Method for evaluating deformability of floating cell

Abstract

The deformability of red blood cells (100) in an electrolyte solution (4) is evaluated using a sensing device (1). In an ion current waveform in which a horizontal axis represents time and a vertical axis represents a pulse-like ion current flowing between an input electrode (2) and an output electrode (3) when the red blood cells (100) pass through a detection channel CH, the symmetry in the horizontal axis direction with respect to the axis Z passing through the minimum value of the ion current and parallel to the vertical axis is determined, and the deformability of the red blood cells (100) is evaluated on the basis of the determination of the symmetry.

Description

Method for evaluating the deformability of suspended cells

[0001] The present invention relates to a method for evaluating the deformability of suspended cells.

[0002] A method using a sensing device has been proposed as a method for evaluating the deformability of red blood cells (Non-Patent Document 1). The evaluation method in Non-Patent Document 1 measures the ionic current flowing through a minute through-hole (detection channel) filled with an electrolyte solution to evaluate the deformability of red blood cells in the electrolyte solution. When red blood cells pass through the detection channel, the flow of the ionic current is obstructed, causing the ionic current to change into a pulse-like shape. The width of the detection channel is narrower than that of the red blood cells, and the red blood cells deform as they pass through the detection channel. When red blood cells harden, they become less deformable, so the time they take to pass through the detection channel increases, and the pulse width of the ionic current becomes longer. The deformability of red blood cells is evaluated based on the pulse width of this ionic current.

[0003] X. Yang, Z. Chen, J. Miao, L. Cui, W. Guan, “High-throughput and label-free parasitemia quantification and stage differentiation for malaria-infected red “Blood cells” Biosens. Bioelectron. 98 (2017) 408-414

[0004] In the conventional evaluation method described above, the width of the detection channel is much narrower than the diameter of a red blood cell. Furthermore, because deformability is evaluated based on the pulse width of the ion current, accurate evaluation requires increasing the length of the detection channel and the pulse width. This poses a problem: the detection channel is prone to clogging with red blood cells. If red blood cells clog the detection channel, it becomes impossible to evaluate the deformability of the red blood cells.

[0005] The present invention has been made in consideration of the above-mentioned circumstances, and its purpose is to provide a method for evaluating the deformability of suspended cells that can evaluate the deformability even using a sensing device that is less likely to be clogged with red blood cells.

[0006] In order to achieve the above-mentioned object, the method for evaluating the deformability of suspension cells according to the present invention is characterized by the following features [1] to [3]: [1] A method for evaluating the deformability of suspension cells using a sensing device comprising: a first tank provided with an input electrode and filled with an electrolyte solution; a second tank provided with an output electrode and filled with the electrolyte solution; and a detection channel communicating between the first tank and the second tank, the method evaluating the deformability of suspension cells in the electrolyte solution using a sensing device comprising: a first axis representing time and a second axis representing a pulsed ionic current flowing between the input electrode and the output electrode when the suspension cells pass through the detection channel; determining symmetry in the first axis direction with respect to a third axis that passes through the minimum value of the ionic current and is parallel to the second axis; and evaluating the deformability of the suspension cells based on the determination of symmetry. [2] The method for evaluating the deformability of suspended cells according to [1], wherein the symmetry is determined based on a comparison between a first time from when the ionic current becomes equal to or less than a threshold value until the ionic current reaches the minimum value and a second time until the ionic current returns from the minimum value to the threshold value. [3] The method for evaluating the deformability of suspended cells according to [1] or [2], wherein the suspended cells are blood cells.

[0007] According to the present invention, it is possible to provide a method for evaluating the deformability of suspended cells, which can evaluate the deformability using a sensing device that is less likely to be clogged with red blood cells.

[0008] The present invention has been briefly described above. The details of the present invention will become clearer by reading the following detailed description of the invention (hereinafter referred to as "embodiments") with reference to the accompanying drawings.

