Biopsy / cytology devices for sampling mammalian cells or tissues

Abstract

A device for isolating cells or tissue from a cavity in a subject, comprising an elongate member arranged to move within a lumen of a needle or catheter, and at least one flexible member arranged at a distal end of the elongate member, the flexible member configured to be in a first constrained configuration within the lumen of the needle or catheter and in a second expanded configuration outside the lumen, the flexible member configured to conform to the internal shape of the cavity into which the device is inserted when in the second expanded configuration.

Description

Technical Field

[0001] The present disclosure generally relates to devices for separating cells within a cavity of a subject and to liquid biopsy / cytodiagnosis for sampling cells or tissue from a cavity of a subject. The biopsy / cytodiagnosis method includes enabling the introduction and manipulation of a device through a hollow catheter or needle to obtain a cell sample.

Background Art

[0002] Pancreatic cancer is currently the fourth leading cause of cancer-related death in the West, and its incidence rate is estimated to be the second highest among all cancers by 2030. The prognosis remains very poor, and since the disease stage is advanced at the time of diagnosis, the 5-year survival rate is only 2% - 9%, the lowest among all cancers. Pancystic lesions are precursors of most pancreatic cancers, and the prevalence of pancystic lesions in the general population can reach 40%. Therefore, there is a great need to appropriately diagnose whether pancreatic cysts are potentially benign or malignant.

[0003] Ultrasonic endoscopy (EUS)-guided fine-needle aspiration (FNA) and subsequent fluid cell analysis are used as diagnostic tools to differentiate between benign, potentially malignant, and malignant pancreatic cysts in cystic lesions. While EUS-FNA yields promising results for solid lesions, it is poor for cystic lesions due to the sparse cellular content in the cystic fluid. The reason for the lack of cells in the cystic fluid is that EUS-FNA removes the cystic fluid content, not the cystic lining or wall, which are normally covered by cells. As a result, the sensitivity of this method ranges from 65% to 95%, and the specificity from 50% to 100%. The average precision is 85%, meaning that in 20% of cases, inconclusive or ambiguous situations arise. This lack of information can prevent cytopathologists from making a diagnosis. To address this issue, a "through-the-needle" surgical cytology brush (EchoBrush, Cook Endoscopy, Winston-Salem, North Carolina) using a 19G needle was developed, yielding diagnostic data in 85.1% of cases. However, due to an increased risk of bleeding, production of these devices was discontinued.

[0004] International Publication No. 2018 / 053402 discloses a pancreatic cyst device deployable into a cyst via a 22-gauge endoscopic ultrasound (EUS) needle. The device is a flexible shaft with a “spiral Q” shaped distal end designed to maximize contact area along the shape of the cyst and a proximal end that can be connected to a handle, allowing the operator to rotate the device within the pancreatic cyst to remove cells from the cyst lining.

[0005] However, the aforementioned devices still carry the risk of damaging the cyst lining, and the spiral-shaped distal end can break during operation and retract into the outer shaft. Furthermore, the flexible shaft is made from nitinol along its entire length, making it costly to manufacture.

[0006] Therefore, it is necessary to improve known devices and methods to address the aforementioned shortcomings. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, the objective of the present invention is to achieve a minimally invasive biopsy / cytology device and method that increases the number of cells or tissues collected from the subject's cavity while reducing tissue damage at the sampling site.

[0008] This objective is achieved in a first embodiment of the present disclosure by providing a device for separating cells or tissue from a cavity in a subject, the device comprising an elongated member disposed to move within the lumen of a needle or catheter, and at least one flexible member disposed at the distal end of the elongated member, the flexible member being configured to be a first constraining configuration within the lumen of the needle or catheter and a second expanding configuration outside the lumen, the flexible member being configured to conform to the internal shape of the cavity into which the device is inserted when in the second expanding configuration.

[0009] By providing a device comprising an elongated member and a flexible member disposed at its distal end, the flexible member is configured to conform to the internal shape of the cavity into which the device is inserted, thereby reducing the risk of tissue damage at the sampling site while increasing the number of cells collected during the sampling procedure.

[0010] In one embodiment, the flexible member is preferably a superelastic wire made of a shape memory alloy. The superelastic wire, and especially shape memory alloys such as nickel-titanium, allows the flexible member to take on a desired shape of the cavity, for example, the internal shape of a cyst, when the device is inserted into the cavity.

[0011] In one embodiment, the flexible member is arranged to form at least one loop in the extended configuration. The loop shape allows the flexible member to conform to a desired shape of the internal cavity in a simple manner. In addition, the loop shape allows the flexible member to be fully extended even if the distance the elongated member needs to be advanced is short.

