Cell sampler unit, cell sampler tool comprising same and cell sampling method

Abstract

A cell sampling unit is disclosed, comprising: a plurality of micropillars for contacting a sample and acquiring cells from the surface of the sample by friction; and a base layer comprising a front side for supporting the plurality of micropillars and the acquired cells, wherein one end of each micropillar is fixed to or extends from the front side of the base layer, and the other end is a free end comprising a contact surface with frictional force; wherein each micropillar has a diameter of approximately 60-140 µm and a height of approximately 60-140 µm; wherein the plurality of micropillars are configured in an array arrangement; the micropillars are arranged at a rate of approximately 1000-1400 micropillars / cm 2 The density distribution between [specific parameters]. Other example embodiments are described herein. In some embodiments, the cell sampling unit is an easy-to-use, convenient, and reliable skin cell sampling method for male HPV screening.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 621,132, filed January 16, 2024, pursuant to 35 USC § 119(e) (35 USC § 119(e)), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to a cell sampler unit, a cell sampler tool including the same, and a cell sampling method. Background Technology

[0003] Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide. Increasing evidence of a link between HPV and anogenital cancers has led to the development of HPV testing for both women and men. Cell sampling is a crucial step in HPV testing because the viral load of HPV in infected tissue is typically very low. To date, different types of samplers have been developed for collecting HPV samples from the cervix of women. However, similar samplers have not yet been developed for men. In many clinical settings, sandpaper and medical swabs are used together for sample collection. This two-step collection method has several drawbacks, such as causing discomfort and anxiety, and more importantly, a low success rate. Therefore, there is an urgent need to develop an easy-to-use genital cell sampler for male HPV DNA testing. Utility Model Content

[0004] In some embodiments, a cell sampler unit, a cell sampler tool comprising the same, and a cell sampling method are provided. In some embodiments, a cell sampler with a novel microstructure is provided for skin cell sampling, such as HPV sampling from male genitalia. In some embodiments, a "Bio-Sandpaper" (BioS) technology is provided, which mimics naturally occurring microstructures, such as bifunctional microstructures that mimic micron- to nanometer-scale bristles in organisms and the micro-patterned hairs on bee legs that capture pollen. Such biomimetic microstructures can readily extract and capture skin cells. To achieve this, in some embodiments, the microstructure contains a plurality of micropillars whose shape and size are optimized to increase the surface area to volume ratio of the biocompatible polymer surface, thereby generating high friction to extract skin cells. In some embodiments, the micropillar spacing and pattern are optimized for efficient capture and release of skin cells. In some embodiments, the biocompatible polymer is tested to check the consistency of microstructure fabrication in the molding system, making the provided sampler feasible for future scale-up manufacturing. In some embodiments, the cell sampler prototype is made from a sampler head and operating rod with a microstructure. In some embodiments, the provided cell sampler is an easy-to-use, convenient, and reliable skin cell sampler for male HPV screening. Attached Figure Description

[0005] Figure 1A This is a photograph showing the mold design of a cell sampler unit with 42 x 35 micropillars according to an example embodiment.

[0006] Figure 1B This is a schematic diagram showing the dimensions of a micropillar with a height of 140 µm and a width of 140 µm on a cell sampler unit, according to an example embodiment.

[0007] Figure 1C This is a photograph showing the main silicon wafer mold used to manufacture a cell sampler unit, according to an example embodiment.

[0008] Figure 1D This is shown according to the example embodiment, 1 x 1.4 cm 2 Photograph of a PDMS cell sampler unit of this size.

[0009] Figure 2A This is a schematic diagram illustrating the steps of manufacturing a cell sampler head unit from PDMS according to an example embodiment.

[0010] Figure 2B This is a photomicrograph showing a front view of a PDMS cell sampler head unit with a microcolumn width of 140 µm under an optical microscope at 100x magnification, according to an example embodiment.

[0011] Figure 2C This is a photomicrograph showing a side view of a PDMS cell sampler head unit with a microcolumn height of 140 µm under an optical microscope at 100x magnification, according to an example embodiment.

[0012] Figure 3A This is a schematic diagram illustrating the steps of manufacturing a cell sampler head from PCL according to an example embodiment.

[0013] Figure 3B This is a photomicrograph showing a front view of a PCL cell sampler head unit with a micropillar width of 140 µm under a stereomicroscope at 80x magnification, according to an example embodiment.

[0014] Figure 3C This is a photomicrograph showing a side view of a PCL cell sampler unit with micropillars of 108 µm in height, under an optical microscope at 100x magnification, according to an example embodiment.

[0015] Figure 4A This is a schematic diagram illustrating the steps of manufacturing a cell sampler head from TPU according to an example embodiment.

[0016] Figure 4B This is a photomicrograph showing a front view of the TPU cell sampler head under an optical microscope at 100x magnification, according to an example embodiment. The micropillar is 140 µm wide.

[0017] Figure 4C This is a photomicrograph showing a side view of the TPU cell sampler head under an optical microscope at 100x magnification, according to an example embodiment. The height of the micropillar is 140 µm.

[0018] Figure 5A This is a schematic diagram illustrating the steps of analyzing a PDMS cell sampler head using a scanning electron microscope (SEM) according to an example embodiment.

[0019] Figure 5B This is an SEM micrograph showing a front view of the PDMS cell sampler head at 100x magnification, according to an example embodiment. The sampler density is >1000 microcolumns / cm². 2 .

[0020] Figure 5C This is a micrograph showing a side view of the PDMS cell sampler under SEM magnification at 100x, according to an example embodiment. The height and width of the micropillar are both 140 µm.

