Target cell capture device

Abstract

The objective is to propose a target cell capture device that uses multiple filters to further improve the capture effect. [Solution] The target cell capture device 1 is divided into an inlet side 7 and an outlet side 9, and a three-dimensional target cell capture filter 3,5 is placed between the inlet side 7 and the outlet side 9, and therefore an intermediate section 11 is also provided. The intermediate section 11 has a structure on the upstream side that sandwiches the three-dimensional target cell capture filter 3,5 of the outlet side 9, and a structure on the downstream side that sandwiches the three-dimensional target cell capture filter 3,5 of the inlet side 7. This makes it possible to significantly improve the capture effect by using an integrated unit in which multiple three-dimensional target cell capture filters are connected in series.

Description

Technical Field

[0001] The present invention relates to a target cell capture device, and more particularly to a target cell capture device having a target cell capture filter with a capture surface for capturing target cells in a liquid.

Background Art

[0002] Selectively extracting target substances (mainly cells, microorganisms, proteins, exosomes, etc.) in a liquid is a very important issue in the fields of life sciences and biotechnology such as medicine, health, and agriculture, and a method for simply, efficiently, and quickly capturing only the target substances is desired.

[0003] FIG. 10 is an exploded perspective view showing a conventional three-dimensional target cell capture device. FIG. 11 is a view showing the three-dimensional target cell capture filter shown in FIG. 10. FIG. 12 is a view showing the three-dimensional target cell capture filter shown in FIG. 10 and other three-dimensional target cell capture filters.

[0004] The inventors of the present application have previously proposed a three-dimensional target cell capture filter for simply, efficiently, and quickly separating and capturing target cells contained in a biological sample such as blood (Patent Document 1, Patent Document 2, Non-Patent Document 1, Non-Patent Document 2). The three-dimensional target cell capture filter deforms three-dimensionally according to the fluid force (pressure distribution by the fluid) (see FIG. 11).

[0005] Furthermore, in Non-Patent Document 2, the inventors also proposed a target cell capture device 50 incorporating the proposed filter internally, as shown in Figure 10. The target cell capture device 50 is divided into an inlet side 51 and an outlet side 53, with a filter 55 placed between the inlet side 51 and the outlet side 53. Three types of filters 55 were used, including those shown in Figures 11 and 12(A), as well as the two types shown in Figures 12(B) and 12(C). As also shown in Figure 2 of Patent Document 2, in Non-Patent Document 2, the filter in Figure 12(C) yielded the best capture results, the filter in Figure 12(B) yielded the next best capture results, and the filter in Figure 12(A) yielded inferior capture results compared to Figures 12(B) and 12(C). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2017-83265 [Patent Document 2] International release WO2020-241365 [Non-patent literature]

[0007] [Non-Patent Document 1] March 5, 2018, Bio-Microsystems Research Meeting, Institute of Electrical Engineers of Japan Research Meeting Materials, pp. 19-21, "Target Cell Capture Using Nucleic Acid Aptamers and Microfilters with Three-Dimensional Deformability," Kenshiro Nakatake, Yuta Nakajima, and 8 others. [Non-Patent Document 2] Talanta Volume 228, 1 June 2021,122239 "Detection of cancer cells in whole blood using a dynamic deformable microfilter and anucleic acid aptamer" Yuta Nakajima, and 10 others [Overview of the project] [Problems that the invention aims to solve]

[0008] However, the target cell capture device using a single filter proposed above has the problem of insufficient capture effectiveness in capturing target cells.

[0009] Therefore, the present invention aims to propose a target cell capture device that uses multiple filters to further improve the capture effect of capturing target cells. [Means for solving the problem]

[0010] The first aspect of the present invention is a target cell capture device comprising: a three-dimensional target cell capture filter having a plurality of notches that allow a portion of the capture surface for capturing target cells in a liquid to elastically deform in a direction perpendicular to the capture surface to form a gap; an inlet side for allowing liquid containing the target cells from the outside to flow into the three-dimensional target cell capture filter; and an outlet side that connects to the inlet side, sandwiching the three-dimensional target cell capture filter together to form an integrated unit, and discharges the liquid that has passed through the three-dimensional target cell capture filter to the outside, characterized in that a plurality of the three-dimensional target cell capture filters are provided between the inlet side connected to the outside and the outlet side connected to the outside. This first aspect is a broader concept that includes both the case of a connected unit of two filters (two filters) as described in the example, in addition to the second aspect described below (specifically, the case of one unit of two filters in the example).