[0012] In one embodiment, at least a portion of the flexible member has a substantial helical shape in which the axis of the helix is ​​substantially parallel to the overall direction of extension of the flexible member. The helical shape roughens the edges, allowing cells to be easily scraped off from the inner lining / wall of the cavity.

[0013] In one embodiment, at least a portion of the flexible member has a serrated structure exhibiting, for example, multiple teeth. Preferably, the serrated structure is achieved by a microstructure machined or arranged on the surface of the flexible member. The serrated structure roughens the edges, increasing wear and facilitating the scraping of cells from the inner lining / wall of the cavity, thereby increasing the cell yield.

[0014] In one embodiment, the elongated member and the flexible member are integrally formed as a monolithic structure, and the diameter of the flexible member is smaller than the diameter of the elongated member. By making the diameter of the flexible member smaller than the diameter of the elongated member, it is possible to double the length of the flexible member in the constrained configuration. Therefore, even if the distance the elongated member is advanced is short, the flexible member can be fully extended into an expanded configuration.

[0015] In one embodiment, an elongated member is provided with a mounting interface at its distal end, and at least one flexible member is attached to the elongated member by the mounting interface. This configuration makes it possible to manufacture the flexible member separately from the elongated member, for example, using a different material or a different process.

[0016] In one embodiment, the mounting interface includes at least one hole for fastening at least one flexible member to an elongated member, a mechanical interlocking mechanism between the elongated member and at least one flexible member, a weld line, an adhesive line, or a combination thereof. Depending on the desired characteristics of the device and / or the intended operating conditions, another option may be selected for fastening. Preferably, the mounting interface is covered with heat-shrink tubing. Protection of the mounting interface by the heat-shrink tubing prevents rupture or delamination of the flexible member from the elongated member.

[0017] In one embodiment, the device further comprises a tubular sheath disposed on the outside of an elongated member and configured to move along the elongated member inside the lumen of a needle or catheter. The tubular sheath protects the flexible member from breakage during forward, movement, and retraction, for example, with respect to a hollow needle into which the device is inserted. Preferably, the tubular sheath is made of a polymer such as polyether, polyamide, polyimide, or polytetrafluoroethylene, PTFE, or a metal such as nickel-titanium or stainless steel.

[0018] In one embodiment, the elongated member and / or the flexible member are surface-treated or coated to reduce the coefficient of friction.

[0019] A second aspect of the present disclosure provides a device for performing fine-needle aspiration, FNA, or fine-needle biopsy, FNB, the device comprising a hollow needle and a device according to the first aspect, which is movably disposed within the lumen of the needle.

[0020] A third aspect of this disclosure relates to a method for collecting cells or tissue from a cavity of a subject by performing fine-needle aspiration, fine-needle aspiration, or fine-needle biopsy, fine-needle biopsy, or fine-needle biopsy, The present invention provides a device comprising a hollow needle, an elongated member movably disposed within the lumen of the needle, and at least one flexible member disposed at the distal end of the elongated member, wherein the flexible member is configured to be in a first constraining configuration within the lumen of the needle and in a second expansion configuration outside the lumen, and when in the second expansion configuration, the flexible member is configured to conform to the internal shape of the cavity into which the device is inserted. Introducing the needle into the cavity of the subject, The method involves advancing an elongated member through the lumen of the needle, such that the flexible member protrudes from the distal end of the needle, takes on a second expansion configuration within the cavity, and advances the elongated member so that it contacts the inner wall of the cavity. The method involves rotating an elongated member so that the expanded flexible member scrapes against the inner wall of the cavity, The slender member is retracted into the lumen of the needle, so that the flexible member becomes the first restraining structure and the slender member is retracted so that it re-enters the distal end of the needle. The process involves drawing liquid from the cavity through the lumen of the needle, A method is provided which includes retracting a needle from a cavity.

[0021] In one embodiment, the device comprises a tubular sheath disposed on the outside of an elongated member, the tubular sheath configured to move along the elongated member within the lumen of a needle or catheter, and the method is as follows: The step of rotating the elongated member involves advancing the tubular sheath through the needle, such that the distal end of the tubular sheath emerges from the distal end of the needle and contacts the expanded flexible member. The process further includes retracting the slender member, followed by retracting the tubular sheath into the needle.

[0022] In one embodiment, the step of introducing a needle into a cavity within a subject is performed using endoscopic ultrasound examination, EUS, and guidance. Thus, sampling can be performed, for example, in a minimally invasive manner as an EUS-FNA / FNB procedure to obtain a sample within the gastric region of the subject.