[0021] Figure 6AThe images show a schematic diagram (left) and a photograph (middle) of a silicon wafer mold having a cell sampler unit with 42 x 35 square micropillars, according to an example embodiment, and a photograph (right) of a PDMS cell sampler head having a cell sampler unit with square micropillars.

[0022] Figure 6B The images shown are front (left) and side (right) photographs of a PDMS sampler with square micropillars, taken under an optical microscope at 100x magnification, according to an example embodiment. The square micropillars are 140 µm high and wide.

[0023] Figure 6C These are schematic diagrams (left) and photographs (middle) of a silicon wafer mold for a cell sampler unit with triangular micropillars, according to an example embodiment, and a photograph (right) of a PDMS cell sampler unit with triangular micropillars.

[0024] Figure 6D These are photomicrographs showing, respectively, a front view (left) and a side view (right) of a PDMS sampler with triangular micropillars under an optical microscope at 100x magnification, according to an example embodiment. The height and width of the triangular micropillars are both 140µm.

[0025] Figure 7 This is a graph showing the frictional force of a cell sampler unit on synthetic skin, measured according to an example embodiment and with reference to ASTM D1894. In this graph, the COF of micropillars of different shapes was tested and compared.

[0026] Figure 8A This is a schematic diagram illustrating the friction generated by microstructures on synthetic skin, measured using a coefficient of friction (COF) tester according to an example embodiment. The figure shows different directions of movement of the triangular micropillars. The frictional force of the cell sampler head on the synthetic skin was measured according to ASTM D1894. The COF of the triangular micropillars in different directions of movement was tested and compared.

[0027] Figure 8B It is displayed according to Figure 8A Example embodiment, a diagram of static COF of triangular micropillars in different directions of movement. Figure 8C It is displayed according to Figure 8A Example embodiment, dynamic COF of triangular micropillar in different moving directions.

[0028] Figure 9A These are micrographs showing micropillars of various heights (60 µm, 100 µm, and 140 µm) prepared according to example embodiments to optimize micropillar height. Friction of the cell sampler on synthetic skin was measured with reference to ASTM D1894.

[0029] Figure 9B It is displayed according to Figure 9A Example embodiments, plots of static COF for micropillars of different heights. Frictional force of the cell sampler on synthetic skin was measured with reference to ASTM D1894.

[0030] Figure 9C It is displayed according to Figure 9A Example embodiments, dynamic COF graphs of micropillars at different heights.

[0031] Figure 10A These are photomicrographs under a 100x optical microscope showing cell samplers with micropillars of various widths (60 µm, 100 µm, and 140 µm) prepared according to example embodiments for optimized micropillar width. The frictional force of the cell samplers on synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different widths was tested and compared.

[0032] Figure 10B It is displayed according to Figure 10A Example embodiments, graphs of static COF for micropillars of different widths.

[0033] Figure 10C It is displayed according to Figure 10A Example embodiments, plots of dynamic COF for micropillars of different widths.

[0034] Figure 11A These are photomicrographs taken under a 100x optical microscope, showing cell samplers with various microcolumn densities prepared according to example embodiments for optimizing microcolumn density. The dimensions of the cell samplers were observed under the 100x optical microscope. The frictional force of the cell samplers on synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different densities was tested and compared.

[0035] Figure 11B It is displayed according to Figure 11A Example embodiments, graphs of static COF of micropillars with different densities.

[0036] Figure 11C It is displayed according to Figure 11A Example embodiments, dynamic COF graphs of micropillars with different densities.

[0037] Figure 12A These are photomicrographs taken under a 100x optical microscope, showing cell samplers with various micropillar patterns prepared according to example embodiments for optimized micropillar patterning. The dimensions of the cell samplers were observed under the 100x optical microscope. The frictional force of the cell samplers on synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different patterns was tested and compared.

[0038] Figure 12B It is displayed according to Figure 12A Example embodiments, static COF diagrams of micropillars with different patterns.

[0039] Figure 12C It is displayed according to Figure 12A Example embodiments, dynamic COF graphs of micropillars with different patterns.

[0040] Figure 13A This is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after one swipe; the image shows large squares of skin cells / corners: (6+10+2+2) / 4 = 5 collected cells = 5 x 0.1 x 10 4 = 5000 skin cells (according to the example embodiment). Cells on the sampler were trypsinized and stained with trypan blue. Cells were quantified using a hemocytometer.

[0041] Figure 13B This is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after three swabs. The image shows large squares of skin cells / corners: (10+10+6+6) / 4 = 8 collected cells = 8 x 0.1 x 10 4 = 8000 skin cells (according to the example embodiment). Other experimental settings and Figure 13A same.

[0042] Figure 13C This is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after 5 swipes. The image shows large squares of skin cells / corners: (15+8+10+7) / 4 = 10 collected cells = 10 x 0.1 x 10 4 = 10,000 skin cells (according to the example embodiment). Other experimental settings and... Figure 13A same.

[0043] Figure 13D This is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after 10 swipes. The image shows large squares of skin cells / corners: (11+10+9+9) / 4 = 9.75 collected cells = 9.75 x 0.1 x 10 4 = 9750 skin cells (according to the example embodiment). Other experimental settings and... Figure 13A same.

[0044] Figure 13EThis is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after 15 swipes. The image shows large squares of skin cells / corners: (15+11+12+14) / 4 = 13 collected cells = 13 x 0.1 x 10⁻⁶ cells. 4 = 13,000 skin cells (according to the example embodiment). Other experimental settings and... Figure 13A same.

[0045] Figure 13F This is a micrograph under a 100x optical microscope, showing the cell collection volume assessment of a PDMS sampler after 20 swipes. The image shows large squares of skin cells / corners: (16+14+18+15) / 4 = 15.75 collected cells = 15.75 x 0.1 x 10 4 = 15,750 skin cells (according to the example embodiment). Other experimental settings and... Figure 13A same.

