Technical Field of the Invention

The invention is related to a microfluidic device that has been designed using magnetic principles in order to sort circulating tumor cells from white blood cells without any labelling procedure.

Known State of the Art (Prior Art)

Circulating Tumor Cells (CTCs) are extremely rare in blood (i.e., 1 CTC versus 1 million white blood cells) and hard to isolate because of the heterogeneity of CTCs in biomarker expression as well. There is no standard method for isolating CTCs from blood cells yet. Different histological and molecular types of tumors express different marker sequences, and even a single distinct histological tumor type has considerable immunological and molecular differences.

The current CTC separation techniques use numerous differences between cells such as size, electric charges, density and expression of cell surface markers. However, these techniques have many limitations in terms of manual sample preparation steps, inconsistent results caused by low specificity and efficiency, and increased cost. To date, an effective and standard method for the sorting of CTCs could not be developed due to their very low presence in blood and their heterogeneous biomarkers.

In the patent document numbered W02015130913A1, a system is described that can separate cells from a heterogeneous cell population in the capillary channel by using the difference in the density of the cells in magnetic media by taking advantage of the difference in positions in which cells are balanced. In the levitation system mentioned in the document, a microscope with a microcapillary channel on it, two neodymium (NdFeB) magnets and two mirrors were used. Cells and the solution (gadolinium-based, non-ionic paramagnetic medium) which has a magnetic effect were loaded into a glass microcapillary tube then placed between two opposite magnets and the cells were observed in a static form. However, since this operation does not occur under flow, it does not allow the cells to be collected, but only allows the positions of the cells to be observed. The patent document numbered WO2017059353A1 is related to the separation of heterogeneous populations with one or more magnets in microfluidic environment based on the magnetic levitation principle. The magnetic levitation platform integrated with the mobile phone camera mentioned in the document is mostly used for counting blood cells; blood counting is performed to observe the development of the disease and the effect of the drug on the disease. In the aforementioned patent, the analysis is made by looking at the cells in a glass capillary tube. In addition, the plastic design shown in the patent does not allow the cells to be observed during the separation, which does not allow the determination of cell densities by separation. The plastic channel used in the aforementioned patent is produced by gluing three plastic sheets together with adhesive tapes on both sides, and the thick and rough channel walls do not allow the cells to be observed between the magnets. Because the glass capillary tube is transparent, it allows monitoring, but it has disadvantages because the glass tube has a single inlet and a single outlet, and the cells displayed at different positions in the channel cannot be collected from different outlets and also the glass tube does not have the design to apply a flow.

There is a need to develop a standard method for sorting CTCs from white blood cells without labeling and collecting the sorted cells, in which samples are not prepared manually, with a high sorting purity and a standard method.

Brief Description of the Invention

The invention provides a microfluidic chip that enables the sorting of circulating tumor cells from white blood cells, which plays a vital role in the early diagnosis, prediction and determination of personalized treatment methods of cancer, and performs this sorting with the principle of magnetic levitation. The aforementioned invention aims to meet the need for the separation and collection of CTCs by developing a new method based on the magnetic levitation principle, which has recently emerged as a very successful method in the biological characterization and monitoring of cells and cellular activities.

With the developed microfluidic chip, it is aimed to sort CTCs from white blood cells at a high rate in a short time like an hour thanks to the high speed (1 mL / hour) flow applied. Invention, the cell sorting platform based on the magnetic levitation principle, unlike the patent document numbered W02015130913A1, enables the sorting of cancer cells from white blood cells under a continuous flow by giving as 1 mL/hour (~ 15 μL / min) flow in the microfluidic channel via a syringe pump. Unlike the patent document numbered W02015130913A1, the sorting in the invention is not in static form but dynamic, and at the end of the 1-hour period, the cells can be directed to different outlets with the flow given to the channel.

Compared with the patent document numbered WO2017059353A1, the main goal of the invention is not blood analysis, but the sorting and collecting of cancer cells from a heterogeneous environment. Unlike the patent documents in the prior art, in the invented system, the channel in which the cells are located is not a glass tube or a plastic channel, but a special microfluidic channel design is offered, where the distance of the channel wall and the position of the separator can be controlled. The design is resistant to flow, has different channel outlets after sorting region, and has an advantage of being transparent that provides monitoring inside the channel.