Brief Description of the Drawings

[0023] Next, the present invention will be described for illustrative purposes with reference to the accompanying drawings. [Figure 1] FIG. shows an endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) path towards a cyst in the pancreas, different steps of operating the device, and a device that interacts with the EUS-FNA outside the human body, used by a device according to an embodiment of the present disclosure. [Figure 2] FIG. shows a photograph of a device according to an embodiment of the present disclosure with a 22G needle exiting, and a scanning electron microscope (SEM) image of a mounting interface in the form of a knot before and after the arrangement of a heat shrink tube. [Figure 3] FIG. shows a cross-sectional view of the operation of a device according to an embodiment of the present disclosure in a cavity of a cyst phantom, a side view photograph of the cyst phantom into which the device is inserted, an axial view of the front and rear parts of the cyst phantom before and after brushing with the device, and the difference between them. [Figure 4] FIG. shows a schematic view of an ex vivo test of a device according to an embodiment of the present disclosure in a porcine small intestine model. [Figure 5a] FIG. shows the absolute cell concentration and brush efficiency from a test performed as shown in FIG. 4. [Figure 5b] FIG. shows the absolute cell concentration and brush efficiency from a test performed as shown in FIG. 4. [Figure 6] FIG. shows a schematic view of an ex vivo test of a device according to an embodiment of the present disclosure in a bovine follicular cyst, and cell counter images without brushing and with brushing. [Figure 7]This figure shows four embodiments of a flexible member disposed at the distal end of an elongated member according to the present disclosure. [Figure 8] This figure shows an enlarged view of a flexible member according to a different embodiment of the present disclosure. [Figure 9] This figure shows different steps for operating a device according to another embodiment of the present disclosure. [Modes for carrying out the invention]

[0024] A detailed description of the device described herein is presented below. In the drawings, similar reference numerals indicate the same or corresponding elements throughout several drawings. It should be understood that these figures are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] In the context of this disclosure, the terms “distal” and “distally” are understood to refer to the location or direction furthest away from the operator when using the device provided by this disclosure. Similarly, the terms “proximal” and “proximally” refer to the location or direction closest to, or toward, the operator when using the device provided by this disclosure.

[0026] Referring to Figure 1, Figure a) shows a schematic diagram of the current endoscopic ultrasound-guided fine-needle aspiration / biopsy (EUS-FNA / FNB) procedure. The endoscope is inserted through the patient's mouth, esophagus, stomach, and small intestine (duodenum) to reach the pancreas, and once the location of the cyst is located using an ultrasound transducer, the operator inserts a 22G needle into the cyst. However, other organs or areas of interest may be targeted, and the endoscope may be inserted through the colon, anus, or rectum. Depending on the procedure, needles of other gauges may also be used.

[0027] Figure 1, diagram b), shows the steps of a procedure to improve cell yield. A flexible member in the form of a loop brush is initially located inside the 22G needle, but when pushed into the cyst, it fits into the lumen within the cyst. By rotating the elongated member, the loop brush is rotated, scraping cells from the cyst wall and releasing the cells into the cystic fluid. The flexible member is then retracted from the needle lumen, and the cystic fluid is aspirated through the needle, and the cystic fluid is collected for downstream cytological / pathological or biochemical, molecular or genetic analysis.

[0028] A load test was conducted to verify the mechanical robustness of the device. As a result, even after 100 repetitions of the operation of introducing the loop brush through a needle, rotating it, and removing it using a slender member 1200 mm in length, the movement of the loop brush was not restricted. Figure 1, photograph c), shows the distal end of the endoscope including the needle and loop brush.

[0029] Tensile tests of the flexible member showed that mechanical failure occurred at a maximum breaking force exceeding 3N, which is comparable to that of a 50m straight Nitinol wire (SI5). The level of mechanical stress during in vivo operation is maintained several orders of magnitude lower than this maximum breaking force.

[0030] Referring now to Figure 2, a device according to one embodiment of the present disclosure is shown. Photograph a) in Figure 2 shows the distal end of the device, where a loop-shaped flexible member is positioned at the distal end of an elongated member referred to in this figure as a guidewire. The flexible member is formed from a thin 50 μm nitinol wire shaped into a loop with a diameter of 1 cm. This loop is secured to a 280 μm thick nitinol elongated member by an overhand knot, as shown in scanning electron microscope (SEM) photograph b) in Figure 2. The nitinol wire is tied to the guidewire through a hole in the guidewire. The hole may have a diameter greater than 0.025 mm and less than 5 mm, and typically less than 0.50 mm. The hole may be located 1 mm or up to 10 mm away from the distal end of the elongated member.

[0031] After knotting, heat shrink tubing is inserted over the knot and shrunk by applying heat, thereby causing the knot to fit into the hole, as shown in the SEM image c) of Figure 2. The heat shrink tubing can have a diameter greater than 0.1 mm and less than 12 mm, and typically greater than 0.2 mm.