[0046] Figure 13G It is a display Figures 13A to 13F The graph of the number of cells collected relative to the number of wipes in the example embodiment shows that the number of cells collected is proportional to the number of wipes. According to the example embodiment, ultrasonic trypsin treatment further improves cell detachment efficiency compared to trypsin alone.

[0047] Figure 13H This is a photograph showing a prototype sampler tool manufactured by adding a polypropylene (PP) handle to the head of a PDMS sampler, according to an example embodiment.

[0048] Figure 14A This is an amplification map of β-globin in Caski cells analyzed by qPCR according to an example embodiment. The Caski cells were collected by a cell sampler and detached by sonicated trypsin treatment. Cell DNA was extracted using Realbest reagent provided by the sponsor.

[0049] Figure 14B This is an amplification map of β-actin in Caski cells analyzed by qPCR according to an example embodiment. The Caski cells were collected by a cell sampler and detached by sonication and trypsinization. Cellular DNA was extracted using Realbest reagent.

[0050] Figure 14C This is an amplification map of GAPDH in Caski cells analyzed by qPCR according to an example embodiment. The Caski cells were collected by a cell sampler and detached by sonication and trypsinization. Cell DNA was extracted using Realbest reagent.

[0051] Figure 14D This is an amplification map of β-globin in human forearm skin cells analyzed by qPCR according to an example embodiment. The human forearm skin cells were collected by a cell sampler and detached by ultrasonic trypsin treatment. Cell DNA was extracted using Realbest reagent.

[0052] Figure 14E This is an amplification map of β-actin in human forearm skin cells analyzed by qPCR according to an example embodiment. The human forearm skin cells were collected by a cell sampler and detached by ultrasonic trypsin treatment. Cell DNA was extracted using Realbest reagent.

[0053] Figure 14F This is an amplification map of GAPDH in human forearm skin cells analyzed by qPCR according to an example embodiment. The human forearm skin cells were collected by a cell sampler and detached by ultrasonic trypsin treatment. Cell DNA was extracted using Realbest reagent.

[0054] Figure 15A This is an amplification diagram of different concentrations of β-actin standard template prepared and analyzed by qPCR according to an example embodiment.

[0055] Figure 15B It is based on Figure 15A Example embodiments show melting curves of different concentrations of β-actin standard templates prepared and analyzed by qPCR.

[0056] Figure 15C It is displayed according to Figure 15A An example embodiment shows a graph of Ct values ​​versus DNA copy number (logarithmic scale) for different concentrations of β-actin standard templates prepared and analyzed by qPCR.

[0057] Figure 15D This is a graph showing the melting curves of human forearm skin cell samples with different concentrations of β-actin standard template prepared and analyzed by qPCR according to an example embodiment.

[0058] Figure 16A These are photographs and micrographs showing Ca Ski cells used and seeded on a glass slide, culture plate, and synthetic skin (to simulate cell collection with HPV DNA) according to an example embodiment. A cell sampler was used to wipe the glass slide and culture plate to collect Ca Ski cells. Ca Ski cells could not be cultured on the synthetic skin, and all cells were suspended in culture medium. HPV-16 was analyzed by qPCR, with β-globin used as an internal control.

[0059] Figure 16BThis is an amplification diagram of HPV-16 and β-globin according to an example embodiment.

[0060] Figure 16C It is based on Figure 16B Melting curve of HPV-16 in an example embodiment.

[0061] Figure 16D It is based on Figure 16B The melting curve of β-globin in an example embodiment.

[0062] Figure 17A The images show a cell sampler with a scaffold (left) and a cell sampler (right) during an accelerated stability test of the cell sampler at 55°C ± 2°C for one month, according to an example embodiment.

[0063] Figure 17B It is displayed according to Figure 17A An example embodiment, a graph of cell sampler weights measured over one month.

[0064] Figure 17C These are photomicrographs showing front views of a scaffolded cell sampler (microcolumn width 140 µm) on day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0065] Figure 17D These are photomicrographs showing the front view of the cell sampler (microcolumn width 140 µm) on day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0066] Figure 17E These are photomicrographs showing side views of a microcolumn (140 µm high) of a scaffolded cell sampler at 100x magnification, on day 0 (left) and day 30 (right), according to an example embodiment.

[0067] Figure 17F These are photomicrographs showing side views of the microcolumn (140 µm high) of the cell sampler at day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0068] Figure 17G This is a micrograph showing the cell collection volume of the cell sampler evaluated according to an example embodiment. The cells on the sampler were trypsinized and stained with trypan blue, and quantified using a hemocytometer under a 100x optical microscope.

[0069] Figure 17HThis is a graph showing the cell collection capacity of the sampler between month 0 and month 1, according to an example embodiment.

[0070] Figure 18A This is a schematic diagram showing the rear dimensions of the example sampler tool according to an example embodiment.

[0071] Figure 18B It is displayed according to Figure 18A An example embodiment, a schematic diagram of the front dimensions of an example sampler tool, wherein the gray area represents the region containing the example sampler unit. Detailed Implementation definition

[0072] As used herein and in the claims, the terms “comprising” (or any related form such as “comprise / comprises”), “including” (or any related form such as “include / includes”), and “containing” (or any related form such as “contain / contains”) mean including the following elements but excluding others. It should be understood that for each embodiment using the terms “comprising” (or any related form such as “comprise / comprises”), “including” (or any related form such as “include / includes”), or “containing” (or any related form such as “contain / contains”), this disclosure / application also includes alternative embodiments in which the terms “comprising”, “including”, or “containing” are replaced with “consistently composed of” or “consistent with”. These alternative embodiments using “consistent with” or “consistently composed of” are to be understood as embodiments of a smaller scope than the “comprising”, “including”, or “containing” embodiments.