[0011] A second aspect of the present invention relates to a target cell capture device comprising: a three-dimensional target cell capture filter having a plurality of notches that allow a portion of the capture surface for capturing target cells in a liquid to elastically deform in a direction perpendicular to the capture surface to form a gap; an inlet side for allowing liquid containing the target cells from the outside to flow into the three-dimensional target cell capture filter; and an outlet side that connects to the inlet side, sandwiching the three-dimensional target cell capture filter together to form an integrated unit, and discharging the liquid that has passed through the three-dimensional target cell capture filter to the outside, wherein the outlet side has a structure that sandwiches the three-dimensional target cell capture filter on its upstream side, and The device comprises an intermediate section having a structure downstream of the inlet section that sandwiches the three-dimensional target cell capture filter, and another three-dimensional target cell capture filter, and is connected in the order of the inlet section, the three-dimensional target cell capture filter, the intermediate section, the other three-dimensional target cell capture filter, and the outlet section to form a single integrated unit, the intermediate section having a structure that allows the liquid containing the target cells to flow into the other three-dimensional target cell capture filter, and the liquid containing the target cells is discharged to the outside by passing in the order of the inlet section, the three-dimensional target cell capture filter, the intermediate section, the other three-dimensional target cell capture filter, and the outlet section from the outside.

[0012] A third aspect of the present invention is that, in the second aspect, a number of the intermediate sections corresponding to the number of other three-dimensional target cell capture filters are used to form a series of multi-stage filter structures, and the whole is made into a single integrated unit. [Effects of the Invention]

[0013] From the perspective of the present invention, unlike the superposition of multilayer filters such as general micropores, it has been found that even with three-dimensional filters that deform three-dimensionally in response to liquid pressure, using multiple filters yields a significantly greater capture effect compared to using a single filter. In particular, existing devices with a single filter unit can be easily expanded into devices with a series-connected unit using multiple filters. This expansion yields results that are more than double when using two filters compared to one filter, and the second-stage filter in particular captures more target cells than a single filter and the first-stage filter, resulting in a significant capture effect and confirming the technical significance of a series-connected multi-stage unit. As a result, it can be developed into specific applications, such as capturing cancer cells, which is useful for early detection of cancer and monitoring postoperative progress. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows a target cell capture device according to an embodiment of the present invention. [Figure 2] This graph shows the results of evaluating 1 mL of blood from cancer patients using a conventional device (Single) and a multilayer microfilter device (Double). [Figure 3] This diagram illustrates the capture state of target cells in the first and second stages when using two filters. [Figure 4] Each of these is a further enlarged view of a part of Figure 3(C). [Figure 5] This figure shows the number of target cells captured in each region from 1 to 9 of the two single filters and the two double filters. [Figure 6] This diagram compares the case of one filter with one unit, the case of two filters with one unit, and the case of one filter with two units (two filters), and shows the graph for the case of one filter with one unit. [Figure 7] This figure shows a table of data corresponding to Figure 6. [Figure 8] A diagram for comparing the case of one unit of one filter, the case of one unit of two filters, and the case of two units of one filter (there are two filters), and a diagram showing graphs for the case of one unit of two filters and the case of two units of one filter (there are two filters). [Figure 9] A diagram showing a table of data corresponding to FIG. 8. [Figure 10] An exploded perspective view showing a conventional three-dimensional target cell capture device. [Figure 11] A diagram showing the three-dimensional target cell capture filter shown in FIG. 10. [Figure 12] A diagram showing the three-dimensional target cell capture filter shown in FIG. 10 and other three-dimensional target cell capture filters.

Mode for Carrying Out the Invention

[0015] Hereinafter, embodiments of the present invention will be described with reference to the drawings. It should be noted that the present invention is not limited to the following embodiments.

Embodiment

[0016] Cancer is the leading cause of death in Japan, but it is said that early detection allows for curative treatment. However, the participation rate in cancer screenings is low due to issues such as the time commitment and cost for patients. Therefore, there is a need for the development of a simple and low-cost cancer screening device. The inventors of this invention have focused on circulating tumor cells (CTCs) present in the blood of cancer patients, aiming to realize minimally invasive cancer screening through blood sampling. The number of CTCs is known to show a high correlation with the stage of cancer progression, making it useful for early detection of cancer and monitoring the progress after surgery. To date, we have developed a microfilter that specifically captures CTCs by targeting proteins expressed on the surface of cancer cells. By using this filter, it is possible to capture and evaluate the small number of cancer cells contained in the blood of actual cancer patients and apply this to cancer diagnosis and treatment. As shown in Figure 10, we have been capturing CTCs using a single microfilter, but it has become clear that it is necessary to capture many more CTCs when applying this to cancer treatment, and there is a problem that a single microfilter cannot capture enough CTCs. Therefore, the following describes the development of a device that uses two microfilters to efficiently and reliably capture a larger number of cancer cells, enabling highly accurate examinations.