[0032] Within the scope of the present invention, other means for attaching the flexible member to the elongated member are conceivable, such as bonding, welding, and mechanical interlocking. The attachment interface may accommodate a heat-shrinkable tube, or it may be without a heat-shrinkable tube, or combined with a heat-shrinkable tube. In one embodiment, the elongated member and the flexible member are formed integrally as a monolithic structure, i.e., as a single part.

[0033] Referring to Figure 3, the operation procedure of the loop brush within the spherical cavity of the in vitro cystic phantom was examined, as shown in Figure 3, diagram a). As shown in Figure 3, photograph b), the flexible member was introduced into the cavity through the lumen of a 22G needle and then rotated. The loop brush was observed to conform to the inner wall of the phantom, and it was found that the flexible member could remove cell-sized talc powder particles from the rigid inner wall of the cavity, thus demonstrating its ability to brush the internal 3D curved surface, as shown in Figure 3, photograph c).

[0034] Currently, there are no animal models of pancreatic cysts that are similar in size to human cysts and mimic cell adhesion in tissue, which is a critical issue for verifying the functionality of the loop brush. Furthermore, pancreatic cysts contain fluids of varying viscosities, with higher viscosity indicating greater malignancy. Therefore, an ex vivo porcine small intestine model was constructed using either saline or glycerol as the liquid culture medium. Referring to Figure 4, the ability of the flexible material to increase the number of cells after brushing was demonstrated. Six intestines, indicated by A-F, were used, and each intestine was cut to produce six test sections, indicated by 1-6. Figure 4, diagram a), shows a schematic diagram of the cutting locations. Figure 4, diagram b) shows schematic cross-sectional diagrams of i) the negative control test, i.e., aspiration without subsequent liquid addition and brushing; ii) the positive control test, i.e., subsequent liquid addition to the first intestine, i.e., brushing with an interdental brush and liquid aspiration; and iii) the sample test, i.e., continuous liquid addition to the second to sixth intestines, i.e., cell collection from the model during brushing with a loop brush and liquid aspiration.

[0035] The cell content after liquid removal was compared for loop brushing, negative control (no brushing), and positive control (brushing with an interdental brush). The results are shown as absolute cell concentration in Figure 5a and as brushing efficiency η in Figure 5b. Samples from the small intestine, shown A-C, were filled with physiological saline, and samples from the small intestine, shown D-F, were filled with glycerol. The dashed horizontal line indicates the limit of detection (LOD) of the cell counter. Cell counts below the LOD are shown just below the LOD line. Error bars indicate standard deviation (SD). *P<0.05. When the Wilcoxon pair test was performed for all 15 repetitions of the negative control and loop brushing, P<0.0001 was found for both media.

[0036] Brush efficiency η was defined as the cell concentration ratio of the loop-brushed sample to the negative control sample. If the negative control sample contained fewer cells than the cell counter's limit of detection (LOD), the LOD value was used instead of the negative control value. All tests showed η > 1. The mean value was η = 11 (n=15) for the saline model and η = 65 (n=15) for the glycerol-containing model. These results indicate that loop brushing can remove cells from the 3D soft tissue surface, and that cells can then be collected by aspiration. After loop brushing, the cell concentrations in the saline and glycerol-filled samples were similar, indicating that loop brushing improves cell collection in cystic fluids of varying viscosities. Differences in cell content were observed in the negative control measurements between the saline and glycerol-filled models. This difference was attributed to differences in liquid viscosity, resulting in a more turbulent packing state for the saline model and a more laminar flow state for the glycerol-filled model. In other words, in a model filled with physiological saline, cells that have already been released are mixed into the liquid, whereas in a model filled with glycerol, such cells are maintained near the intestinal wall.

[0037] It was further confirmed that the force applied to the tissue by the flexible member during typical operation is in the range of approximately 0.1 mN to 0.7 mN (SI7). The use of the loop brush was also confirmed in a model of the endoscopic pathway to a cyst, mimicking the curves of the gastrointestinal system. No adverse effects were observed when the loop brush was rotated against the lumen of a pig small intestine model at 60 rPm for 1 hour.

[0038] Referring here to Figure 6, we tested the ability of ex-vivo bovine follicular cyst models to increase cell count after brushing. As shown in Figure 6, a), the experimental procedure began with a liquid sample without brushing (negative control), followed by loop brushing, and then a second liquid sample. Figure 6, photograph b), shows bovine ovaries with and without follicular cysts. Figure 6, photographs c) and d), show color-enhanced cell counter images of the negative control sample (c) and loop-brushed sample (d) and their corresponding cell counts.

[0039] Loop brushing resulted in at least a tenfold increase in cell concentration compared to the negative control. This efficiency estimate is conservative, as the presence of cell clusters after brushing makes it difficult to count all cells in the sample. These results demonstrate that the flexible member can be successfully manipulated within the cyst through the lumen of a 22G needle, allowing for brushing and dispersion of cells within the cystic fluid environment. The flexible member shows potential for successful operation during pancreatic adenocarcinoma diagnosis, as it can gently abrade and disperse cells in two or more types of tissue.