[0073] For clarity, “comprising,” “including,” “containing,” and “having,” as well as any related forms, are open-ended terms that allow for additional elements or features beyond the specified essential elements, while “consisting of” is a closed-ended term that is limited to the elements listed in the claims and excludes any elements, steps, or ingredients not specified in the claims.

[0074] As used herein, the singular forms “a / an” and “the” are intended to also include the plural forms unless the context clearly indicates otherwise. When a range is mentioned in the specification, the range is understood to include every discrete point within the range. For example, 1 to 7 means 1, 2, 3, 4, 5, 6, and 7.

[0075] As used herein, the term “about” is understood to mean within the normal tolerance range in the field and not exceeding ±10% of the specified value. For example only, about 50 refers to values ​​from 45 to 55, including all values ​​in between. As used herein, the phrase “about” also includes the specific value; for example, about 50 includes 50.

[0076] It should be understood that terms such as “top,” “bottom,” “middle,” “side,” “length,” “inner,” “outer,” “internal,” “external,” “vertical,” and “horizontal” that may be used herein describe reference points only and do not limit the present invention to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” and “third” identify only one of the multiple parts, components, and / or reference points disclosed herein, and likewise do not limit the present invention to any particular configuration or orientation.

[0077] Although specific embodiments are mentioned in the description, this disclosure should not be construed as limiting it to the embodiments set forth herein. Numbered Examples

[0078] Example 1. A cell sampling unit comprising: a plurality of micropillars for contacting a sample, such as skin or another surface, and acquiring cells from the surface of the sample, such as skin or another surface, by friction; and a base layer comprising a front side for supporting the plurality of micropillars and the acquired cells, wherein one end of each micropillar is fixed to or extends from the front side of the base layer, and the other end is a free end comprising a contact surface with frictional force; wherein each micropillar has a diameter of approximately 60-140 µm and a height of approximately 60-140 µm; wherein the plurality of micropillars are configured in an array arrangement; the micropillars are arranged at approximately 1000-1400 micropillars / cm 2 Density distribution between them.

[0079] Example 2. A cell sampling unit as described in Example 1, wherein the height of each microcolumn is 60 µm, 100 µm or 140 µm.

[0080] Example 3. A cell sampling unit as described in any of the preceding examples, wherein the sample, such as skin or other surface, is derived from human skin, such as skin from male reproductive organs for the detection of human papillomavirus (HPV) in men.

[0081] Example 4. A cell sampling unit as described in any of the preceding examples, wherein the overall length of the cell sampling unit is approximately 1-2 cm and the width is approximately 1-3 cm.

[0082] Example 5. A cell sampling unit as described in any of the preceding examples, wherein each micropillar is a cylinder, triangular prism, square prism, hexagonal prism, or pentagonal prism.

[0083] Example 6. A cell sampling unit as described in any of the preceding examples, wherein each micropillar and / or the substrate is made of a biocompatible polymer material, such as polydimethylsiloxane (PDMS), thermoplastic polyurethane (TPU), and / or polycaprolactone (PCL).

[0084] Example 7. A cell sampling unit for detecting human papillomavirus (HPV) in men, the cell sampling unit comprising a plurality of micropillars for contacting a sample and acquiring cells from the surface of the sample through friction; and a base layer comprising a front side for supporting the plurality of micropillars and the acquired cells, wherein one end of each micropillar is fixed to or extends from the front side of the base layer, and the other end is a free end comprising a contact surface with frictional force; wherein each micropillar is cylindrical with a diameter of approximately 60-140 µm and a height of approximately 100-140 µm; wherein the plurality of micropillars are configured in an array arrangement; the micropillars are arranged at approximately 1000 micropillars / cm 2 The density distribution; and each micropillar and / or the substrate is made of polydimethylsiloxane (PDMS).

[0085] Example 8. A cell sampling unit for detecting human papillomavirus (HPV) in men as described in Example 7, wherein the height of each microcolumn is 60 µm, 100 µm or 140 µm.

[0086] Example 9. A cell sampling tool comprising: a cell sampling unit as described in any one of Examples 1-8; and an actuator; wherein a base layer of the cell sampling unit is attached to or extends from the actuator, such that the cell sampling unit rubs against the sample to obtain cells under the action of an external force applied to the actuator.

[0087] Example 10. A cell sampling method, the method comprising the following steps: rubbing a sample with a cell sampling unit as described in any one of Examples 1-8 or a cell sampling tool as described in Example 9 to obtain cells therefrom.

[0088] Example 11. The method as described in Example 10, wherein at least 5,000 cells are obtained from the cell sampling unit.

[0089] Example 12. The method as described in Example 10 or 11, wherein more than 25,000 cells are obtained.

[0090] Example 13. The method of any one of Examples 10-12, wherein the friction sample includes the following steps: placing the cell sampling unit or the cell sampling tool on a sample surface, such as skin or other surface, the free end facing the sample surface, such as skin or other surface, and rubbing the sample back and forth under the action of an external force to collect the cells.

[0091] Example 14. The method as described in any one of Examples 10-13, wherein the sample is human skin, such as male genital skin for human papillomavirus (HPV) detection, or skin or surface of other animals or humans.

[0092] Example 15. The method of any one of Examples 10-14, the method further comprising the following steps: immersing the cell sampling unit or the cell sampling tool in the extraction solution to detach the cells from the sampling unit and to cause the cells to detach from the sampler under external force; and separating the detached cells from the extraction solution by centrifugal force, removing the supernatant to obtain a cell precipitate that can be used for DNA extraction.