[0017] Figure 1 shows a target cell capture device according to an embodiment of the present invention. Figure 1(A) is an exploded perspective view. Figure 1(B) shows an example of an actual product.

[0018] The target cell capture device 1 is a CTC detection device and comprises three-dimensional, dynamically deformable microfilters 3 and 5 for specifically capturing cancer cells, and a resin housing 6 for gripping the microfilters 3 and 5 and delivering the sample. The resin housing 6 has an upstream inlet side 7, a downstream outlet side 9, and an intermediate side 11 located between the two. Microfilter 3 is sandwiched between the inlet side 7 and the intermediate side 11, and microfilter 5 is sandwiched between the intermediate side 11 and the outlet side 9.

[0019] The inlet side 7 has a structure similar to the inlet side 51 shown in Figure 10. The outlet side 9 also has a structure similar to the outlet side 53 shown in Figure 10. On the other hand, the intermediate section 11 has a structure similar to the microfilter 5 side of the outlet side 9 on the microfilter 3 side, and a structure similar to the microfilter 3 side of the inlet side 7 on the microfilter 3 side on the microfilter 5 side. In other words, by incorporating the intermediate section 11, which is a component that can hold one more filter between the inlet side 51 and the outlet side 53 of the conventional housing shown in Figure 10, it is possible to create a device that can mount two filters 3 and 5.

[0020] Figure 2 is a graph showing the results of evaluating 1 mL (milliliter) of blood from cancer patients using a conventional device (Single) and a multilayer microfilter device (Double).

[0021] The experimental method is described below. A neutraavidin was modified onto the microfilter. Next, a terminally biotinylated anti-EpCAM aptamer was introduced into either a sample solution of healthy blood spiked with human breast cancer cells (MDA-MB-453) or blood from a cancer patient, and this aptamer bound to EpCAM expressed on the surface of the cancer cells. Because biotin and avidin bind strongly, their interaction allows for the specific capture of cancer cells on the filter. The sample solution and the blood from cancer patients were then delivered to a conventional device and a multilayered device, respectively. Subsequently, the captured cancer cells were immobilized on the filter, immunostained, and the number of captured cells was counted. The results showed that the multilayered device (Double) captured approximately three times more CTCs than the conventional device (Single). This remarkable effect is attributed to the significantly greater capture effect of the second-stage filter compared to the filter of the conventional device (Single) and the first-stage filter of the device (Double), as explained below.

[0022] Figure 3 illustrates the capture state of target cells in the first and second stages when using two filters. Figure 3(A) shows the capture state of target cells in the first stage, Figure 3(B) shows the capture state of target cells in the second stage-5, Figure 3(C) is a magnified view of a part of Figure 3(B), and Figure 3(D) shows the filter divided into nine regions, and clearly indicates the position of position 5 on the filter, as shown in Figure 3(B) is position 5. Figure 4 shows further magnified views of parts of Figure 3(C), illustrating an example of the staining results for Merge, DAPI, Cytokeratin, and CD45. The "Merge" in Figure 4 is a composite of the "DAPI," "Cytokeratin," and "CD45" images to its right. Figure 5 shows the number of target cells captured in each region from 1 to 9 of two single filters and two double filters.

[0023] First, as can be seen from Figure 3, there is a difference between the capture results of the first stage shown in Figure 3(A) and the capture results of the second stage shown in Figures 3(B) and 3(C). The capture results of the second stage shown in Figures 3(B) and 3(C) are significantly larger than those of the first stage shown in Figure 3(A). Taking this into consideration, we prepared two devices each of a single filter and a double filter and conducted verification. When using a single filter, 9 CTCs were captured in each case, while when using two filters, 125 CTCs and 71 CTCs were captured in each case. As shown in Figure 5, the results showed that the second stage of the double filter captured significantly more target cells than both the single filter and the first stage of the double filter. We have performed CTC capture using the blood of approximately 50 cancer patients using the single filter device, but there have been no cases in which more than 10 CTCs were captured, so it is clear that a groundbreaking capture effect has been achieved. From the above, it has been shown that the number of CTCs captured increases dramatically by using a multi-layered microfilter.

[0024] Figure 6 is a diagram for comparing the case of one unit of one filter, one unit of two filters, and two units of one filter (two filters in total), and shows the graph for the case of one unit of one filter. Figure 7 is a table of data corresponding to Figure 6. Figure 8 is a diagram for comparing the case of one unit of one filter, one unit of two filters, and two units of one filter (two filters in total), and shows the graphs for the case of one unit of two filters and two units of one filter (two filters in total). Figure 9 is a table of data corresponding to Figure 8.

