Cell sampling unit and cell sampling tool

Abstract

Disclosed are a cell sampling unit and a cell sampling tool, the cell sampling unit comprising: a plurality of microcolumns for contacting a sample and obtaining cells from a surface of the sample by friction; and a base layer comprising a front side for supporting the plurality of microcolumns and acquired cells, in which one end of each microcolumn 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 having a frictional force; wherein each microcolumn has a diameter of about 60-140 [mu] m and a height of about 60-140 [mu] m; wherein the plurality of microcolumns are configured in an array arrangement; the microcolumns are distributed at a density between about 1000 to 1400 microcolumns / cm2. Other example embodiments are described herein. In certain embodiments, the cell sampling unit is an easy to use, convenient and reliable skin cell sampling for male HPV screening.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell sampling unit and a cell sampling tool. BACKGROUND

[0002] Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the world. The growing evidence of the link between HPV and anogenital cancers has led to the development of HPV testing for women and men. Cell sampling is a critical step in HPV testing, as the viral load of HPV in infected tissue is often very low. To date, different types of samplers have been developed for the collection of HPV samples from the cervix of women. However, no similar sampler has 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 disadvantages, such as causing discomfort and anxiety, and more importantly, a low collection success rate. Therefore, there is an urgent need to develop an easy-to-use genital cell sampler for male HPV DNA. SUMMARY

[0003] In certain embodiments, a cell sampling unit, a cell sampling tool and a cell sampling method are provided. In certain embodiments, a cell sampler with a novel microstructure is provided for skin cell sampling, such as male genital HPV sampling. In certain embodiments, a "Bio-Sandpaper" (BioS) technology is provided, which is similar to naturally occurring microstructures, such as a bifunctional microstructure that mimics the micron to nanometer scale structured bristles in the body of a bee and the micro-patterned hair on the legs of a bee that captures pollen. Such biomimetic microstructures can easily pick up and capture skin cells. To achieve this, in some embodiments, the microstructure contains a number of micro-pillars that will be optimized in shape and size to increase the surface-to-volume ratio of the biocompatible polymer surface, resulting in high friction to pick up skin cells. In some embodiments, the micro-pillar spacing and pattern are optimized for efficient capture and release of skin cells. In some embodiments, biocompatible polymers are tested to check the consistency of microstructure fabrication in a casting system, such that the provided sampler is feasible for future scale-up manufacturing. In some embodiments, a cell sampler prototype is made from a sampler head with microstructure and a handle. In some embodiments, the provided cell sampler is an easy-to-use, convenient and reliable skin cell sampler for male HPV screening. BRIEF DESCRIPTION OF DRAWINGS

[0004] Figure 1A is a photograph showing a mold design of a cell sampling unit with 42 x 35 micro-pillars, according to an example embodiment.

[0005] Figure 1BFigure 1 is a schematic showing the micro-pillar dimensions of 140 pm in height and 140 pm in width on the cell sampling unit according to example embodiments.

[0006] Figure 1C Figure 2 is a photograph showing the master silicon wafer mold for manufacturing the cell sampling unit according to example embodiments.

[0007] Figure 1D Figure 3 is a photograph showing the PDMS cell sampler head unit of 1 x 1.4 cm 2 size according to example embodiments.

[0008] Figure 2A Figure 4 is a schematic showing the steps of manufacturing the cell sampler head unit from PDMS according to example embodiments.

[0009] Figure 2B Figure 5 is a micrograph showing the front view of the PDMS cell sampler head unit with micro-pillars of 140 pm in width under an optical microscope at 100x magnification according to example embodiments.

[0010] Figure 2C Figure 6 is a micrograph showing the side view of the PDMS cell sampler head unit with micro-pillars of 140 pm in height under an optical microscope at 100x magnification according to example embodiments.

[0011] Figure 3A Figure 7 is a schematic showing the steps of manufacturing the cell sampler head from PCL according to example embodiments.

[0012] Figure 3B Figure 8 is a micrograph showing the front view of the PCL cell sampler head unit with micro-pillars of 140 pm in width under a stereomicroscope at 80x magnification according to example embodiments.

[0013] Figure 3C Figure 9 is a micrograph showing the side view of the PCL cell sampler head unit with micro-pillars of 108 pm in height under an optical microscope at 100x magnification according to example embodiments.

[0014] Figure 4A Figure 10 is a schematic showing the steps of manufacturing the cell sampler head from TPU according to example embodiments.

[0015] Figure 4B Figure 11 is a micrograph showing the front view of the TPU cell sampler head under an optical microscope at 100x magnification according to example embodiments. The width of the micro-pillars is 140 pm.