[0093] Example 16. The method of any one of Examples 10-15, the method further comprising the step of rubbing the sample, such as human skin, 1-20 times to obtain 5,000-50,000 cells or more. Example Methods, Sampler Head Optimization, and Results 1. Fabrication of an example cell sampler head with biomimetic microstructures

[0094] In the examples below, the cell sampler unit may be referred to as a cell sampler head, sampling head, or sampler tip. A silicon wafer mold is chosen as the master mold for the example cell sampler head. Such a mold has a smooth surface, a high melting point, and dimensions accurate to tens to hundreds of micrometers, making it suitable for manufacturing cell sampler heads using different biocompatible polymers. As shown in Figure 1, the master negative mold consists of microstructures (at least 1400 micropillars / 1.4 cm). 2 The cell sampler head is made of silicon wafers. It is cast from PDMS. The microstructure dimensions of the PDMS-made cell sampler head are certified by the supplier using a white light interferometer. The height and width of the supplier-made cell sampler head are approximately 140 µm. 2. Evaluate the consistency of the microstructure of the example cell sampler head manufactured using a biocompatible polymer.

[0095] In this example, three example biocompatible polymers, including TPU, PDMS, and PCL, were tested as example cell sampler heads. To manufacture the PDMS cell sampler head, a silicone elastomer kit was used. Figure 2A Mix 10 parts of silicone base material with 1 part of curing agent until homogeneous. Remove air bubbles from the solution by placing it in a desiccator for approximately 30 minutes. Then, pour the clear solution onto the silicon wafer master mold. Place the mold containing PDMS in a desiccator to remove air bubbles from the micropillar pores for 18 hours, allowing the PDMS to completely fill the micropillar pores. Then, place the mold in a 65°C oven for 2 hours and cut it into 1 x 1.4 cm pieces. 2 The size of the micropillars was determined. The height and width were measured using an optical microscope. Both the height and width were approximately 140 µm. Figure 2B and 2C ).

[0096] For the PCL cell sampler head, place the PCL solid on the master mold and incubate in an 80°C vacuum oven for 18 h. Figure 3A Allow the sampler head to cool to room temperature. Carefully remove the PCL sampler head with a scalpel and cut it into pieces approximately 1 x 1.4 cm. 2 The size of the micropillars was determined. The height and width of the micropillars were measured using an optical microscope. The width of the micropillars was approximately 140 µm. Figure 3B ), with a height of approximately 108 µm ( Figure 3C ).

[0097] To manufacture the TPU cell sampler head, TPU particles were placed on the master mold and incubated in a 200°C vacuum oven for 18 hours. Figure 4A Allow the sampler head to cool to room temperature. Carefully remove the TPU sampler head with kitchen shears and cut it into 1 x 1.4 cm pieces. 2 The size of the micropillars was determined. The height and width of the micropillars were measured using an optical microscope. Both the height and width of the micropillars reached 140 µm. Figure 4B and 4C ).

[0098] Micropillars made of PDMS can achieve a height and width of 140 µm. PDMS cell sampler tips are soft and flexible, making them comfortable to wipe on the forearm. It is the best among the three testing materials. Even after incubation in a vacuum oven, the height of PCL micropillars did not reach 140 µm. This is likely due to its higher viscosity in the liquid state compared to PDMS and TPU. Furthermore, PCL sampler tips are brittle and difficult to wipe on the forearm. It is not considered a suitable material for sampler tips. Although sampler tips made of TPU meet the target size requirements, TPU is difficult to remove from the mold. TPU is somewhat difficult to wipe on the forearm. The production of TPU sampler tips also requires high temperatures. Compared to PDMS, TPU is the second preferred material for sampler tips. The results indicate that PDMS is a relatively better choice for cell sampler tip materials due to its texture and ease of production. 3. The microstructure of the cell sampler head was characterized by scanning electron microscopy (SEM).

[0099] like Figure 5A As shown, the example PDMS cell sampler head was cut and fixed onto a metal stage. After gold coating, the microstructure was characterized by SEM, and ten regions of interest (ROIs) for each sample were examined. The height and width of the micropillars were 140 µm. Figure 5B and Figure 5C The density is also greater than 1000 columns / cm³, and detailed information for each measurement is listed in Table 1. 2 . Table 1. Microstructure characterization using scanning electron microscopy, with ten regions of interest (ROIs) examined for each sample. The height and width of all ROIs were approximately 140 µm. 4. Optimize the shape, size, density, and pattern of the micropillars on the cell sampler head and measure the coefficient of friction on a skin model.

[0100] a) Optimize the shape of the micropillars

[0101] In addition to cylinders, square and rectangular cylinders were also prepared. Square and triangular master molds were also made. Figure 6A and 6C The micropillars are 140 µm in height and width, and are designed to have at least 1400 micropillars per 1.4 cm column. 2 ( Figure 6B and 6D ).

[0102] The frictional force generated by the microstructure on the synthetic skin was measured using a coefficient of friction (COF) tester (Labthink MXD-02) and expressed as COF. 8N synthetic skin was used as a human forearm and was fixed to the sliding surface of the tester. The PDMS sampler head was secured to the center of the sled with double-sided tape, and the weight of the sled with the sampler head was recorded. The test speed was set to 150 mm / min. Figure 7 As shown, the frictional force of the cell sampler head on synthetic skin was measured according to ASTM D1894. The COF of different shaped micropillars was tested and compared. Typically, the frictional force of the sampler head against human forearm skin is approximately 0.2–0.5 N. The order of frictional force is as follows: triangular micropillars > circular micropillars > square micropillars > flat surfaces (no micropillars).