It appears that no previous technical document offers a system that allows sorting of CTCs from white blood cells without labeling, under continuous flow with magnetic levitation within the microfluidic chip.

Capillary glass is not used in the design of the invention, instead, a microfluidic channel (1) made of glass and polymer with one inlet and two outlets is used, which allows the cells to be visualized and analyzed, and at the same time the cells can be sorted. In the invention, cells with different densities are collected by the effect of flow from different outlets, and thus cancer cells can be sorted from blood cells and become a preliminary step for the next diagnostic and therapeutic analyzes.

In addition, in the device design presented in the invention, the separable density range for cells can be changed by simply sliding the channel (1) on the horizontal axis, without changing the magnet positions and the concentration of the paramagnetic solution. In this way, it is possible to sort different cells and particles by using only one microfluidic channel (1) design.

The microfluidic channel (1) used in the invented system has been specially designed by the people to whom the invention belongs. The glass capillary tube used in the prior art is a standard tube with certain dimensions and it is not suitable for use in cell sorting experiments since it is not possible to change the dimensions of the channel width or length. For this reason, a microfluidic channel made of glass (11) and polymer is designed in which the desired width, wall thicknesses and the position of the separator (12) can be controlled in the invention. The mentioned polymer can be a silicone-based elastomer. Polydimethylsiloxane (PDMS), which is preferably silicone-based elastomers, is used in the invention. In the invention system, which is produced to sort cancer cells from blood cells, the microfluidic channel (1) is produced by creating a mold with the desired channel width and the position of the separator (12) at high resolution with soft lithography and reproducing it using polydimethylsiloxane (double agent polymer) and then adhering it to the glass (11) surface with oxygen plasma. There is a glass (11) surface on the lower and upper surfaces of the microfluidic channel (1).

With the advantage of the invention of four mirrors (6) placed at an angle of 45° on the microfluidic chip and the transparency of the microfluidic channel (1), the cells can be sorted under flow and collected at different outlets and can be monitored simultaneously. Two neodymium magnets (4,5) the same poles of which face each other are placed at the top and bottom of the channel. Thus, with this way, a channel design that is thin enough to be able to enter between two magnets and the thin glass (11) surface that allows monitoring of cells.

The invented device is the first product to show the sorting of cancer cells on a microfluidic chip with the help of magnetic forces without using a labelling method. In addition, with the proposed invention and method, it is possible to design and develop a single-use, independent of the labeling technique and therefore inexpensive platform instead of complex, expensive devices that require specially trained personnel to sort cancer cells from blood cells.

The most important goal of the invention is to sort cancer cells without labeling them with antibodies. Thanks to this feature, cancer cells can be sorted independently from the antigens on their surface. When the same type of cancer cells with different surface antigens are considered, the proposed method has the ability to sort a larger population of cancer cells from blood cells.

The magnetic levitation principle used in the invention has never been used before to sort and collect cells under flow. In this context, thanks to the invention, with the necessary infrastructure, it will also contribute to the applications of this technology in different fields. The invention can be used in cancer diagnosis and tracking the cancer by sorting CTCs in the blood. In addition, the sorted cells can be used in the development of personalized treatment methods to fight with cancer in a more effective way.

The invention will also allow the development of different cell separation projects. Identifying and separating microorganisms such as viruses, fungi and yeasts without labeling them in the blood and applying a specific treatment to the patient with respect to identified microorganisms; separating drug-resistant microorganisms and developing more effective drugs for them; as an alternative to the amniocentesis method carrying significant risks, separating the fetal cells in the circulation of pregnant women and performing prenatal genetic tests on these separated cells without any risk are among these studies.

Definition of the Figures of the Invention Figure 1: Visualization of forces acting on cells in the magnetic levitation platform.

Figure 2: Magnetic levitation setup consisting of two magnets, four mirrors and a PDMS microfluidic channel.