[0025] Referring to Figures 6 and 7, this data shows the results of an experiment conducted by spiking cancer cells into the blood of healthy individuals. Here, 1000 cancer cells were spiked into the blood, and the data is from one unit of a single filter (labeled "Single" in Figure 7), which is an existing device. Single 1 had only one filter stage and captured 63 cells, Single 2 also had only one filter stage and captured 151 cells, and Single 3 also had only one filter stage and captured 109 cells, with an average of 107.7 cells. In conclusion, approximately 10% of the spiked cells were captured.

[0026] Referring to Figures 8 and 9, this data also shows the results of experiments conducted by spiking cancer cells into the blood of healthy individuals. Here, 100 cancer cells were spiked into the blood, and the results were obtained using a device with two filters in one unit (labeled "double" in Figures 8 and 9) and a device with two units, each with one filter (labeled "linked" in Figures 8 and 9). Both the double and linked devices captured over 100 cells, and even when comparing the average, both showed a higher capture efficiency compared to the single device in Figures 6 and 7. In other words, it was shown that using multiple filters compared to one filter resulted in a higher capture efficiency. The inventors believe that the reason for capturing more than 100 cells was an error during cell preparation. Specifically, after visually confirming that there were 50 cells in 5 μL (microliters) of culture medium, the sample was spiked into blood at a rate of 10 μL (microliters), and the inventors believe that an error of about 10% is not unreasonable.

[0027] Looking further at Figures 8 and 9, the trend is that, when broken down, high capture was observed in the second stage in the double configuration, and in the first stage in the linked configuration. The first stage of the linked configuration can be seen as being similar to the single configuration, and if so, the significance of the second stage in the double configuration is also demonstrated to some extent from this data. In other words, as the number of filters is increased by linking, the number of captures decreases downstream, while increasing the number of filters within a single unit, such as by making the double configuration into a triple configuration, may result in a higher number of captures, suggesting that the technical significance of the second stage in the double configuration may be high. Furthermore, given that the length increases with linked configurations, the double configuration is more compact and therefore more significant as a device.

[0028] In addition, the three-dimensional target cell capture filters 3 and 5 of the target cell capture device 1 shown in Figure 1 were used in the verification experiment using those shown in Figure 12(C). However, other filters may be used in addition to those shown in Figures 12(A) and 12(B), and the two filters do not need to be the same. [Explanation of symbols]

[0029] 1...Target cell capture device, 3,5...Three-dimensional target cell capture filter, 11...Intermediate section

Claims

1. A three-dimensional target cell capture filter having multiple notches that allow a portion of the capture surface for capturing target cells in a liquid to elastically deform in a direction perpendicular to the capture surface, thereby forming a gap, The three-dimensional target cell capture filter has an inlet side into which a liquid containing the target cells from the outside is introduced, In a target cell capture device, the three-dimensional target cell capture filter is sandwiched together with the inlet side and connected to the inlet side to form an integrated unit, and the device also includes an outlet side for discharging the liquid that has passed through the three-dimensional target cell capture filter to the outside, A target cell capture device characterized by having a plurality of three-dimensional target cell capture filters between the inlet side connected to the outside and the outlet side connected to the outside.

2. A three-dimensional target cell capture filter having multiple notches that allow a portion of the capture surface for capturing target cells in a liquid to elastically deform in a direction perpendicular to the capture surface, thereby forming a gap, The three-dimensional target cell capture filter has an inlet side into which a liquid containing the target cells from the outside is introduced, In a target cell capture device, the three-dimensional target cell capture filter is sandwiched together with the inlet side and connected to the inlet side to form an integrated unit, and the device also includes an outlet side for discharging the liquid that has passed through the three-dimensional target cell capture filter to the outside, An intermediate section having a structure on its upstream side that sandwiches the three-dimensional target cell capture filter on the outlet side, and a structure on its downstream side that sandwiches the three-dimensional target cell capture filter on the inlet side, Equipped with other three-dimensional target cell capture filters, The inlet side, the three-dimensional target cell capture filter, the intermediate section, the other three-dimensional target cell capture filter, and the outlet side are connected in that order to form a single integrated unit. The aforementioned intermediate section has a structure that allows the liquid containing the target cells to flow into the other three-dimensional target cell capture filter. A target cell capture device in which the liquid containing the target cells is discharged to the outside by passing, in order, from the outside, through the inlet side, the three-dimensional target cell capture filter, the intermediate section, the other three-dimensional target cell capture filter, and the outlet side.

3. The target cell capture device according to claim 2, wherein a number of the intermediate sections corresponding to the number of other three-dimensional target cell capture filters are used to form a multi-stage filter structure in series, and the whole is made into a single integrated unit.

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