[0009] FIG. 1 is a schematic cross-sectional view of a sensing device used in the method for evaluating the deformability of suspended cells of the present invention. FIG. 2(A) is a schematic diagram of a detection channel used in a conventional method for evaluating the deformability of red blood cells, and FIG. 2(B) is a graph showing the ion current flowing through a sensing device using the detection channel shown in FIG. 2(A). FIG. 3(A) is a schematic diagram of a detection channel used in the method for evaluating the deformability of red blood cells of the present embodiment, and FIGS. 3(B) and 3(C) are graphs showing the ion current flowing through a sensing device using the detection channel shown in FIG. 3(A). FIG. 4 is a graph showing the results of measuring the ion current with an ammeter when normal red blood cells and malaria-infected red blood cells are placed in the electrolyte solution of the sensing device shown in FIG. 1. FIG. 5(A) is an explanatory diagram illustrating the passage of normal red blood cells through the detection channel, and FIG. 5(B) is an explanatory diagram illustrating the passage of malaria-infected red blood cells through the detection channel. FIG. 6 is an explanatory diagram for explaining the evaluation of the symmetry of the ion current waveform in another embodiment. Fig. 7 is a graph showing the results of measuring the ion current with an ammeter when old red blood cells and GA red blood cells are placed in the electrolyte solution of the sensing device shown in Fig. 1. Fig. 8 is a graph showing the results of measuring the ion current with an ammeter when normal red blood cells, old red blood cells, and GA red blood cells are placed in the electrolyte solution of the sensing device shown in Fig. 1. Fig. 9 is a graph showing the ion current waveforms simulated for each Young's modulus of the cell membrane.

[0010] Specific embodiments of the present invention will be described below with reference to the accompanying drawings. The method for evaluating the deformability of suspended cells according to the present invention is a method for evaluating the deformability of suspended cells in an electrolyte solution using a sensing device 1 shown in FIG. 1 . Here, suspended cells are cells that can float in an electrolyte solution, and include cultured cells that grow in a suspended state in a medium, as well as adherent cells that have been processed to become suspended. Preferably, the cells are blood cells, and include not only cells with nuclei such as white blood cells, but also cells without nuclei such as red blood cells. In the following embodiment, an example of evaluating the deformability of red blood cells as suspended cells will be described.

[0011] For convenience of explanation, the "height direction D1," "length direction D2," and "width direction D3" are defined below as shown in Figure 1 etc. The "length direction D1" is defined as the direction connecting a first tank 5 and a second tank 6, which will be described later, and the "height direction D1," "length direction D2," and "width direction D3" are perpendicular to one another.

[0012] First, a sensing device 1 used in the method for evaluating red blood cell deformability (hereinafter simply referred to as the "evaluation method") of this embodiment will be described. As shown in Fig. 1, the sensing device 1 includes a first tank 5 provided with an input electrode 2 and filled with an electrolyte solution 4, a second tank 6 provided with an output electrode 3 and filled with the electrolyte solution 4, a detection channel CH communicating with the first tank 5 and the second tank 6, an inlet portion 7, and an outlet portion 8.

[0013] The input electrode 2 and the output electrode 3 are provided in the shape of a long cylinder. The longitudinal direction of the input electrode 2 and the output electrode 3 is arranged along the height direction D1, and the input electrode 2 and the output electrode 3 are arranged side by side in the length direction D2. One end of the input electrode 2 is immersed in the electrolyte solution 4 in the first tank 5, and the other end protrudes from the first tank 5. One end of the output electrode 3 is immersed in the electrolyte solution 4 in the second tank 6, and the other end protrudes from the second tank 6. A low DC voltage output from a DC power supply 10 is applied between the input electrode 2 and the output electrode 3. In addition, an ammeter 11 is provided between the input electrode 2 and the output electrode 3, and the ammeter 11 can measure the ion current flowing between the input electrode 2 and the output electrode 3.