Figure 3: The principle of sorting of cells with different densities under flow inside a microfluidic channel in the magnetic levitation platform. Figure 4: A. Microfluidic channel with one inlet and two outlets, B. PDMS upper and lower channel wall thicknesses at the section taken from the separator part of the microfluidic channel.

Figure 5: Microfluidic chip and magnet positions. A. The situation where the microfluidic channel is in the same position as the magnets, B. The situation where the microfluidic channel is at y = 250 pm away from the magnets, C. The microfluidic channel is at y = 500 pm away from the magnets, D. The microfluidic channel is at y = 1000 away from the magnets.

Figure 6: Separable density range according to magnet positions within the microfluidic channel. Figure 7: Sorting efficiencies of microparticles with 1.02 g/mL and 1.09 g/mL densities at different Gd (15 mM, 30 mM, 60 mM) concentrations and under different different flow rates (5 μL/dk, 10 μL/dk, 15 μL/dk, 20 μL/dk).

Figure 8: Sorting efficiencies of U-937 cell line in the upper channel at different cell and Gd concentrations. Figure 9: Sorting efficiencies of MDA-MB-231 cell line in the upper channel at different cell and Gd concentrations. Definitions of the parts forming the invention

1. Microfluidic channel

2. The upper wall of the microfluidic channel

3. The lower wall of the microfluidic channel

4. Top magnet

5. Bottom magnet

6. Mirror

7. Upper channel

8. Lower channel

9. Outlet of the upper channel

10. Outlet of the lower channel

11. Glass

12. Seperator

13. Inlet

Detailed Description of the Invention

“The sample to be sorted”, in which contains cells aimed to be sorted, is given to the invented microfluidic device, and the cells equilibrated at different heights according to their densities can be sorted and collected under a flow. The collected cells can be used in cancer diagnosis or molecular tests to monitor success of treatment.

The "sample to be sorted" mentioned in the invention can be blood, serum or a plasma sample. The cells in the sample to be sorted mentioned in the invention can be cancer cells, circulating tumor cells, red blood cells, white blood cells, stem cells, platelets and microorganisms such as viruses, bacteria, yeast. The term "cell solution" defines the sample to be sorted mixed with the paramagnetic solution. With the invention, it is possible to separate all the mentioned cell groups from the cell mixture/sample.

For this purpose, Polydimethylsiloxane (PDMS) microfluidic chip is produced by soft lithography method. While the lower and upper wall thicknesses of this chip are kept thin (<800 pm (in the 200-800 pm range, preferably in the range of 400-600 pm)) by favor of the mold, high magnetic field has been obtained. Keeping the distance less than <2 mm between the upper and lower magnets increases the magnetic field values, which allows analysis with a less concentrated paramagnetic solution. High concentrations of paramagnetic solutions affect the viability of the cells and this may cause a decrease in sorting efficiency.

The microfluidic channel has one inlet and two outlets. The separator having a fixed thickness (thinner than the size of the channel) and length, and a pointed end in the outlet section passes through the middle point of the channel, which is the separation point for cells at different heights, and thus allows the cells to be directed to different outlets and collected from different outlets. The position of the separator is important in terms of separating the cells/directing them to different outlets. The thickness of the separator can be changed by considering the size of the channel and the density of the cells to be sorted. The thickness of the separator (12) mentioned in the invention is in the range of 25-200 pm, preferably 100 pm.

In the invention application, the "sample to be sorted" is cancer and blood cells, and the sample (cancer and blood cells) to be sorted is given from the channel inlet (13) with pumping and reaches the equilibrium height depending on their density along the channel and at the outlet section cancer cells are directed from above the separator and the blood cells are directed from below the separator to different outlets. With the invention, by using two strong magnetic neodymium magnets (N52) fixed with the parts surrounding the microfluidic chip, high magnetic forces can be created, and the sorting of cancer cells and blood cells that have different densities is performed at high flow rates (1 mL/hour (may range from 0.1-2 mL / hour)). In this respect, the invention is an innovative product within the scope of the studies conducted for the early diagnosis of cancer.