[0103] The coefficient of friction (COF) of cell sampler heads of different shapes was calculated using the software "MXD-02 coefficient of friction tester". Static friction time was defined as 0-10 seconds, while dynamic COF was calculated based on 10-60 seconds. Figure 7 As shown in C. The static and dynamic COF of the sampler head are shown in Table 2. The percentage increase in COF is calculated as follows: [(COF 微柱 - COF 平坦表面 ) / COF 平坦表面 *100%]. Compared to flat surfaces without microstructures, circular micropillars showed a 57.9% and 32.8% increase in static and dynamic COF, respectively. Triangular micropillars showed an increase of over 50% in both static and dynamic COF, while square micropillars showed an increase of less than 20%. Based on the COF results, circular and triangular micropillars are relatively better choices for sampler heads.

[0104] For triangular micropillars, different directions of movement can affect COF and cell collection capacity. COF was tested on synthetic skin using a COF meter for lateral, forward, and backward movement of the triangular micropillars. Figure 8A The frictional force of the cell sampler head on synthetic skin was measured according to ASTM D1894. The COF (coefficient of friction) varied from 0.199 to 0.238 in different directions of movement, with the highest static COF observed when moving backward. Figure 8B The dynamic COF value ranges from 0.198 to 0.289, while moving forward displays the highest value. Figure 8C ). Table 2. COF of cell sampler heads of different shapes measured using a coefficient of friction (COF) tester, referring to ASTM D1894. The percentage increase in COF is defined as: [(COF...] 微柱 – COF 平坦表面 ) / COF 平坦表面 *100%]. b) Optimize the size of the micropillars

[0105] To optimize the size of the micropillars, micropillars with heights of 60 µm, 100 µm, and 140 µm were prepared. Figure 9A The 140µm high micropillars showed the highest static COF value. Figure 9B Micropillars with heights of 100 µm and 140 µm showed the highest dynamic COF (). Figure 9C In addition, micropillars with widths of 60 µm, 100 µm, and 140 µm were prepared. Figure 10A The 140 µm wide micropillars showed the highest static COF value. Figure 10B ), while the 100 µm wide micropillars showed the highest dynamic COF ( Figure 10C ). c) Optimize the density of micropillars

[0106] Micropillars with densities of 1000, 1200, and 1400 micropillars / cm were prepared. 2 Cell sampler head ( Figure 11A 1000 microcolumns / cm 2 The highest static COF and dynamic COF were observed, and the microcolumn density was negatively correlated with the dynamic COF. Figure 11B and 11C ). d) Optimize the pattern of the micropillars

[0107] For patterning, a cell sampler head with a micropillar array and a cross pattern was fabricated. Figure 12A Array patterns show higher static COF and dynamic COF than cross patterns. Figure 12B and 12C ).

[0108] In summary, cell samplers 1, 3 and 4 all exhibited high dynamic COF values ​​on synthetic skin (Table 3). Table 3. Dynamic friction coefficients of samplers of different sizes. n = 3. 5. Evaluate the ability of the cell sampler prototype to collect skin cells from the human forearm.

[0109] The sampler tool prototype was manufactured by adding a polypropylene (PP) handle to the sampler head. Figure 13H In short, place the master silicon mold inside the soft silicon mold. Insert the PP handle into the soft mold and pour PDMS into the mold. Degas the mold in a desiccant and incubate at 65°C as described in Section 2. Then cut the sampler head to 1 x 1.4 cm. 2 Size.

[0110] Wipe the forearm 1, 3, 5, 10, 15, and 20 times. Place the sampler tip in a 50 ml tube, add 2 ml of trypsin to completely submerge the sampler tip, and then incubate at 37°C for 3 min. Then remove the sampler tip from the solution and centrifuge the solution at 3000 rcf for 5 min. Carefully remove the supernatant using a pipette. Add 50 µl of Tris-buffered saline (TBS) to the bottom of the tube, followed by 50 µl of trypan blue. Transfer 10 µl of the solution to a hemocytometer and count the cells under an optical microscope. Measure the average number of cells (Cells) on the four large squares at the corners. Figure 13A -F). Our data indicate that approximately 5000–15000 cells were collected after varying numbers of wipings. The number of cells collected was directly proportional to the number of wipings (-F). Figure 13G ).

[0111] To further improve cell detachment, sonication with trypsin was performed. After collecting skin cells, the sampler tip was placed in a 15 ml tube, and 2.5 ml of trypsin was added to completely submerge the sampler tip. The tube was then incubated and sonicated at 37°C in a temperature-controlled sonicator for 10 min. The sampler tip was then removed from the solution, and approximately 1.4 ml of sample solution was transferred to a 1.5 ml tube. The sample solution was centrifuged at 20,000 rcf for 5 min. The supernatant was carefully removed using a pipette, and the remaining solution (approximately 1.1 ml) in the 15 ml tube was transferred to a 1.5 ml tube. The sample solution was centrifuged again at 20,000 rcf for 5 min. After carefully removing the supernatant, 50 µl of TBS was added to the precipitate. Then, 50 µl of trypan blue was added to the 1.5 ml tube. 10 µl of the solution was transferred to a hemocytometer, and the cell count was performed under an optical microscope. Approximately 25,000–48,000 cells were collected through various wiping cycles. Therefore, ultrasonic trypsin treatment is used as a cell detachment method.

[0112] The cell collection capabilities of sampler heads 1, 3, and 4 were examined. The cell collection volumes of sampler heads 1, 3, and 4 were comparable. Sampler head 1 is currently in use, therefore it was selected for further development. 6. Assess the amount of genomic DNA extracted from skin cells collected using a prototype cell sampler.