[0040] Current results demonstrate the functionality, mechanical robustness, simple procedure, and low force on tissue of the flexible member in a gastrointestinal tract model, indicating compatibility with current EUS-FNA techniques. Future studies should aim to evaluate the safety of the procedure, particularly regarding potential damage to surrounding tissues and brushing of intracystic septa, i.e., wall structures that separate the cystic cavity into two or more compartments.

[0041] Referring here to Figure 7, four embodiments of the loop-shaped flexible member according to this disclosure are shown. The flexible member is positioned at the distal end of the elongated member. The diameter of the elongated member may be greater than 0.1 mm and less than 12 mm, and is typically greater than 0.2 mm. The length of the elongated member may be as short as 1 cm, and is typically greater than 3 cm, and may be up to 2 m. During operation, the elongated member may be used to guide the flexible member. For example, the flexible member may be introduced into the cavity, rotated inside, and removed from the cavity when the elongated member moves forward, rotates, and retracts within the needle lumen, respectively. The elongated member may be operated by hand or by a mechanical / robot device. Operation may be performed on-site or remotely.

[0042] The diameter of the flexible member may be greater than 0.025 mm and less than 4 mm, typically less than 0.5 mm, and more preferably less than 0.21 mm. The diameter of the loop may be greater than 1 mm, typically greater than 2 mm, less than 8 cm, and typically less than 3 cm.

[0043] In the first embodiment, the flexible member comprises a single loop, which is substantially circular in an expanded configuration not constrained within the lumen of a needle or catheter. An exemplary needle may have an inner diameter greater than 0.1 mm and less than 6 mm, typically less than 5.5 mm. An exemplary catheter may have an inner diameter greater than 0.33 mm and less than 12 mm, typically less than 9 mm. In the second embodiment, the flexible member comprises two or more loops, for example, two loops as shown herein. In the third embodiment, the flexible member comprises at least one serrated portion, comprising a plurality of teeth or dots in a zigzag pattern. The serrated edge of the loop increases the abrasive action of the flexible member against the wall of a cyst or other cavity, thereby increasing the cell yield. The serrated edge may be formed by the macrostructure of the flexible member, for example, the flexible member is formed with a serrated structure. In the fourth embodiment, the flexible member is wound into a coil to form a helical shape, and then a loop is formed such that the axis of the helix is ​​substantially parallel to the overall extending direction of the flexible member, i.e., the contour of the loop shape.

[0044] Referring now to Figure 8, magnified views of the surface of a flexible member according to different embodiments of the present disclosure are shown. In one embodiment, the surface of the flexible member may be substantially smooth, as in the upper left embodiment of Figure 7. In another embodiment, the surface of the flexible member may have a microstructure, as in the lower left embodiment of Figure 7, thereby increasing wear and thus cell yield. The microstructure may be formed by notches or recesses on the surface of the flexible member, as shown in the center view of Figure 8. In one embodiment, the microstructure is formed by one or more cylindrical structures having a plurality of protrusions, e.g., studs, and the microstructure is inserted onto the flexible member. The cylindrical structure may be rigid and may locally restrict the flexibility of the flexible member. The flexible member can bend in the portion between the cylindrical structures, thus improving the conformal behavior of the flexible member with respect to the tissue wall of the cavity.

[0045] Referring now to Figure 9, the steps of the procedure for operating a device according to one embodiment of the present disclosure are shown. In step a), a needle including a device comprising an elongated member and a flexible member in a constrained configuration inside the lumen of the needle is introduced into a cavity of a subject, for example, a cyst. In step b), the elongated member is advanced distally through the lumen of the needle. As a result, the flexible member exits the lumen of the needle and transitions into an expanded configuration, where the flexible member substantially conforms to the internal shape of the cyst and contacts the inner wall. In step c), a tubular sheath is advanced on the elongated member inside the lumen of the needle until the distal end of the tubular sheath contacts the expanded flexible member. The tubular sheath can be made from a polymer such as polyether, polyamide, polyimide, or polytetrafluoroethylene, PTFE, or from a metal such as nickel-titanium or stainless steel, and protects the flexible member from the sharp facets of the needle. In step d), the elongated member is rotated inside the needle lumen, causing the flexible member to rotate inside the cyst and wear away cells from the inner wall. In step e), the elongated member is retracted proximally, allowing the flexible member to re-enter the needle and transition to a restrained configuration. At this stage, the tubular sheath is maintained beyond the needle tip so that the flexible member does not come into contact with the needle during retraction. In step f), the tubular sheath is retracted proximally into the needle lumen. Finally, in step g), the fluid in the cyst is aspirated along with the cells removed by the abrasive action of the flexible member. The fluid contents in the cavity may be spontaneously excreted by the organism (e.g., by urination). The fluid contents may also be removed by catheterization.