[0016] Figure 4Cis a photomicrograph showing a side view of a PDMS cell sampler head under an optical microscope at 100x magnification, according to an example embodiment. The height of the micro-pillars is 140 pm.

[0017] Figure 5A is a schematic diagram showing the steps of analyzing a PDMS cell sampler head by scanning electron microscopy (SEM), according to an example embodiment.

[0018] Figure 5B is a SEM photomicrograph showing a front view of a PDMS cell sampler head under a SEM at 100x magnification, according to an example embodiment. The density of the sampler is >1000 micro-pillars / cm 2 .

[0019] Figure 5C is a photomicrograph showing a side view of a PDMS cell sampler under a SEM at 100x magnification, according to an example embodiment. The height and width of the micro-pillars are both 140 pm.

[0020] Figure 6A are a schematic diagram (left) and a photograph (middle) of a silicon wafer mold of a cell sampling unit with 42 x 35 square micro-pillars, and a photograph of a PDMS cell sampler head with square micro-pillars, according to an example embodiment, respectively.

[0021] Figure 6B are photomicrographs showing a front view (left) and a side view (right) of a PDMS sampler with square micro-pillars under an optical microscope at 100x magnification, according to an example embodiment. The height and width of the square micro-pillars are both 140 pm.

[0022] Figure 6C is a schematic diagram (left) and a photograph (middle) of a silicon wafer mold of a cell sampling unit with triangular micro-pillars, and a photograph of a PDMS cell sampling unit with triangular micro-pillars, according to an example embodiment.

[0023] Figure 6D are photomicrographs showing a front view (left) and a side view (right) of a PDMS sampler with triangular micro-pillars under an optical microscope at 100x magnification, according to an example embodiment. The height and width of the triangular micro-pillars are both 140 pm.

[0024] Figure 7 is a graph showing the friction force of a cell sampling unit on synthetic skin measured with reference to ASTM D1894, according to an example embodiment. In this graph, the COF of different shaped micro-pillars are tested and compared.

[0025] Figure 8Ais a graph showing the friction generated by the microstructure on synthetic skin measured with a coefficient of friction (COF) tester, according to example embodiments. Different directions of movement of the triangular micro-pillars are shown. The friction force of the cell sampler head on synthetic skin was measured with reference to ASTM D1894. COF of different directions of movement of the triangular micro-pillars were tested and compared.

[0026] Figure 8B is a graph showing the static COF of different heights of micro-pillars, according to example embodiments. Figure 8A Figure 8C is a graph showing the dynamic COF of different heights of micro-pillars, according to example embodiments. Figure 8A

[0027] Figure 9A is a micrograph showing micro-pillars of various heights (60 pm, 100 pm, and 140 pm) prepared for optimizing the micro-pillar height, according to example embodiments. The friction force of the cell sampler on synthetic skin was measured with reference to ASTM D1894.

[0028] Figure 9B is a graph showing the static COF of different heights of micro-pillars, according to example embodiments. The friction force of the cell sampler on synthetic skin was measured with reference to ASTM D1894. Figure 9A

[0029] is a graph showing the dynamic COF of different heights of micro-pillars, according to example embodiments. Figure 9C Figure 9A is a micrograph showing the cell sampler with micro-pillars of various widths (60 pm, 100 pm, and 140 pm) prepared for optimizing the micro-pillar width, under an optical microscope at 100x magnification, according to example embodiments. The friction force of the cell sampler on synthetic skin was measured with reference to ASTM D1894. COF of different width samplers were tested and compared.

[0030] Figure 10A is a graph showing the static COF of different widths of micro-pillars, according to example embodiments.

[0031] Figure 10B Figure 10A is a graph showing the dynamic COF of different widths of micro-pillars, according to example embodiments.

[0032] Figure 10C is a graph showing the dynamic COF of different widths of micro-pillars, according to example embodiments. Figure 10A

[0033] 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.

[0034] Figure 11B It is displayed according to Figure 11A Example embodiments, graphs of static COF of micropillars with different densities.

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

[0036] 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.

[0037] Figure 12B It is displayed according to Figure 12A Example embodiments, static COF diagrams of micropillars with different patterns.

[0038] Figure 12C It is displayed according to Figure 12A Example embodiments, dynamic COF graphs of micropillars with different patterns.

[0039] Figure 13A These are photomicrographs taken under a 100x optical microscope, showing the cell collection volume assessed by a PDMS sampler after one swipe. The images show large squares of skin cells / corners: (6+10+2+2) / 4 = 5 collected cells = 5 x 0.1 x 10⁻⁶ cells. 4 =5000 skin cells (according to the example embodiment). Cells on the sampler are triedpsinized and stained with trypan blue. Cells are quantified using a hemocytometer.