[0113] Genomic DNA was extracted from cells collected using a cell sampler using a commercially available kit (Qiagen). The sampler was held and rubbed against the forearm 20 times. The sampler tip was placed in a 50 ml tube, and 2 ml of trypsin was added to the sampler tip. The tube was incubated at 37°C for 3 min. The sampler tip was then removed from the solution, and the solution was centrifuged at 3000 rcf for 5 min. The supernatant was carefully removed using a pipette. 200 µl of PBS, 20 µl of proteinase K, and 200 µl of buffer AL were added to the tube. The solution was mixed thoroughly and incubated at 56°C for 10 min with oscillation at 300 rpm. The solution was cooled to room temperature, and 200 µl of ethanol was added to the solution. The solution was transferred to a DNeasy Mini spin column and placed in a 2 ml collection tube. The sample was centrifuged at 6000 rcf for 1 min. The flow-through was discarded into the collection tube. The spin column was then placed in a new 2 ml collection tube. Add 500 µl of buffer AW1 to the spin column and centrifuge at 6000 rcf for 1 min. Discard the flow-through in a collection tube. Place the spin column in a new 2 ml collection tube and add 500 µl of buffer AW2 to the spin column. Centrifuge the column at 20000 rcf for 3 min. Discard the flow-through and transfer the spin column to a new 1.5 ml tube. Elute the DNA by adding 50 µl of buffer AE to the center of the spin column membrane. Incubate the column at room temperature (15°C–25°C) for 1 min. Centrifuge the spin column at 6000 rcf for 1 min.

[0114] DNA quantification was performed using NanoDrop (Implen NP80) via UV absorbance at 260 nm. Briefly, 1 µl of buffer AE was pipetted into NanoDrop and calibrated as a blank. Then, 1 µl of filtrate was pipetted into NanoDrop for DNA measurement at a concentration of ng / mL. As shown in Table 4, when cells were detached from the cell sampler head using trypsin, the amount of DNA extracted per cell sampler head was greater than 4 ng / µl, while when cell detachment was performed using trypsin combined with sonication, the amount of DNA extracted per cell sampler head increased to 5.67 ng / µl. Table 4. The sampler head was used to swab human forearm skin and detach skin cells using trypsin or trypsin combined with ultrasound. Genomic DNA was extracted from the cells collected by the cell sampler head using a commercially available kit (Qiagen). The DNA concentration of the extraction filtrate was quantified using UV absorbance at 260 nm. 7. The copy number of housekeeping genes in the extracted DNA was assessed using real-time quantitative PCR.

[0115] After genomic DNA was extracted using Realbest reagent (provided by the sponsor), copies of the housekeeping gene present in the sample were obtained by real-time quantitative PCR (qPCR). Briefly, the collected cells were detached using trypsin and sonication. The collected cell pellet was then resuspended in 50 µl of Realbest reagent and incubated at 98°C for 30 min with shaking at 300 rpm. The sample solution was centrifuged at 8000 rpm for 5 min. 40 µl of the supernatant was transferred to a new tube. The extracted DNA was mixed with the qPCR master mixture and the primers for the housekeeping gene. The sample was then processed in a qPCR thermal cycler. A plasmid containing the housekeeping gene was used to construct a standard curve. The copy number of the housekeeping gene in the extracted DNA sample was calculated based on the generated standard curve.

[0116] Initially, β-globin was proposed as a housekeeping gene. However, β-globin was detected in CaSki cells but not in forearm skin cells. Other common housekeeping genes, β-actin and GAPDH, were also tested. Both β-actin and GAPDH were detected in both CaSki cells and forearm skin cells. Figure 14A-14F β-actin has a higher CT value than GAPDH, and was therefore selected as a housekeeping gene for internal control in human skin cells.

[0117] Calculate the copy number of housekeeping genes in cells collected from human forearm skin. A β-actin standard template was used to construct a standard curve. Figure 15A and 15C The melting curve confirmed the specificity of the β-actin standard. Figure 15B ).

[0118] After swabbing the human forearm 20 times with a sampler, the gene copy number of the housekeeping gene in the extracted DNA samples was calculated (Table 5). The CT values ​​of all samples were approximately 30-31, and the average gene copy number was approximately 2200 / µl. Table 5. Human β-actin gene copy number collected from human forearm and analyzed by qPCR. 8. Using cell lines with HPV DNA to simulate the collection of cells with HPV DNA

[0119] Since it was impossible to obtain HPV-infected cells from healthy volunteers, an alternative method was used. CaSki cells, a previously HPV-infected human cancer cell line, were used. The cells were packed at 5 x 10⁻⁶ cells / day. 4 cells / mm 2 The cells were seeded at a density similar to that of epidermal cells in the human forearm ( ). Figure 16A Ca Ski cells could not be cultured on synthetic skin, and all cells were suspended in culture medium. The sampler was used to wipe the slides and culture plates 20 times. Cells were detached by sonication and trypsin treatment. DNA was extracted using a Qiager DNA extraction kit. HPV-16 was analyzed by qPCR, with β-globin as an internal control. Individual Ca Ski cells served as a positive control. On the amplification map, the CT values ​​for both HPV-16 and β-globin were below 40, indicating the presence of HPV DNA. Figure 16B The melting curve also confirmed the specificity of targeting HPV DNA. Figure 16C ). Table 6. HPV-16 positive cell line Ca Ski cells were seeded on slides and culture plates to simulate cell collection with HPV DNA. HPV-16 was analyzed by qPCR, with β-globin used as an internal control. 9. Accelerated stability testing of the cell sampler was conducted at 55°C ± 2°C for 1 month, and its cell collection capacity was evaluated.