[0046] In one embodiment, the aspirated liquid contents can be pushed out of the needle lumen by advancing the elongated member and the flexible member distally. In one embodiment, the liquid contents may be pushed out of the needle lumen by pushing the wire forward inside the needle, for example, a stylet. In one embodiment, the liquid contents can be pushed out of the needle lumen by using air or liquid through a syringe. The flexible member can be cut after liquid removal and then used for cytological, pathological, biological, or chemical analysis. The liquid contents can then be used for cytological, pathological, biological, or chemical analysis. Cytological analysis is, for example, cell morphology analysis. Pathological analysis is, for example, histological morphology analysis. Chemical analysis is, for example, protein analysis, glycoprotein analysis, enzyme analysis, DNA analysis, cancer marker analysis, and immunoassay.

[0047] In one embodiment, the surfaces of the elongated members, flexible members, and / or tubular sheaths may be treated or coated to reduce the coefficient of friction, thereby facilitating forward, backward, and rotational movement.

[0048] Manufacturing of loop brushes In one embodiment, the loop brush consists of three main components: a 280 μm nitinol elongated member (stylet wire from Expect™ Slimline EUS-FNA device M00555510, Boston Scientific, USA) tied together to form a loop; a 50 μm nitinol wire (NiTi#1 wire 0.002" ± 0.0001" straight annealed light oxide, Fort Wayne Metals, Ireland); and a polyethylene terephthalate medical heat shrink tubing (103-0510, Nordson Medical, USA) with a pre-shrink inner diameter of 460 μm to protect the knot area.

[0049] To fabricate the loop brush, a 150 μm hole was drilled through the distal end of a slender member. The slender member was inserted through a 2 cm section of heat-shrink tubing, without covering the hole. Next, the same end of a 50 μm nitinol wire was passed through the hole twice to form a loop. The loop was wrapped around a 1 cm diameter cylinder of ice, and an overhand knot was made using both ends of the 50 μm nitinol wire. This positioned the knot relative to the slender member, resulting in a loop diameter of 1 cm. The cylinder on the ice was then allowed to melt at room temperature for 20 minutes, and the excess wire end used to facilitate the knot was cut off, leaving approximately 1 cm of wire end. Finally, the heat-shrink tubing was pushed forward by hand to cover the nitinol wire end and the knot, and the tubing was heated at 70°C for 30 seconds to shrink it. The heat-shrink tubing had two functions. In other words, it had the function of protecting the knot by preventing it from becoming untied while the loop brush was moving, and the function of holding a portion of the loop wire linearly inside the heat shrink tube to form a loop shape outside the tube.

[0050] [In vitro cyst phantom and brushing test] 2 x 2 x 2 cm 3 A cyst model was fabricated by milling a PMMA cube to obtain a spherical cavity with a diameter of 1 cm. The PMMA cube was divided into two parts: the anterior part contained half of the sphere, and the posterior part contained the other half. A 5 mm hole was made in the posterior part to create a phantom entrance. Figure 3b shows an image of the brush inside the cyst phantom.

[0051] Preparation for the brushing test was performed by applying Scottch® permanent double-sided tape (2346832, Office Depot, Sweden) to the sphere for 1 minute, removing the tape, placing Dialon® talc powder (7322338361053, Apotea, Sweden) on the surface, and binding it to the cyst phantom.

[0052] Excess talc powder was removed by making the two cavities collide with each other 20 times with the cavities facing each other. Prior to brushing, each part of the cube was fixed in a position holder, and the internal cavity was imaged using a Leica M205 C microscope with the cavity facing the microscope. These two parts were then assembled with two alignment pins and secured with a clamp. The loop brush was inserted into a 22G subcutaneous injection needle (4710007040, Henke-Sass Wolf, Germany) and held in place in front of the phantom entrance. To facilitate the test, the elongated loop brush member was secured by a wire holder on the outside of the needle. The wire holder was soldered to the shaft of a robot zone 101rpm 12VDC motor (638194, ServoCity, Sweden).

[0053] Cavity brushing was performed by running the motor at 60 rpm for 1 minute. After brushing, both parts of the sphere were separated and imaged again. Camera settings were kept constant throughout all image acquisition.

[0054] To measure the difference between the pre- and post-brushing images, the post-brushing images were aligned to the pre-brushing images using Python code (SI4). Following image alignment, all images were converted to 16-bit grayscale, and images representing the pre- and post-brushing image differences were obtained for each spherical portion. This was done using the difference function of ImageJ (version 1.52a, National Institutes of Health, USA).