The separation provided by the invention is not in static form, it is dynamic, and at the end of the 1-hour period, the cells can be directed to different outlets with the flow given to the channel. As shown in Figure 1 and Figure 4, PDMS microfluidic channel produced by soft lithography method. By keeping the thickness of the lower wall (3) and the upper wall (2) thin (200-800 pm (preferably 400-600 pm)) with a mold obtained from the three-dimensional printer, the upper and lower magnets (4 and 5) are enabled to affect the channel more and thus a high magnetic field is obtained in the channel. In this way, the magnetic force applied each particle or cell is kept high. In the magnetic levitation platform, microparticles/cells in the paramagnetic solution will move towards the middle of the two magnets, which is the point where the magnetic field is the minumum in the channel. The middle point of the magnets (as the levitation height), different from the levitation height at the tip of the separator, allows the cells to be collected from different outlets depending on their density after equilibrium.

In the magnetic levitation method, the negative magnetic susceptibility difference between the cells and the paramagnetic solution, in which the cells are mixed, levitates the cells to different height levels in the magnetic field. This height depends on the density of the cells but is independent of their size. This method is the only method in which cells can be observed and identified without using the labelling method while measuring cell densities down to the single cell level. Using this method, it can be observed that each cell type has a unique levitation, i.e, a unique density profile. During the levitation of cells inside the channel between the magnets, whose same poles face each other, the negative magnetic susceptibility difference between the cell and the solution moves the cells towards a small magnetic field strength, i.e. B (magnetic flux density. At the point where the magnetic force (F _M ) acting on the cell is equal to the buoyancy force (F _G ) of the fluid, the cell remains stationary. This point reached by the cell depends on the density of the cell.

Magnetic principles: The net force (F _Net ) acting on a particle (or cell) in the liquid under the influence of the magnetic force until a particle (or cell) comes to the balance position in the system, is the resultant of magnetic force (F _M ), drag force (F _D ) and gravity force (F _G ) (equation 1 set). In this system, inertial force (F _t ) can be disregarded due to the low Reynolds number in microfluidic magnetophoresis and and Brownian force (F _B ) is also discarded due to the fact that it only affects the movement of sufficiently small particles (approximately <10 nm) significantly.

(1)

The F _M applied on the particle depends on; B: magnetic flux density (Tesla, T), V: del operator in: magnetic dipole values (equation 2). B decreases as it is away from the surface of the magnet. From here, the magnetic dipole can be reached at the low magnetic field inside the paramagnetic salt solution or the ferrofluid (equation 3). (3)

In this equation; F _M value can be expressed by arranging the equation as V: particle volume, μ ₀ : permeability of free space (1.2566 x10 ^-6 kg •m •A ^-2 •s ^-2 ), and D _x : magnetic susceptibility difference (X _p -X _m ) of the particle and the surrounding medium (equation 4). (4)

Magnetic flux values in three dimensions can be obtained by expanding in the cartesian coordinate system:

(5)

The hydrostatic buoyancy force F _G , which is an another force applied on the particle, is calculated depending on V: particle volume, ^Δ ρ: bulk density difference between the particle and the surrounding medium ( ρ _P - ρ _M ), g: gravitational acceleration (9.8 m s ^-2 ) (equation 6). (6)

The cell will remain stationary at the point where the magnetic force is equal to the hydrostatic buoyancy force. But if a perpendicular flow to magnetic forces is applied during magnetic levitation, cells will start to drag (Figure 1). The fluid drag force F _D , which is another force applied on a spherical particle at determined conditions, alters depending on R (particle diameter), η (dynamic viscosity), f _d (drag coefficient), and v _p (particle velocity) (equation 7). (6)

In the tests, cell lines grown in normal cell culture (to model the sample to be sorted) are given to the PDMS microfluidic channel (1) from the inlet (13) as shown in Figure 4 with the help of a syringe pump. As shown in Figure 2, in order to increase the magnetic effect applied to the microfluidic channel (1) and to focus the magnetic effect only on the channel, 2 neodymium magnets, in the form of a rectangular prism, as the upper magnet (4) and the lower magnet (5), were placed in the invention. A PDMS microfluidic channel (1) and 4 mirrors (6) placed at an angle of 45°, which allows monitoring, are assembled together with plastic parts produced by a three-dimensional printer. PDMS polymer was chosen as a material of the channel because of its transparency, resistance to pressure and the most suitable polymer for soft lithography method. The mentioned polymer can be a silicone-based elastomer. Preferably, polydimethylsiloxane (PDMS), one of silicone-based elastomers, is used in the invention. Different flexible and transparent polymers such as Ecoflex, which is compatible with soft lithography, can also be used in the fabrication of the microfluidic channel of the invention.