[0014] In this embodiment, the first tank 5 and the second tank 6 are separated by an insulating film 9, and a detection channel CH, an inlet portion 7, and an outlet portion 8 are provided in the insulating film 9. The detection channel CH is provided between the inlet portion 7 and the outlet portion 8. The detection channel CH has a flow path length L1 along the longitudinal direction D2. The inlet portion 7 and the detection channel CH communicate with each other via the end of the detection channel CH on the inlet portion 7 side. The outlet portion 8 and the detection channel CH communicate with each other via the end of the detection channel CH on the outlet portion 8 side. The detection channel CH has a rectangular cross section, and has the same height and width W1 from the end on the inlet portion 7 side to the end on the outlet portion 8 side in the longitudinal direction D2.

[0015] The inlet section 7 connects the first tank 5 to the detection channel CH. The outlet section 8 connects the detection channel CH to the second tank 6. The inlet section 7 and the outlet section 8 are provided at the same height from one end to the other in the longitudinal direction D2. The inlet section 7 and the outlet section 8 are provided symmetrically in the longitudinal direction D2 with the detection channel CH at the center, and the width of the inlet section 7 and the outlet section 8 decreases as they approach the detection channel CH.

[0016] Next, a method for evaluating the deformability of red blood cells in an electrolyte solution 4 using the sensing device 1 shown in Fig. 1 will be described. When a low voltage is applied between the input electrode 2 and the output electrode 3, an ionic current from the input electrode 2 flows through the detection channel CH toward the output electrode 3. At this time, as shown in Figs. 2 and 3, when red blood cells in the electrolyte solution 4 pass through the detection channel CH, the flow of the ionic current is obstructed, the ionic current temporarily decreases, and a pulsed ionic current is detected. The conventional evaluation methods described in this embodiment and the background art evaluate the deformability of red blood cells based on the ionic current.

[0017] Next, the inventors considered conventional evaluation methods. Red blood cells 100 are disk-shaped, with a diameter of approximately 7 μm to 8 μm and a height of 2 μm to 3 μm. As shown in FIG. 2, the detection channel CH used in conventional evaluation methods has a width W2 and height of 5 μm (W2 = 5 μm), which are significantly smaller than the diameter of the red blood cells 100, and a flow path length L2 of 45 μm (L2 = 45 μm). The conventional detection channel CH has a narrow width W2 and a long flow path length L2, which poses a problem of being easily clogged by the red blood cells 100.

[0018] In the conventional evaluation method, the width W2 of the detection channel CH is narrowed and the flow path length L2 is lengthened, and when the deformability of the red blood cells 100 decreases, it becomes difficult for the red blood cells 100 to pass through the detection channel CH. This is utilized to evaluate the deformability of the red blood cells 100 based on the pulse width T2 of the ion current. This pulse width T2 of the ion current depends on the above-mentioned deformability, but does not depend on the repair ability, so there is a problem in that the repair ability cannot be evaluated.

[0019] As a result of intensive research to solve these problems, the inventors have concluded that, as shown in Figure 3(A), if the flow path length L1 is made shorter than the length L2 (= 45 μm) of the conventional detection channel CH, or preferably if the width W1 of the detection channel CH is made wider than the width W2 (= 5 μm) of the conventional detection channel CH, clogging of the red blood cells 100 will be reduced, and the time it takes for the ion current to reach the minimum value from the steady state value will depend on the deformation ability, and the time it takes for the ion current to return to the steady state value from the minimum value will depend on the repair ability, making it possible to evaluate the repair ability.

[0020] When entering the detection channel CH, the red blood cell 100 deforms from a normal disk shape to an elliptical disk shape that matches the width W1 of the detection channel CH. Furthermore, when exiting the detection channel CH, the red blood cell 100 restores its shape from an elliptical disk to a disk shape. At this time, if the restoration ability becomes lower than the deformation ability, the red blood cell 100 will have difficulty restoring its shape from an elliptical disk to the normal disk shape. For this reason, the red blood cell 100 exits the detection channel CH in an elliptical disk shape that is less likely to inhibit the ionic current, and therefore the time it takes for the ionic current to return from the minimum value to the steady value is shorter than the time it takes for the ionic current to reach the minimum value from the steady value.