[0040] 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 10⁻⁶ cells. 4 = 8000 skin cells (according to the example embodiment). Other experimental settings and... Figure 13AThe same.

[0041] Figure 13C is a micrograph under optical microscope at 100x magnification showing cell collection assessment of PDMS sampler swabbed 5 times, the picture shows large blocks of skin cells / corners: (15+8+10+7) / 4 = 10 collected cells = 10 x 0.1 x 10 4 = 10000 skin cells (according to example embodiments). Other experimental settings were the same. Figure 13A

[0042] Figure 13D is a micrograph under optical microscope at 100x magnification showing cell collection assessment of PDMS sampler swabbed 10 times, the picture shows large blocks 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 example embodiments). Other experimental settings were the same. Figure 13A

[0043] Figure 13E is a micrograph under optical microscope at 100x magnification showing cell collection assessment of PDMS sampler swabbed 15 times, the picture shows large blocks of skin cells / corners: (15+11+12+14) / 4 = 13 collected cells = 13 x 0.1 x 10 4 = 13000 skin cells (according to example embodiments). Other experimental settings were the same. Figure 13A

[0044] Figure 13F is a micrograph under optical microscope at 100x magnification showing cell collection assessment of PDMS sampler swabbed 20 times, the picture shows large blocks of skin cells / corners: (16+14+18+15) / 4 = 15.75 collected cells = 15.75 x 0.1 x 10 4 = 15750 skin cells (according to example embodiments). Other experimental settings were the same. Figure 13A

[0045] Figure 13G is a graph showing the amount of collected cells versus swabbing times for example embodiments of Figures 13A-F , indicating that the amount of cell collection is proportional to the number of swabbing times. According to example embodiments, the ultrasonic trypsin treatment further improves the cell detachment efficiency compared to trypsin alone.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Figure 15C is a graph showing Ct values of different concentrations of beta actin standard template prepared and analyzed by qPCR versus DNA copy number (log scale) according to an example embodiment. Figure 15A

[0056] Figure 15D is a graph showing melt curves of human forearm skin cell samples with different concentrations of beta actin standard template prepared and analyzed by qPCR according to an example embodiment.

[0057] Figure 16A is a photograph and micrograph showing Ca Ski cells according to an example embodiment, plated on glass slides, culture plates, and synthetic skin (to simulate cell collection with HPV DNA). Cell scrapers were used to wipe the glass slides and culture plates to collect Ca Ski cells. Ca ski cells were unable to be cultured on synthetic skin and all cells were suspended in culture medium. HPV-16 was analyzed by qPCR with beta globin as an internal control.

[0058] Figure 16B is a graph showing amplification of HPV-16 and beta globin according to an example embodiment.

[0059] Figure 16C is a melt curve of HPV-16 according to an example embodiment. Figure 16B

[0060] Figure 16D is a melt curve of beta globin according to an example embodiment. Figure 16B

[0061] Figure 17A is a photograph showing a cell scraper with a scaffold (left) and a cell scraper (right) during a 1 -month cell scraper accelerated stability test at 55 °C ± 2 °C according to an example embodiment.

[0062] Figure 17B is a graph showing cell scraper weight measured over 1 month according to an example embodiment. Figure 17A

[0063] Figure 17C is a micrograph showing a front view of a cell scraper with a scaffold (micropost width of 140 pm) at 0 days (left) and 30 days (right) under an optical microscope at 100x magnification according to an example embodiment.

[0064] Figure 17D ​​​​Figure 1 is a micrograph showing a front view of a cell sampler (micropost width of 140 pm) at day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0065] Figure 17E Figure 2 is a micrograph showing a side view of a micropost of a cell sampler with scaffolds (micropost height of 140 pm) at day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0066] Figure 17F Figure 3 is a micrograph showing a side view of a micropost of a cell sampler (micropost height of 140 pm) at day 0 (left) and day 30 (right) under an optical microscope at 100x magnification, according to an example embodiment.

[0067] Figure 17G Figure 4 is a micrograph showing the cell collection amount of an evaluated cell sampler, according to an example embodiment. Cells on the sampler were trypsinized and stained with trypan blue and quantified using a hemocytometer under an optical microscope at 100x magnification.

[0068] Figure 17H Figure 5 is a graph showing that the cell collection capacity of the sampler was comparable between month 0 and month 1, according to an example embodiment.

[0069] Figure 18A Figure 6 is a schematic diagram showing the back side dimensions of an example sampler tool, according to an example embodiment.