Before transfering cells, cells at the specified concentration were prepared in fetal bovine serum (FBS) with an agent containing the gadolinium ion (Gd). This agent, with its paramagnetic and water soluble structure, is a highly effective substance in the diagnosis of cancerous tissues using magnetic resonance imaging (MRI). The Gd concentration can change the levitation height of the cells and the speed of cells to reach this height. Apart from the Gd ion, different paramagnetic ions (such as metal manganese ((Mn ²⁺ )) used in MRIs can also be used.

The sorting process of cells includes the following steps and is described in detail in the following sections:

1. The sample to be sorted is mixed with Gd, so that the final solution contains Gd at a concentration of 30 mM (can range from 10-100 mM).

2. The cell solution with 1 mL (the amount of cell solution may be in the range of 0.1-10 mL) containing the sample to be sorted and Gd ions is drawn into the syringe and placed on the syringe pump.

3. The tip of the syringe is connected to the microfluidic chip inlet (13) with the help of a capillary tubing. In order to collect the cells sorted from the upper channel outlet (9) and the lower channel outlet (10), these outlets are connected to two tubes with the help of capillary tubing. 4. A maximum of 2 mL/hour (preferably 1 mL/hour) flow is started from the syringe pump and the cell solution is delivered to the PDMS microfluidic channel (1).

5. After the entire cell solution is given to the PDMS microfluidic channel (1), the sorted cells are collected from the tubes connected to the upper channel outlet (9) and lower channel outlet (10).

Optimization of Gd Concentrations and Flow Rates of Cells to the Channel

In an application of the invention, the flow rate of the cells introduced into the channel and the Gadavist concentration are optimized to separate efficiently cancer cells from white blood cells. For this, instead of cancer cells and white blood cells, microparticles that have similar densities with these cells were used. Microparticles were used in the invention only for optimization, to mimic cancer cells and white blood cells.

The densities of breast cancer cells are 1.044 ± 0.018 g / mL and their radius are 8.92 ± 1.64 pm, the density of white blood cells is 1.088 ± 0.005 g / mL and their radius is 4.52 ± 0.60 pm (Tekin et al., PNAS 2015,112: 28). For example, as shown in Figure 3, when the solution containing cells with 1.06 g/ mL and 1.09 g/mL densities is delivered to the PDMS microfluidic channel (1) using a syringe pump, cells with lower density (1.06 g/mL) moving in the flow direction will remain above the separator (12) and will be collected from the upper channel outlet (9) of the channel. Since the cells with higher density (1.09 g/mL) remain below the separator (12), they will be collected from the lower channel outlet (10).

Accordingly, in order to observe the movement and separation of the cells in the magnetic levitation platform and to optimize the system, particles with two different densities (1.02 g/mL and 1.09 g/mL) and diameters of 10 pm and 20 pm were used, and sorting efficiencies of those density microparticles in phosphate buffered saline (PBS) were calculated (Figure 7) at different Gadavist concentrations (15 mM, 30 mM, 60 mM) and flow rates (5 μL/min, 10 μL/min, 15 μL/min, 20 μL/min). The sorting efficiency is calculated as the ratio of the number of microparticles collected from the upper channel outlet to the number of microparticles collected from the lower and upper channel outlets. Flow rate and Gd concentration are factors affecting the sorting efficiency. Namely; changing the Gd concentration changes the final levitation height of the particles, and increasing the flow rate causes the particles to reach the separator before they reach the final levitation height. Gd concentration and flow velocity range that result in sorting efficiency above 90% were determined. Microparticles with 1.02 g/mL density were collected from the upper channel outlet with a sorting efficiency of 93.11% in a medium containing 30 Mm Gd with 15 μL/min flow rate. Under the same conditions, 14.13% of the 1.09 g/mL density particles were collected from the upper channel.