[0021] Furthermore, when the red blood cell 100 is normal and has the same deformation ability and repair ability, the deformation when it enters the detection channel CH and the repair when it leaves the detection channel CH are similar, so as shown in Figure 3 (B), the ion current waveform, with the horizontal axis (first axis) representing time and the vertical axis (second axis) representing ion current, is thought to be symmetrical in the horizontal axis direction (first axis) with respect to axis Z (third axis) which passes through the minimum value of the ion current and is parallel to the vertical axis.

[0022] Furthermore, it was considered that if an abnormality occurs in the red blood cell 100, causing a deviation in the deformability and repair ability, the ionic current waveform will not be symmetrical in the horizontal direction with respect to the axis Z, as shown in Figure 3(C). In other words, if the repair ability is lower than the deformability, the axis Z of the ionic current waveform will be biased to the right (the side where time passes), as shown in Figure 3(C). It was considered that this could be used to evaluate the red blood cell 100 based on the symmetry of the ionic current waveform in the horizontal direction with respect to the axis Z.

[0023] To verify this idea, the following test 1 was carried out.

[0024] [Test 1] The sensing device 1 used a detection channel CH with a width W1 of 5 μm and a channel length L1 of 10 μm. Normal red blood cells 100 and malaria-infected red blood cells 100 were contained in the electrolyte solution 4 of this sensing device 1. Phosphate-buffered saline (PBS) was used as the electrolyte solution 4. Normal red blood cells 100 are flexible and easily deformable (high deformability), and easily recover from deformation (high recovery ability). On the other hand, it is known that malaria-infected red blood cells 100 lose their flexibility due to the formation of a nucleus 200 (see Figure 5) derived from the malaria parasite within the red blood cells 100. It is also known that red blood cells 100 nearing the end of their lifespan also lose their flexibility. Malaria-infected red blood cells 100 were visualized using a fluorescent nuclear stain under a fluorescence microscope, and the ion currents of normal red blood cells 100 passing through the detection channel CH and malaria-infected red blood cells 100 passing through the detection channel CH were measured. A very small amount of fluorescent nuclear stain was used, so as not to cause any effects such as coagulation of the red blood cells. In this measurement, a voltage of 1.0 V was applied between the input electrode 2 and the output electrode 3 using a 1.0 V DC power supply 10, and the ion current sampling rate was set to 200 kHz. The measurement results are shown in Figure 4. In Figure 4, the ion currents of normal red blood cells 100 and malaria-infected red blood cells 100 are offset on the vertical axis to make the graph easier to read.

[0025] It was found that the ionic current waveform when a normal red blood cell 100 passed through the detection channel CH was symmetrical in the horizontal direction with respect to the axis Z, as shown by the dashed line in Fig. 4. This is thought to be because normal red blood cells 100 have a high repair ability, as shown in Fig. 5(A).

[0026] In contrast, it was found that the ionic current waveform when a malaria-infected red blood cell 100 passed through the detection channel CH is not symmetrical in the horizontal direction with respect to the axis Z, but is asymmetrical, as shown by the solid line in Figure 4. The axis Z of the ionic current waveform of the malaria-infected red blood cell is biased to the right side of the drawing along the horizontal axis (i.e., toward the passage of time). This is thought to be because the repair ability of the malaria-infected red blood cell 100 is lower than that of normal red blood cells.

[0027] Next, the present inventors conducted the following Test 2 to confirm the reproducibility of the above results.

[0028] [Test 2] The detection channel CH of the sensing device 1 had a width W1 of 10 μm and a flow path length L1 of 10 μm. Normal red blood cells 100 were contained in the electrolyte solution 4 of this sensing device 1. Phosphate-buffered saline was used as the electrolyte solution 4. The ionic current was measured for 2,000 pulses as the normal red blood cells 100 passed through the electrolyte solution 4. In this measurement, a voltage of 1.0 V was applied between the input electrode 2 and the output electrode 3 using a 1.0 V DC power supply 10, and the sampling rate of the ionic current was set to 200 kHz. As a result of the measurement, it was confirmed that the most frequently occurring ionic current waveform of the 2,000 pulses was symmetrical in the horizontal direction with respect to the axis Z.