[0070] Figure 18B Figure 7 is a schematic diagram showing the front side dimensions of an example sampler tool, according to an example embodiment of Figure 18A where the grey area represents the area containing the example sampler unit. DETAILED DESCRIPTION

[0071] Definitions

[0072] As used herein and in the claims, the terms “comprising” (or any

[0073] For the purposes of clarity, “comprise,” “include,” “contain” and “have” and any variations thereof are open-ended terms that refer to elements or features that are not exclusive, but that also include, without limitation, additional elements or features not expressly listed or otherwise described. “Consisting essentially of’ is a closed term that excludes additional elements or features that would materially alter the composition or method.

[0074] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. In referring to ranges of values, the range is understood to include each individual 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 be within the normal tolerances of the art and no more than ±10% of the stated value. By way of example only, about 50 means from 45 to 55, including all values in between. As used herein, the phrase “about” a particular value also includes the particular value, e.g., about 50 includes 50.

[0076] It should be understood that terms such as "top," "bottom," "intermediate," "side," "length," "inner," "outer," "internal," "external," "outboard," "vertical," "horizontal," and the like, as can be used herein, merely describe points of reference and do not limit the present application to any particular orientation or configuration. Further, terms such as "first," "second," "third," and the like, merely identify one of multiple parts, components, and / or points of reference, and likewise do not limit the present application to any particular configuration or orientation.

[0077] While the description refers to particular embodiments, the disclosure should not be interpreted as limited to the embodiments set forth herein.

[0078] Numbered Embodiments

[0079] Embodiment 1. A cell sampling unit comprising: a plurality of microcolumns for contacting a sample, such as skin or other surface, and acquiring cells from the surface of the sample, such as skin or other surface, by abrasion; and a base layer comprising a front side for supporting the plurality of microcolumns and the acquired cells, wherein one end of each microcolumn 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 abrasion force; wherein the diameter of each microcolumn is about 60-140 pm, and the height is about 60-140 pm; wherein the plurality of microcolumns are configured in an array arrangement; the microcolumns are distributed with a density of about 1000-1400 microcolumns / cm 2 .

[0080] Embodiment 2. The cell sampling unit according to embodiment 1, wherein the height of each microcolumn is 60 pm, 100 pm, or 140 pm.

[0081] Embodiment 3. The cell sampling unit according to any of the preceding embodiments, wherein the sample, such as skin or other surface, is from human skin, such as skin from male genital organs for male human papillomavirus (HPV) testing.

[0082] Embodiment 4. The cell sampling unit according to any of the preceding embodiments, wherein the overall length of the cell sampling unit is about 1-2 cm, and the width is about 1-3 cm.

[0083] Embodiment 5. The cell sampling unit according to any of the preceding embodiments, wherein the microcolumns are cylinders, triangular prisms, square prisms, hexagonal prisms, or pentagonal prisms.

[0084] Example 6. The cell sampling unit according to any one of the preceding examples, wherein each micropost and / or the base layer is made of a biocompatible polymer material, such as polydimethylsiloxane (PDMS), thermoplastic polyurethane (TPU), and / or polycaprolactone (PCL).

[0085] Example 7. A cell sampling unit for male human papillomavirus (HPV) detection, comprising a plurality of microposts for contacting a sample and acquiring cells on a surface of the sample by rubbing; and a base layer comprising a front side for supporting the plurality of microposts and acquired cells, wherein one end of each micropost 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 rubbing force; wherein each micropost is cylindrical with a diameter of about 60-140 pm and a height of about 100-140 pm; wherein the plurality of microposts are configured in an array arrangement; the microposts are distributed with a density of about 1000 microposts / cm 2 ; and wherein each micropost and / or the base layer is made of polydimethylsiloxane (PDMS).

[0086] Example 8. The cell sampling unit according to example 7, wherein the height of each micropost is 60 pm, 100 pm, or 140 pm.

[0087] Example 9. A cell sampling tool comprising: a cell sampling unit according to any one of examples 1-8; and a manipulator; wherein 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 acquire cells under an external force applied to the manipulator.

[0088] Examples

[0089] Methods, Sampler Head Optimization, and Results

[0090] 1. Fabrication of a demonstration cell sampler head with biomimetic microstructures

[0091] In the following examples, the cell sampling unit can be referred to as a cell sampler head, sampling head, or sampler head. A silicon wafer mold was chosen as the master mold for the demonstration cell sampler head. Such a mold is smooth in surface, high in melting point, and precise in size to tens to hundreds of micrometers, suitable for fabricating cell sampler heads using different biocompatible polymers. As shown in FIG. 1, the master negative mold has microstructures (at least 1400 microposts / 1.4 cm 2The sampler head is made of silicon wafers. It is cast from PDMS. The microstructure dimensions of the PDMS-made sampler head are certified by the supplier using a white light interferometer. The height and width of the supplier-made sampler head are approximately 140 μm.