[0120] The stability of the cell sampler prototype will be evaluated under accelerated aging conditions. Under accelerated aging conditions at 55°C (above 25°C), the aging factor is 23 = 8. The cell sampler prototype (with and without scaffolding) will be tested. Figure 17A Cells were stored in a temperature-controlled chamber at 55°C ± 2°C and ambient humidity for one month (31 days), and cell collection capacity was assessed as described in Section 5 (Ultrasonic Trypsin Treatment). The weight of the cell samplers was measured continuously for one month, and the weight of all samplers remained stable throughout the month. Figure 17B The dimensions of the micropillars on the sampler were monitored. No changes in the width and height of the samplers were observed after one month in all samplers. Figure 17C , 17D 17E and 17F). Width and height of all samplers ( Figure 17C and Figure 17D All samples were kept at approximately 140 µm. The cell collection volume was assessed. Cells on the sampler were trypsinized and stained with trypan blue. Cells were quantified using a hemocytometer under an optical microscope. Figure 17H No significant difference in the cell collection capacity of the sampler was observed between month 0 and month 1. 10. Example Sampling Tool

[0121] Figure 13C An example sampling tool including a handle and a cell sampling unit is shown. The sampler tool prototype is manufactured by attaching a polypropylene (PP) handle to the PDMS sampler head.

[0122] Figure 18A and 18B Another example sampler tool containing any of the example cell sampler units as described herein is shown. In this example, the example cell sampler unit and the manipulator are formed as a single unit. The gray area represents the region containing the example sampler unit, located at the front end of the manipulator. The manipulator has two curved sections to facilitate a user gripping the manipulator to apply external force to the manipulator, thereby applying it to the sampler unit. in conclusion

[0123] In summary, examples of a biocompatible polymer-based cell sampler unit and a cell sampler have been developed that can be used to efficiently and painlessly collect cells for male HPV testing. In some embodiments, the provided cell sampler is small and flexible. In some embodiments, the cell sampler head is approximately 1 x 1.4 cm in size. 2 In some embodiments, the sampling surface contains > 1000 micropillars / cm². 2 The micropillars are arranged in a regular array, with each micropillar having a height and width of <150 µm. Due to their novel design, the cell sampler can collect a sufficient number of cells for downstream HPV analysis. After cell collection, the cell sampler is enzymatically digested to detach the cells. The detached cells will be used for DNA extraction and further qPCR analysis.

[0124] The example cell sampler provided is an easy-to-use, convenient, and reliable sampling device capable of both self-sampling and remote sampling. Given specified sizes and microstructures, the sampler can be used not only for HPV testing but also for other diagnostic tests, such as those for dermatology.

Claims

1. A cell sampling unit comprising: Multiple micropillars, the multiple micropillars being used to contact a sample and acquire cells from the surface of the sample through friction; and The base layer includes a front side for supporting the plurality of micropillars and the acquired cells. Each micropillar has one end fixed to or extending from the front side of the base layer, and the other end is a free end containing a contact surface with friction. Each micropillar has a diameter of approximately 60-140 µm and a height of approximately 60-140 µm. The plurality of micropillars are configured in an array arrangement; the micropillars are arranged at a density of approximately 1000-1400 micropillars / cm. 2 Density distribution between them.

2. The cell sampling unit of claim 1, wherein the sample is derived from human skin, such as skin from male reproductive organs for the detection of human papillomavirus (HPV) in men.

3. The cell sampling unit as claimed in claim 1, wherein the overall length of the cell sampling unit is approximately 1-2 cm and the width is approximately 1-3 cm.

4. The cell sampling unit as described in claim 1, wherein each micropillar is a cylinder, triangular prism, square prism, hexagonal prism, or pentagonal prism.

5. The cell sampling unit of claim 1, wherein each micropillar and / or the substrate is made of a biocompatible polymer material, such as polydimethylsiloxane (PDMS), thermoplastic polyurethane (TPU), and / or polycaprolactone (PCL).

6. A cell sampling unit for detecting human papillomavirus (HPV) in men, the cell sampling unit comprising... Multiple micropillars are used to contact the sample and acquire cells on the surface of the sample through friction; as well as The base layer includes a front side for supporting the plurality of micropillars and the acquired cells. Each micropillar has one end fixed to or extending from the front side of the base layer, and the other end is a free end containing a contact surface with friction. Each micropillar is cylindrical, with a diameter of approximately 60-140 µm and a height of approximately 100-140 µm. The plurality of micropillars are configured in an array arrangement; the micropillars are arranged at approximately 1000 micropillars / cm. 2 The density distribution; and Each micropillar and / or the substrate is made of polydimethylsiloxane (PDMS).

7. A cell sampling tool, comprising: The cell sampling unit as described in any one of claims 1-6; and Operator; The base layer of the cell sampling unit is attached to or extends from the manipulator, such that the cell sampling unit rubs against the sample to obtain cells under the action of an external force applied to the manipulator.

8. A cell sampling method, the method comprising the following steps: Cells are obtained by rubbing a sample with the cell sampling unit as described in any one of claims 1-6 or the cell sampling tool as described in claim 7.

9. The method of claim 8, wherein the friction sample comprises the following steps: The cell sampling unit or the cell sampling tool is placed on the sample surface, with the free end facing the sample surface, and the sample is rubbed back and forth under the action of an external force to collect the cells.

10. The method of claim 8 or claim 9, wherein the sample is human skin, such as male genital skin used for human papillomavirus (HPV) detection in men.

11. The method of any one of claims 8-10, further comprising the following steps: The cell sampling unit or the cell sampling tool is immersed in the extraction solution to detach the cells from the sampling unit and to induce the cells to detach from the sampler under external force; and The detached cells are separated from the extract by centrifugation, and the supernatant is removed to obtain a precipitate that can be used for DNA extraction.

12. The method of any one of claims 8-10, further comprising the following steps: Rub the sample, such as human skin, 1-20 times to obtain 5,000-50,000 cells or more.

Images