[0029] Malaria-infected red blood cells 100 with an infection rate of 91.3% were also included in the electrolyte solution 4 of the sensing device 1. The ionic current was measured for 2000 pulses as the malaria-infected red blood cells 100 passed through. As a result, it was confirmed that the most frequent ionic current waveform of the 2000 pulses was asymmetric in the horizontal direction relative to the axis Z.

[0030] From the above-described test, it was found that the symmetry of the ionic current waveform in the horizontal direction relative to the axis Z can be determined, and the deformability of the red blood cells 100 can be evaluated based on this symmetry. Utilizing this, in the method for evaluating the red blood cells 100 of this embodiment, the symmetry of the ionic current waveform in the horizontal direction relative to the axis Z can be determined, and the deformability of the red blood cells 100 can be evaluated based on this symmetry.

[0031] Next, a method for determining the symmetry of an ionic current waveform will be described. As shown in FIG. 3 , symmetry is determined based on a comparison between a first time T11, which is the time from when the ionic current falls below the threshold value Ith until the ionic current reaches its minimum value, and a second time T12, which is the time from when the ionic current returns from its minimum value to the threshold value Ith. If the difference (T11-T12) or ratio (T11 / T12) between the first time T11 and the second time T12 is equal to or less than a predetermined value, the ionic current waveform is evaluated as symmetrical, and no abnormality has occurred in the red blood cell 100. On the other hand, if the difference (T11-T12) or ratio (T11 / T12) between the first time T11 and the second time T12 exceeds a predetermined value, the ionic current waveform is evaluated as asymmetrical, and the red blood cell 100 is evaluated as having hardened. The threshold value Ith is set equal to or less than the steady-state value of the ionic current and greater than its minimum value. Preferably, setting the threshold value Ith equal to half the peak height H of the ionic current facilitates evaluation of symmetry.

[0032] When the ionic current is determined to be asymmetric, if the first time T11 is greater than the second time T12 as shown in Figure 3(C), the axis Z of the ionic current waveform is biased to the right, and the repair ability of the red blood cells 100 is evaluated to be lower than their deformability. However, because there are variations and measurement errors in the first times T11 and T12, the ionic current waveforms when a large number of red blood cells 100 pass through may be measured and their average may be calculated.

[0033] According to the above-described embodiment, the symmetry of the ion current waveform is determined, and the deformability of red blood cells is evaluated based on the symmetry. This makes it possible to evaluate the deformability of red blood cells even when using a sensing device 1 with a wide detection channel and a long flow path length, i.e., one that is less likely to be clogged by red blood cells.

[0034] According to the above-described embodiment, the symmetry of the ionic current waveform is determined based on a comparison between the first time T11 and the second time T12, which makes it possible to easily determine the symmetry of the ionic current waveform.

[0035] The present invention is not limited to the above-described embodiments, and can be appropriately modified, improved, etc. Furthermore, the material, shape, size, number, location, etc. of each component in the above-described embodiments are arbitrary and not limited as long as they can achieve the present invention.

[0036] According to the above-described embodiment, the symmetry of the ionic current waveform is determined based on a comparison between the first time T11 and the second time T12, but this is not limiting. For example, as shown in Fig. 6, the symmetry of the ionic current may be determined based on a comparison of the difference or ratio between an integral value S1 of the difference between the ionic current at the first time T11 and the threshold value Ith and an integral value S2 of the difference between the ionic current at the second time T11 and the threshold value Ith. Specifically, if the difference exceeds 0, it is determined that there is asymmetry, and if the ratio exceeds 1, it is determined that there is asymmetry.