[0092] 2. Evaluate the consistency of the microstructure of the example cell sampler head manufactured using a biocompatible polymer.

[0093] 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, ensuring the PDMS completely fills 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 ).

[0094] For the PCL cell sampler head, place the PCL solid on the master mold and incubate in an 80°C vacuum oven for 18 hours. 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 ).

[0095] To manufacture the TPU cell sampler head, TPU particles were placed on the master mold and incubated in a vacuum oven at 200°C 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 1x1.4cm 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 ).

[0096] The height and width of the micro-pillars made of PDMS can reach 140 pm. The PDMS cell sampler head is soft and elastic, and it is comfortable to wipe the forearm. It is the best of the three test materials. The height of the PCL micro-pillars does not reach 140 pm, even after incubation in a vacuum oven. This can be because its viscosity is higher in the liquid state compared to PDMS and TPU. In addition, the PCL sampler head is brittle and difficult to wipe on the forearm. It is not considered a material for the sampler head. Although the sampler head made of TPU can meet the target size of the sampling head, it is difficult to remove TPU from the mold. It is a bit difficult to wipe TPU on the forearm. The production of the TPU sampler head also requires high temperature. Compared with PDMS, TPU is the second priority material for the sampler head. The results show that PDMS is a relatively better choice for cell sampler head material due to texture and ease of production.

[0097] 3. Characterize the microstructure of the cell sampler head by scanning electron microscopy (SEM).

[0098] As Figure 5A shown in Figure 5B , the example PDMS cell sampler head was cut and fixed on a metal stage. After gold coating, the microstructure was characterized by SEM, and ten regions of interest (ROIs) were examined for each sample. The height and width of the micro-pillars were 140 pm Figure 5C , and the detailed information of each measurement is listed in Table 1. The density was also greater than 1000 pillars / cm 2 .

[0099]

[0100] Table 1. Characterize the microstructure by scanning electron microscopy and examine ten ROIs for each sample. The height and width of all ROIs are approximately 140 pm.

[0101] 4. Optimize the shape, size, density, and pattern of the micro-pillars on the cell sampler head and measure the coefficient of friction on the skin model

[0102] a) Optimize the shape of the micro-pillars

[0103] In addition to cylinders, square and rectangular pillars were also prepared. Square and triangular master molds Figure 6A and 6C were made. The height and width of the micro-pillars were 140 pm, and they were designed to be at least 1400 micro-pillars / 1.4 cm 2 ( Figure 6B and 6D ).

[0104] 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).

[0105] 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 the figure. 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 increases of 57.9% and 32.8% in static and dynamic COF, respectively. Triangular micropillars showed increases of over 50% in both static and dynamic COF, while square micropillars showed increases of less than 20%. Based on the COF results, circular and triangular micropillars are relatively better choices for sampler heads.

[0106] For triangular micropillars, different directions of movement can affect COF and cell collection capacity. The COF of triangular micropillars during lateral, forward, and backward movement was tested on synthetic skin using a COF analyzer. 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, with the highest value displayed when moving forward. Figure 8C ).

[0107] static friction coefficient

[0108]

[0109] Dynamic coefficient of friction

[0110]

[0111]

[0112] Table 2. COF of cell sampler heads of different shapes were measured with a coefficient of friction (COF) tester according to ASTM D1894. The percentage increase of COF was defined as: [(COF 微柱 -COF 平坦表面 ) / COF 平坦表面 *100%].

[0113] b) Optimization of the size of the microposts

[0114] To optimize the size of the microposts, microposts with a height of 60 pm, 100 pm, and 140 pm were prepared (Figure 2a). The microposts with a height of 140 pm showed the highest static COF value (Figure 2b). The microposts with a height of 100 pm and 140 pm showed the highest dynamic COF (Figure 2c). In addition, microposts with a width of 60 pm, 100 pm, and 140 pm were prepared (Figure 2d). The microposts with a width of 140 pm showed the highest static COF value (Figure 2e), while the microposts with a width of 100 pm showed the highest dynamic COF (Figure 2f). Figure 9A ). The microposts with a height of 100 pm and 140 pm showed the highest dynamic COF ( Figure 9B ). In addition, microposts with a width of 60 pm, 100 pm, and 140 pm were prepared ( Figure 9C ). The microposts with a width of 140 pm showed the highest static COF value ( Figure 10A ), while the microposts with a width of 100 pm showed the highest dynamic COF ( Figure 10B ). Figure 10C