[0055] [Pig enteric cyst model] Pork small intestines were removed immediately after death by Skovde Slakteri AB (Skovde, Sweden) and bagged in DMEM (10313021, Thermoti Fisher Scientific, Sweden)-based culture medium containing 10% FBS (Gibco™ 10270106, Fisher Scientific, Sweden) and 1% penicillin streptomycin (15070063, Thermoti Fisher Scientific, Sweden) to prevent tissue degradation. The small intestines were stored at 4°C for up to 24 hours after death until use.

[0056] For each experiment, the small intestine was cut using a scalpel and divided into six 8cm-long segments, starting approximately 3cm from the pylorus. These segments were then cut open axially to fully expose the rumen side of the small intestine. All exposed pieces were washed with tap water for 1 minute and gently rubbed by hand to remove any remaining living tissue. Each intestinal piece was placed in a container of tap water, and the tissue was kept for up to 20 minutes until the experiment.

[0057] A brushing test was performed using a hemispherical mold with a radius of 2.5 cm to hold the intestinal sample. The mold had five 1 mm diameter drill holes connected to a vacuum line, ensuring that the intestinal sample was held in the correct position with the lumen facing outwards (Figure 4b).

[0058] After fixing the tissue in a mold, 300 μL of 0.9% physiological saline (786-561, G-Biosciences, Sweden) or glycerol (G9012, Sigma-Aldrich, Sweden) was placed on the tissue, and a test without brushing (negative control) was performed. After allowing the liquid to stand for 1 minute, 100 μL of the liquid was carefully aspirated using a pipette, taking care not to touch the intestinal wall. This method was performed prior to all loop brushing and positive control experiments to determine the number of cells that would spontaneously be discharged into the medium due to cutting, washing, processing, and / or placement of the medium on the sample. Therefore, a negative control test was performed for each intestinal section before the loop brushing test on the same intestinal section. Loop brushing was performed on 5 of the 6 intestinal sections. After the negative control test, the liquid volume was maintained throughout the test by initiating loop brushing by adding an additional 100 μL of liquid to the tissue section. After the negative control test, positive control brushing was performed on the remaining tissue sections. Positive control brushing was also initiated by adding an additional 100 μL of fluid to the tissue section. An interdental brush (Dentalux, Sweden) was used as the positive control brush. This brush was gently pressed against the intestinal lumen, and then 0.5 cm from the center of the intestinal section. 2 In the specified region, the brush was moved back and forth axially for 1 minute without rotation. After brushing, 100 μL of liquid was aspirated again.

[0059] [Bovine follicular cyst model] The bovine reproductive system was removed immediately after death by Skovde slakteri, bagged in a DMEM-based medium containing 10% FBS and 1% penicillin-streptomycin to prevent tissue degradation, and stored at 4°C for 24 hours before excision.

[0060] One ovary without a follicular sac and one ovary containing a follicular sac were excised from the bovine reproductive system using a scalpel and fixed in a petri dish (Figure 5b). The experimental design shown in Figure 5a) was carried out as follows: First, the follicular cyst was punctured and 1.5 mL of fluid was aspirated using a 22G subcutaneous injection needle and syringe. Second, the follicular cyst was punctured again using a 22G subcutaneous injection needle with a pre-assembled loop brush, the loop brush was then introduced, manually rotated at approximately 60 rpm for 1 minute, and the brush was removed. Finally, 1.5 mL of cystic fluid was aspirated through the needle using a syringe. After homogenization, 100 μL aliquots were taken from the 1.5 mL sample for further cell analysis.

[0061] [Liquid sample processing] The aspirated liquid samples were transferred to a test tube (0030120086, Eppendorf, Sweden), then mixed with 500 μL of StemPro™ Accutase™ (A1110501, Thermofischer Scientific, Sweden). The mixture was then aspirated and gently homogenized with a pipette by dispensing at 2-second intervals for 30 seconds, and incubated at 37°C for 5 minutes. Next, the samples (except for the bovine) were filtered using a 70 μm cell strainer (431751, CorningQR, Netherlands), and distal separation was performed using a Micro Star 17R distal separator (VWR, Sweden) at 11000 rpm for 4 minutes. The supernatant was removed, and a fluorophore solution of physiological saline containing 0.05% v / v Hoechst 33342 (H3570, Thermofischer Scientific, Sweden) was added to each sample. The positive control was mixed with 100 μL of solution, and the negative control and loop-brushing samples were mixed with 50 μL of solution to match the appropriate pellet size. Finally, the samples were wrapped in aluminum foil for 20 minutes, then unwrapped and prepared for cell counting.