[0037] According to the above-described embodiment, the inlet 7 and the outlet 8 are provided symmetrically in the longitudinal direction, and the ion current waveform when a normal red blood cell 100 passes through the detection channel CH is symmetrical in the horizontal direction, but this is not limited to this. The inlet 7 and the outlet 8 do not have to be symmetrical in the longitudinal direction. In this case, the red blood cell 100 can be evaluated by comparing the symmetry in the horizontal direction of the ion current waveform when a normal red blood cell 100 passes through the detection channel CH with the symmetry in the horizontal direction of the ion current waveform when the red blood cell 100 to be evaluated passes through the detection channel CH.

[0038] According to the above-described embodiment, it was confirmed by [Test 1] and [Test 2] that the ionic current waveform is asymmetric when a malaria-infected red blood cell 100 passes through the detection channel CH. However, malaria infection is not the only cause of red blood cell hardening. For example, it is known that red blood cells 100 can also harden due to diseases such as diabetes. It is also known that red blood cells 100 can harden due to deterioration over time.

[0039] The present inventors conducted the following Tests 3 and 4 to confirm whether the ionic current waveform becomes asymmetric even when red blood cells become stiff due to causes other than malaria infection.

[0040] [Test 3] The detection channel CH of the sensing device 1 had a width W1 of 4 μm and a flow path length L1 of 10 μm. Red blood cells 100 that had deteriorated over time several days or more after blood collection were impregnated into the electrolyte solution 4 of the sensing device 1. The ionic current was measured when the deteriorated red blood cells 100 passed through the detection channel CH. Furthermore, red blood cells 100 (hereinafter abbreviated as "GA red blood cells 100") hardened using glutaraldehyde (hereinafter abbreviated as "GA"), a known reagent for hardening cells, were impregnated into the electrolyte solution 4 of the sensing device 1. The ionic current waveform was measured when the GA red blood cells 100 passed through the detection channel CH. The results are shown in FIG. 7.

[0041] In more detail, 100 red blood cells (hematocrit value 50%, frozen for several days or more after collection) that had deteriorated over time were washed twice (1500 G, 5 minutes of centrifugation) with phosphate buffered saline (PBS solution), which is electrolyte solution 4, and the washed 4 × 10 ６ A PBS solution was prepared in which 2 μL of the PBS solution in which the aged erythrocytes 100 were suspended was placed in the first tank 5 and the second tank 6, which had already been filled with the PBS solution, and the ionic current waveform was measured when the aged erythrocytes 100 passed through the detection channel CH.

[0042] In addition, 2 μL of a PBS solution containing 200 μL of suspended red blood cells 100 was added with 2 μL of 10% GA to give a 0.1% GA concentration, and this aliquot was placed into the first tank 5 and the second tank 6, which were already filled with PBS solution, and the ionic current waveform was measured when the GA red blood cells 100 passed through the detection channel CH.

[0043] 7, it was confirmed that the ion current waveform when the aged red blood cell 100 passed through the detection channel CH was asymmetric, with the first time T11 being greater than the second time T12. It was also confirmed that the ion current waveform when the GA red blood cell 100 passed through the detection channel CH was asymmetric, with the first time T11 being greater than the second time T12.

[0044] [Test 4] The detection channel CH of the sensing device 1 had a width W1 of 2 μm and a flow path length L1 of 10 μm. Normal red blood cells 100 from a healthy individual were placed in the electrolyte solution 4 of the sensing device 1. The ionic current was measured when the normal red blood cells 100 passed through the detection channel CH. Additionally, red blood cells 100 that had deteriorated over time, which had been collected for several days or more, were placed in the electrolyte solution 4 of the sensing device 1. The ionic current was measured when the deteriorated red blood cells 100 passed through the detection channel CH. Furthermore, red blood cells 100 immersed in glucose (GC; blood glucose) (hereinafter abbreviated as "GC red blood cells") were placed in the electrolyte solution 4 of the sensing device 1 as a model of diabetic red blood cells 100. The ionic current was measured when the GC red blood cells 100 passed through the detection channel CH. The results are shown in Figure 8.