In order to model the patient's blood (the sample to be sorted), microparticles were prepared in a medium containing fetal bovine serum (FBS), and were introduced into the microfluidic channel at 30 mM Gd (may be in the concentration range of 10-100 mM) in the solution. The solution in which Gd was added gained paramagnetic property, and microparticles with 1.02 g/mL and 1.09 g/mL densities were collected from the upper channel at ~ 90% and 5% efficiency, respectively, under 15 μL / min flow. The flow rate at which the results are obtained (15 μL/min) is close to the flow rate desired to be studied (1 mL/hour). Also, microparticle sorting efficiencies in PBS and FBS are similar.

In order to model the patient's blood (the sample to be sorted), three different concentrations of white blood cells (U-937 macrophage cell line, 10 ⁷ cells/mL, 10 ⁶ cells/mL and 10 ⁵ cells/mL) and cancer cells (MDA-MB-231 breast cancer cell line, 10 ³ cells/mL, 10 ² cells/mL and 10 cells/mL) were selected and separated at different Gd concentrations (20 mM, 30 mM and 40 mM) under 1 mL/hr flow, and their efficiency was calculated (Figures 8-9). The optimum Gd concentration selected as a result of microparticles sorting experiments gave results compatible with cell sorting experiments. Sorting efficiency in 30 mM Gd was 2.17% at a concentration of 10 ⁷ U-937/mL, 9.50% at a concentration of 10 ⁶ U-937/mL, and 13.20% at a concentration of 10 ⁵ U-937/mL. Likewise, in 30 mM Gd, at 10 ³ MBA-MB-231/mL concentration 88.38%, at 10 ² MBA-MB-231/mL concentration 83.83% and at 10 MBA-MB- 231/mL concentration 66.75% sorting efficiencies have been observed.

In addition, with the invention, the next goal may be to increase the pull of white blood cells to the lower magnet in order to achieve the sorting efficiency above 90%, that is, to collect almost all of the white blood cells from the lower channel. For this, it can be ensured that the white blood cells labeled with magnetic nanoparticles coated with the CD45 antibody that will allow them to bind to the antigens on the surface can be collected with high efficiency from the lower channel outlet (10). Namely; CD45 will bind to the white blood cells and change the magnetic susceptibility of the white blood cells, thereby allowing the white blood cells to accumulate at the lower channel (8), increasing the levitation height difference between cancer cells. This will increase the sorting efficiency.

As a result of the optimizations mentioned above, suitable Gd concentration and flow rate for sorting were determined as 30 mM and maximum 1 ml/hour, respectively. However, when sorting with the invention, the Gd concentration may be in the range of 10-100 mM, and the rate of flow of the cell solution into the channel may be in the range of 0.1-2 ml/hour. However, to reach same sorting efficiency at different Gd concentrations, the positions of the magnets with respect to each other can be changed. As shown in Figure 5, the microfluidic chip can be positioned in different ways according to the magnets, and the sortable density value in the channel can be changed without changing the amount of Gd and the distance between the magnets. As the distance of the microfluidic chip to the magnets increases on the horizontal axis, the magnetic force acting on the microparticles/cells changes, and this has an effect on guiding microparticles/cells to different outlets of the channel under flow. In cell sorting experiments, the horizontal distance of the PDMS microfluidic channel (1) to the magnets is positioned as y = 0 pm (Figure 5 A.). According to the simulation results, it was observed that when the distances of the magnets were kept constant and only the microfluidic chip was moved from the outer frame of the magnets in the horizontal direction as y = 250 pm (Figure 5 B.), y = 500 pm (Figure 5 C.) and y = 1000 pm (Figure 5 D.), the value of the sortable density decreased (Figure 6). Thus, by using the same microfluidic chip, cells can be sorted in different density ranges by changing the position of the microfluidic chip on the horizontal axis with respect to the magnets, without changing the position of the magnets relative to each other and the paramagnetic solution concentration.