 30-40, 50-60, 60-70, 70-80, or 80-90 degrees.
In some embodiments, the electrodes (200) may be used to selectively collect, concentrate, and detect intracellular components from purified cells trapped in microstreaming vortices (190). In some embodiments, the plurality of cells (160) may be lysed by the electrodes (200), pumping a lysing buffer into the system, or a combination thereof. In some embodiments, the intracellular component may comprise DNA, RNA, protein, a small molecule, or a combination thereof. In some embodiments, the electrodes (200) may be used for Polymerase Chain Reaction (PCR) heating of concentrated nucleic acids.
Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of” or “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of” or “consisting of” is met.
The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.
This application is a non-provisional and claims benefit of U.S. Provisional Application No. 63/234,931 filed Aug. 19, 2021, the specification of which is incorporated herein in its entirety by reference. This application is a continuation-in-part and claims benefit of U.S. application Ser. No. 17/340,581 filed Jun. 7, 2021, which is a continuation-in-part and claims benefit of U.S. Non-Provisional application Ser. No. 16/547,152 filed Aug. 21, 2019, now U.S. Pat. No. 11,052,395, which claims benefit of U.S. Provisional Application No. 62/720,829 filed Aug. 21, 2018, the specifications of which are incorporated herein in their entirety by reference. This application is a continuation-in-part and claims benefit of PCT Application No. PCT/US2021/027945 filed Apr. 19, 2021, which claims benefit of U.S. Provisional Application No. 63/011,426 filed Apr. 17, 2020, the specification of which are incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63234931 | Aug 2021 | US | |
| 62720829 | Aug 2018 | US | |
| 63011426 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17340581 | Jun 2021 | US |
| Child | 17891889 | US | |
| Parent | 16547152 | Aug 2019 | US |
| Child | 17340581 | US | |
| Parent | PCT/US21/27945 | Apr 2021 | US |
| Child | 16547152 | US |