[0045] More specifically, a PBS solution containing healthy human blood (immediately after blood collection) was used as the electrolyte solution 4 containing normal red blood cells 100. As in the above [Test 3], the electrolyte solution 4 containing the red blood cells 100 was prepared by dissolving 4 × 10 ６ 200 μL of PBS solution containing 100 GC red blood cells suspended at 1 / mL was used. A glucose solution was also prepared by adding glucose (final concentration 20%) to a PBSA solution (PBS + 1% human serum albumin). The glucose solution and the PBS solution containing 100 GC red blood cells suspended in 1:1 were mixed and stirred at 36.5°C for 5 hours. The mixture was then washed twice with PBS (centrifugation at 1500 G for 5 minutes) to prepare electrolyte solution 4 containing 100 GC red blood cells.

[0046] As shown in Figure 8, the ion current waveform when a normal red blood cell 100 passes through the detection channel CH is such that the first time T11 is approximately equal to the second time T12, confirming that they are the target. The electrolyte solution 4 containing normal red blood cells 100 also contains white blood cells (8-15 μm) with larger diameters than the red blood cells 100 (7-8 μm), but no clogging of the detection channel CH due to these cells was observed in measurements of several hundred pulses. This is thought to be because whole blood contains several million red blood cells 100 per μL, while white blood cells are present at a concentration of tens of millions per μL, less than 1 / 1000 of the red blood cells 100. Therefore, most white blood cells stochastically become red blood cells 100, preventing clogging that would interfere with measurement.

[0047] Furthermore, it was confirmed that the ion current waveform when a red blood cell 100 that has deteriorated over time and a GC red blood cell 100, which is a model of a diabetic red blood cell 100, passed through the detection channel CH was asymmetric, with the first time T11 being greater than the second time T12.

[0048] These results demonstrate that this method, even with the simple evaluation method of measuring whole blood in a PBS solution, can evaluate the decreased ability of red blood cells 100 to change over time or due to glycation, compared to normal red blood cells 100.

[0049] Furthermore, in the above-described embodiment, the red blood cells 100 are evaluated, but the present invention is not limited to this. A similar evaluation may be performed on floating cells other than red blood cells (e.g., blood cells such as white blood cells and platelets).

[0050] To confirm whether the above-described method for evaluating deformability can be applied to suspended cells other than red blood cells, the inventors performed a simulation using the finite element method (FEM) to determine the ionic current waveforms of suspended cells for each Young's modulus of the cell membrane. In this simulation, the detection channel CH had a width W1 = 10 μm and a flow path length L1 = 10 μm. Suspended cells were modeled as having a cell diameter of 8 μm and a cell membrane thickness of 80 nm. The water pressure in the first tank 5 was set to 10 Pa, and the water pressure in the second tank 6 was set to 0 Pa. Using the Navier-Stokes equations and the stress-strain relationship for an incompressible fluid, a fluid-structure interaction (FSI) calculation was performed, and the current value I was calculated from the resulting cell shape-time model using the following equation (1): I=Eσ∫A(D4)dD (1) E: voltage (1.0 V) applied to the input electrode 2 and the output electrode 3 σ: electrical conductivity of the electrolyte solution 4 (1.73 S / m) A(D): cross-sectional area of ​​the detection channel CH at position D in the longitudinal direction D2, excluding suspended cells

[0051] The simulation results are shown in Figure 9. As shown in the figure, the higher the Young's modulus, the lower the repair ability, and the lower the repair ability, the more the curve tilts to the right. In other words, because the ionic current waveform deforms according to the Young's modulus of the cell membrane, it was confirmed that the above-mentioned method for evaluating deformability can be applied to suspended cells other than red blood cells.

[0052] In the above-described embodiment, the detection channel CH has a rectangular cross section, but this is not limited thereto. The detection channel CH may have any shape that allows suspended cells such as red blood cells to pass through, and may also have a circular cross section.