[0115] c) Optimization of the density of the microposts

[0116] Microposts heads with a density of 1000, 1200, 1400 microposts / cm 2 were prepared (Figure 3a). The microposts head with 1000 microposts / cm 2 showed the highest static COF and dynamic COF, and the micropost density was negatively correlated with the dynamic COF (Figure 3b and 3c). Figure 11A Figure 11B 11C

[0117] d) Optimization of the pattern of the microposts

[0118] For the patterning, microposts heads with micropost array and cross pattern were prepared (Figure 4a). The array pattern showed higher static COF and dynamic COF than the cross pattern (Figure 4b and 4c). Figure 12A Figure 12B 12C

[0119] In summary, cell samplers 1, 3 and 4 all showed high dynamic COF values on synthetic skin (Table 3).

[0120] ​​​​​​​

[0121]

[0122] Table 3. Dynamic coefficient of friction for different size samplers. n = 3.

[0123] 5. Evaluate the ability of the cell sampler prototype to collect skin cells from the human forearm.

[0124] The sampler tool prototype was fabricated by adding a polypropylene (PP) handle to the sampler head Figure 13H ). Briefly, the master silicon mold was placed in a soft silicon mold. The PP handle was inserted into the soft mold and PDMS was poured into the mold. The mold was degassed in a desiccator and incubated at 65 °C as described in Section 2. The sampler head was then cut to a size of 1 x 1.4 cm 2 .

[0125] The forearm was swabbed 1, 3, 5, 10, 15, and 20 times with it. The sampler head was placed in a 50 ml tube, 2 ml of trypsin was added to fully immerse the sampler head, followed by incubation at 37 °C for 3 min. The sampler head was then removed from the solution and the solution was centrifuged at 3000 ref for 5 min. The supernatant was carefully removed using a pipette. After adding 50 μΐ of Tris-buffered saline (TBS) to the bottom of the tube, 50 μΐ of trypan blue was added. 10 μΐ of the solution was transferred onto a hemocytometer and the number of cells was counted under an optical microscope. The average number of cells Figure 13A -F) was measured on the four large squares in the corners. Our data showed that about 5000-15000 cells were collected via different numbers of swabbing. The number of cells collected was directly proportional to the number of swabbing Figure 13G .

[0126] To further improve cell detachment, ultrasonic trypsin treatment was performed. After collecting the skin cells, the sampler head was placed in a 15 ml tube, 2.5 ml of trypsin was added to fully immerse the sampler head, followed by incubation and sonication in a temperature-controlled ultrasonicator at 37 °C for 10 min. The sampler head was then removed from the solution and about 1.4 ml of the sample solution was transferred to a 1.5 ml tube. The sample solution was centrifuged at 20000 ref for 5 min. The supernatant was carefully removed using a pipette and the remaining solution (about 1.1 ml) in the 15 ml tube was transferred to a 1.5 ml tube. The sample solution was again centrifuged at 20000 ref for 5 min. After carefully removing the supernatant, 50 μΐ of TBS was added to the precipitate. Then 50 μΐ of trypan blue was added to the 1.5 ml tube. 10 μΐ of the solution was transferred onto a hemocytometer and the number of cells was counted under an optical microscope. About 25000-48000 cells were collected via different numbers of swabbing. Thus, ultrasonic trypsin treatment was used as a cell detachment method.

[0127] The cell collection capacity of the sampler heads 1, 3, 4 was examined. The cell collection amount was comparable between the sampler heads 1, 3 and 4. Sampler head 1 was in use, so sampler head 1 was selected for further development.

[0128] 6. Assess the amount of genomic DNA extracted from the skin cells collected by the cell sampler prototype.

[0129] Genomic DNA was extracted from the cells collected by the cell sampler using a commercially available kit (Qiagen). The sampler was held and swiped 20 times on the forearm. The sampler head was placed in a 50 ml tube and 2 ml of trypsin was added to the sampler head. The tube was incubated at 37 °C for 3 min. The sampler head was then removed from the solution and the solution was centrifuged at 3000 ref for 5 min. The supernatant was carefully removed using a pipette. 200 μΐ of PBS, 20 μΐ of proteinase K and 200 μΐ of buffer AL were added to the tube. The solution was mixed well and incubated at 56 °C for 10 min with shaking at 300 rpm. The solution was cooled to room temperature and 200 μΐ 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 ref for 1 min. The flow-through was discarded in the collection tube. The spin column was then placed in a new 2 ml collection tube. 500 μΐ of buffer AW1 was added to the spin column and centrifuged at 6000 ref for 1 min. The flow-through was discarded in the collection tube. After placing the spin column in a new 2 ml collection tube, 500 μΐ of buffer AW2 was added to the spin column. The column was centrifuged at 20000 ref for 3 min. The flow-through was discarded and the spin column was transferred to a new 1.5 ml tube. The DNA was eluted by adding 50 μΐ of buffer AE to the center of the spin column membrane. The column was incubated at room temperature (15-25 °C) for 1 min. The spin column was centrifuged at 6000 ref for 1 min.