[0062] [Cell Counter Analysis] Cell counting was performed using the Countess II FL automated cell counter (Thermotifiser Scientific, Sweden) in combination with the EVOS™ photocube and DAPI (AMEP4650, Thermofisher Scientific, Sweden). All samples were gently homogenized with a pipette, and 10 μL of each sample was placed on a Countess™ cell counting chamber slide (C10228, Thermofisher Scientific, Sweden). Intestinal cell counting was performed using the DAPI photocube with settings of size 4–14 μm, brightness 0–255 a.u., circularity 0.67, and autofocus enabled. Cell counting of follicular cyst samples was also performed using the DAPI photocube, with settings of size 6–29 μm, brightness 0–255 a.u., circularity 0.78, and autofocus enabled. Finally, dilution calculations were performed for both the intestinal and follicular cyst samples, based on a final volume of 50 μL.

[0063] [Statistical analysis] The results of intestinal cell counting were applied to a one-sided Wilcoxon matched-pair signed-rank test with a significance level of 5% to test for statistical significance between the use and non-use of the loop brush. This was achieved using GraphPad Prism8 software (GraphPad, CA, USA).

[0064] [Conclusion] A loop brush designed to acquire cells from the inner wall and hollows of pancreatic cysts in conjunction with EUS-FNA was successfully tested. No disadvantages were found when introducing, rotating, and removing the loop brush from the cyst via a 22G needle. Cellular contents in fluids recovered from ex vivo cyst models were compared before and after brushing with the loop brush. When low-viscosity water was used as the fluid in the soft tissue-like lumen, the cell content increased up to 54-fold, when high-viscosity glycerol was used, it increased up to 174-fold, and at least 10-fold in bovine ovarian cysts. The loop brush proved to be a powerful, minimally invasive, all-around tool that can be used in conjunction with EUS-FNA and can brush cells from cysts in soft and hard cyst models using cellulosic or viscous fluids.

[0065] The loop brush experiment was performed using a brush connected to a DC motor, similar to the in vitro cyst phantom test. The distal end of the brush was gently pressed against the intestinal lumen and rotated at 60 rpm for 1 minute in a 0.5 cm² area in the center of the intestinal section. Then, 100 μL of liquid was aspirated using a pipette. Between each experiment, used intestinal sections were discarded, the mold was washed with ethanol, and then washed with cleanroom paper.

[0066] Preferred embodiments of a device for separating cells or tissues from a subject cavity, as described herein, have been described. However, those skilled in the art should understand that these can be modified within the scope of the appended claims without departing from the spirit of the invention.

[0067] The alternative embodiments or parts of the embodiments described above can be freely combined without departing from the spirit of the present invention, as long as the combination is not contradictory.

Claims

1. A device for performing fine-needle aspiration, FNA, or fine-needle biopsy, FNB, Hollow needle and A slender member is movably disposed inside the lumen of the needle, At least one flexible member disposed at the distal end of the elongated member and Equipped with, The flexible member has a first restraining configuration within the lumen of the needle, and following the advance of the elongated member in the needle, the flexible member has a second expanding configuration outside the lumen, and when the flexible member is in the second expanding configuration, it is arranged to form at least one loop. The device further comprises a tubular sheath disposed on the outside of the elongated member, the tubular sheath configured to contact, on the outside of the lumen, a loop that moves along the elongated member and expands inside the lumen of the needle. device.

2. The device according to claim 1, wherein the flexible member is a superelastic wire made of a shape memory alloy.

3. The device according to claim 1 or 2, wherein the loop is substantially circular in the second extended configuration.

4. The device according to any one of claims 1 to 3, wherein at least a portion of the flexible member has a substantially helical shape, and the axis of the helix is ​​substantially parallel to the overall direction of extension of the flexible member.

5. The device according to any one of claims 1 to 4, wherein at least a portion of the flexible member has a sawtooth structure.

6. The device according to claim 5, wherein the sawtooth structure is a microstructure on the surface of the flexible member.

7. The device according to any one of claims 1 to 6, wherein the elongated member and the flexible member are integrally formed as a monolithic structure, and the diameter of the flexible member is smaller than the diameter of the elongated member.

8. The device according to any one of claims 1 to 6, wherein the elongated member is provided with a mounting interface at its distal end, and the at least one flexible member is attached to the elongated member via the mounting interface.

9. The device according to claim 8, wherein the mounting interface includes at least one hole for fastening the at least one flexible member to the elongated member, a mechanical interlocking mechanism between the elongated member and the at least one flexible member, a weld line, an adhesive line, or a combination thereof.

10. The device according to claim 8 or 9, wherein the mounting interface is covered with heat-shrink tubing.

11. The device according to any one of claims 1 to 10, wherein the tubular sheath is made from a polymer such as polyether, polyamide, polyimide, or polytetrafluoroethylene, PTFE, or a metal such as nickel-titanium or stainless steel.

12. The device according to any one of claims 1 to 11, wherein the elongated member and / or the flexible member are surface-treated or coated to reduce the coefficient of friction.

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