[0053] In the above-described embodiment, a detection channel CH with a width W1 of 5 μm or 10 μm and a flow path length L1 of 10 μm was used, but this is not limited to this. The detection channel CH is designed to match the size of suspended cells. For example, it is possible to determine the width W1 at which suspended cells can pass without clogging, and then design the flow path length L1 so that the symmetry of the ionic current waveform can be determined at the determined width W1. In the case of red blood cells, the width W1 may be designed to be 5 μm to 30 μm, and the flow path length L1 may be designed to be 1 μm to 30 μm. More preferably, the width W1 may be designed to be 5 μm to 20 μm, and the flow path length L1 may be designed to be 1 μm to 20 μm. More preferably, the width W1 may be designed to be 5 μm to 10 μm, and the flow path length L1 may be designed to be 1 μm to 10 μm.

[0054] Here, the features of the embodiments of the method for evaluating the deformability of suspended cells according to the present invention described above will be briefly summarized and listed below in [1] to [3].

[0055] [1] A method for evaluating the deformability of suspended cells, using a sensing device (1) comprising: a first tank (5) provided with an input electrode (2) and filled with an electrolyte solution (4); a second tank (6) provided with an output electrode (3) and filled with the electrolyte solution; and a detection channel (CH) connecting the first tank and the second tank, to evaluate the deformability of suspended cells in the electrolyte solution, the method comprising: determining symmetry in a first axis direction with respect to a third axis that passes through the minimum value of the ionic current and is parallel to the second axis in an ionic current waveform in which the first axis represents time and the second axis represents a pulsed ionic current that flows between the input electrode and the output electrode when the suspended cells pass through the detection channel; and evaluating the deformability of the suspended cells based on the determination of symmetry. [2] The method for evaluating the deformability of suspended cells according to [1], wherein the symmetry is determined based on a comparison between a first time from when the ionic current becomes equal to or less than a threshold value until the ionic current reaches the minimum value and a second time until the ionic current returns from the minimum value to the threshold value. [3] The method for evaluating the deformability of suspended cells according to [1] or [2], wherein the suspended cells are blood cells.

[0056] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

[0057] This application is based on a Japanese patent application (Patent Application No. 2023-099478) filed on June 16, 2023, the contents of which are incorporated herein by reference.

[0058] According to the present invention, it is possible to provide a method for evaluating the deformability of suspended cells, which can evaluate the deformability even when using a sensing device that is less likely to be clogged with red blood cells. The present invention, which has this effect, is useful for methods for evaluating the deformability of suspended cells.

[0059] 1 Sensing device 2 Input electrode 3 Output electrode 4 Electrolyte solution 5 First tank 6 Second tank 100 Red blood cells (suspended cells) CH Detection channel Ith Threshold T11 First time T12 Second time Z axis (third axis)

Claims

1. A first tank is provided with input electrodes and filled with an electrolyte solution, A second tank is provided with an output electrode and filled with the electrolyte solution, A detection channel connecting the first tank and the second tank, An inlet portion connecting the first layer and the detection channel, It comprises an outlet section that connects the second layer and the detection channel, The inlet and outlet portions are provided at the same height from one end to the other in the longitudinal direction that connects the first and second tanks, and are provided such that their width decreases as they approach the detection channel. A method for evaluating the deformability of suspended cells in an electrolyte solution, comprising using a sensing device to evaluate the deformability of suspended cells, In an ion current waveform where the first axis represents time and the second axis represents the pulsed ion current flowing between the input electrode and the output electrode when the suspended cells pass through the detection channel, the symmetry in the first axis direction with respect to the third axis parallel to the second axis, passing through the minimum value of the ion current, is determined. Based on the determination of the symmetry, the deformability of the floating cells is evaluated. A method for evaluating the deformability of suspension cells.

2. In the method for evaluating the deformability of suspension cells according to claim 1, The symmetry is determined based on a comparison between a first time, from the time when the ion current falls below a threshold, until the ion current reaches the minimum value, and a second time, from the time when the ion current returns from the minimum value back to the threshold. A method for evaluating the deformability of suspension cells.

3. In the method for evaluating the deformability of suspension cells according to claim 1 or 2, The aforementioned floating cells are blood cells. A method for evaluating the deformability of suspension cells.