[0130] The quantification of DNA was determined by UV absorbance at 260 nm using a NanoDrop (Implen NP80). Briefly, 1 μΐ of buffer AE was pipetted into the NanoDrop and blanked. Then 1 μΐ of the filtrate was pipetted into the NanoDrop for DNA measurement in ng / μΐ concentration. As shown in Table 4, the amount of DNA extracted per cell sampler head was higher than 4 ng / μΐ when trypsin was used to detach the cells from the cell sampler head, whereas the amount of DNA extracted per cell sampler head increased to 5.67 ng / μΐ when trypsin was used for cell detachment in combination with ultrasonication.

[0131]

[0132] Table 4. Sampler head swabbed human forearm skin and cells were detached from the skin with trypsin or trypsin with sonication. Genomic DNA was extracted from the cells collected from the cell sampler head by a commercial kit (Qiagen). DNA concentration of the extract filtrate was quantified by UV absorbance at 260 nm.

[0133] 7. Evaluation of the copy number of the housekeeping gene of the extracted DNA by real-time quantitative PCR

[0134] After genomic DNA was extracted by Realbest reagent (provided by the sponsor), the copy of the housekeeping gene present in the sample was obtained by real-time quantitative PCR (qPCR). Briefly, the collected cells were detached with trypsin with sonication. The collected cell pellet was then resuspended with 50 μΐ 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 μΐ of the supernatant was transferred to a new tube. The extracted DNA was mixed with the qPCR master mix and primers of the housekeeping gene. Then, the sample was processed in a qPCR thermal cycler. A plasmid containing the housekeeping gene was used to establish a standard curve. The gene copy number of the housekeeping gene in the extracted DNA sample was calculated according to the generated standard curve.

[0135] Initially, beta globin was proposed as the housekeeping gene. However, beta globin was detected in Ca Ski cells but not in forearm skin cells. Other common housekeeping genes, beta actin and GAPDH, were also tested. Both beta actin and GAPDH were detected in Ca Ski cells and forearm skin cells. Figures 14A-14F ). The CT value of beta actin was higher than that of GAPDH, so it was chosen as the housekeeping gene for the internal control of human skin cells.

[0136] The gene copy number of the housekeeping gene of the cells collected from human forearm skin was calculated. The beta actin standard template was used to establish a standard curve Figure 15A and 15C ). The melting curve confirmed the specificity of the beta actin standard Figure 15B ).

[0137] After the human forearm was swabbed 20 times with the sampler, the gene copy number of the housekeeping gene in the extracted DNA sample was calculated (Table 5). The CT value of all samples was about 30-31, and the average gene copy number was about 2200 / μΐ.

[0138]

[0139]

[0140] Table 5. Gene copy number of human beta actin collected from human forearm and analyzed by qPCR.

[0141] 8. Cell collection with cell lines having HPV DNA

[0142] Since it was not possible to obtain HPV infected cells from healthy volunteers, an alternative approach was taken. A human cancer cell line previously infected with HPV, Ca Ski cells, was used. Cells were seeded onto slides, plates, and synthetic skin at a density of 5 x 10 4 cells / mm 2 , which is similar to the epidermal cell density of a human forearm Figure 16A . Ca Ski cells were unable to be cultured on synthetic skin and all cells were suspended in media. The sampler was wiped across the slides and plates 20 times. Cells were detached by ultrasonic trypsinization. DNA was extracted using a Qiagen DNA extraction kit. HPV-16 was analyzed by qPCR with beta globin as an internal control. Ca Ski cells alone were used as a positive control. On the amplification plot, the CT values for both HPV-16 and beta globin were below 40, indicating the presence of HPV DNA Figure 16B . Melting curves also confirmed the specificity of the HPV DNA targeting Figure 16C .

[0143]

[0144] Table 6. HPV-16 positive cell line, Ca Ski cells, were seeded onto slides and plates to simulate cell collection with HPV DNA. HPV-16 was analyzed by qPCR with beta globin as an internal control.

[0145] 9. Cell sampler accelerated stability testing at 55°C ± 2°C for 1 month and evaluation of cell collection ability.

[0146] The stability of the cell sampler prototypes will be evaluated under accelerated aging conditions. The aging factor is equal to 23 = 8 under accelerated aging conditions at 55°C, which is higher than the ambient temperature of 25°C. The cell sampler prototypes (with / without stand, Figure 17A ) were stored in a temperature-controlled room at 55°C ± 2°C, 100% atmospheric humidity for 1 month (31 days) and evaluated for cell collection ability as described in Section 5 (ultrasonic trypsinization). The weight of the cell sampler was measured for 1 month and the weight of all samplers remained stable throughout the month Figure 17B . The dimensions of the microposts on the sampler were monitored. No changes in the width and height of the samplers were observed after 1 month in all samplers Figure 17C , 17D, 17E and 17F). The width and height of all samplers Figure 17C and Figure 17D were kept at approximately 140 pm. Cell samples were evaluated for cell collection amount. Cells on the samplers were trypsinized and stained with trypan blue. Cells were quantified under an optical microscope with a hemocytometer. According to Figure 17H , no significant difference in cell collection capacity of the samplers was observed between month 0 and month 1.

[0147] 10. Example sampling tool

[0148] Figure 13C An example sampling tool comprising a handle and a cell sampling unit is shown. The sampler tool prototype was fabricated by attaching a polypropylene (PP) handle to a PDMS sampler head.

[0149] Figure 18A and 18B Another example sampler tool containing any of the example cell sampling units as described herein is shown. In this example, the example cell sampling unit is formed as one integral piece with the manipulator. The gray area represents the area containing the example sampler unit, which is located at the front end of the manipulator. The manipulator has two curved sections to facilitate the user to hold the manipulator to exert an external force on the manipulator, and thus on the sampler unit.

[0150] Conclusion

[0151] In summary, examples of biocompatible polymer-based cell sampling units and cell samplers provided can be used to collect cells for male HPV testing efficiently and painlessly. In some embodiments, the provided cell samplers are small and flexible. In some embodiments, the cell sampler head is about 1 x 1.4 cm 2 in size. In some embodiments, the sampling surface contains a regular array of >1000 microposts / cm 2 , each with a height and width < 150 pm. Due to the novel design, the cell sampler is able to collect a sufficient number of cells for downstream analysis of HPV. After the cells are collected, the cell sampler will be enzymatically digested to detach the cells. The detached cells will be used for DNA extraction and further qPCR analysis.

[0152] The provided example cell samplers are an easy-to-use, convenient, and reliable sampling device that can be used for self-sampling or remote sampling. With the specified size and microstructure, the samplers can not only be applied to HPV testing, but also to other diagnostic tests, such as dermatological diseases.

Claims

1. A cell sampling unit comprising: a plurality of micro-pillars for contacting a sample and acquiring cells from a surface of the sample by rubbing; and a base layer comprising a front side for supporting the plurality of micro-pillars and acquired cells, wherein one end of each micro-pillar 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 having a rubbing force; wherein each micro-pillar has a diameter of about 60-140 pm and a height of about 60-140 pm; wherein the plurality of microposts are configured in an array arrangement; the microposts are distributed at a density between about 1000-1400 microposts / cm 2 .

2. The cell sampling unit of claim 1, wherein each micro-pillar has a height of 60 pm, 100 pm, or 140 pm.

3. The cell sampling unit of claim 1, wherein the sample is from human skin.

4. The cell sampling unit of claim 3, wherein the human skin is skin of a male genital organ.

5. The cell sampling unit of claim 1, wherein the cell sampling unit has an overall length of 1-2 cm and a width of 1-3 cm.

6. The cell sampling unit of claim 1, wherein each micro-pillar is a cylinder, a triangular prism, a quadrangular prism, a hexagonal prism, or a pentagonal prism.

7. A cell sampling unit for male human papillomavirus (HPV) testing, the cell sampling unit comprising: a plurality of micro-pillars for contacting a sample and acquiring cells on a surface of the sample by rubbing; a plurality of micro-pillars for contacting a sample and acquiring cells from a surface of the sample by rubbing; and a base layer comprising a front side for supporting the plurality of micro-pillars and acquired cells, wherein one end of each micro-pillar 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 having a rubbing force; wherein each micro-pillar is cylindrical, has a diameter of 60-140 pm, and a height of 100-140 pm; wherein the plurality of microposts are configured in an array arrangement; the microposts are distributed at a density of about 1000 microposts / cm 2 ; and wherein each micro-pillar and / or the base layer is made of polydimethylsiloxane (PDMS).

8. The cell sampling unit of claim 7, wherein each micro-pillar has a height of 60 pm, 100 pm, or 140 pm.

9. A cell sampling tool comprising: a cell sampling unit according to any one of claims 1-8; and a manipulator; wherein 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 acquire cells under an external force applied to the manipulator.

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