WO2025152897A1

WO2025152897A1 - Cell sampler units, cell sampler tools comprising the same and methods of cell sampling

Info

Publication number: WO2025152897A1
Application number: PCT/CN2025/072058
Authority: WO
Inventors: Chun Hay Ko; Chun Fai Wong; Kwan Wun TSANG
Original assignee: Neo Health Hk Ltd
Current assignee: Neo Health Hk Ltd
Priority date: 2024-01-16
Filing date: 2025-01-13
Publication date: 2025-07-24
Anticipated expiration: 2026-07-16
Also published as: CN223601478U

Abstract

A cell sampling unit, comprising: a plurality of micropillars for contacting a sample and obtaining cells from surface of the sample by friction; and a base layer comprising a front side for supporting the plurality of micropillars and the cells obtained, wherein one end of each micropillar is fixed to or extending from the front side of the base layer and the opposing end is a free end, the free end comprising a contacting surface having friction; wherein each micropillar has a diameter of about 60-140 μm and a height of about 60-140 μm;wherein the plurality of micropillars are configured in an array arrangement; the micropillars are distributed with a density of between: about 1000-1400 micropillars/cm². Other example embodiments are described herein. In certain embodiments, the cell sampling unit is easy-to-use, convenient and reliable skin cell sampling for HPV screening in men.

Description

CELL SAMPLER UNITS, CELL SAMPLER TOOLS COMPRISING THE SAME AND METHODS OF CELL SAMPLING

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Application having Serial No. 63/621,132 filed January 16, 2024, the entire contents of which is/are hereby incorporated by reference herein.

FIELD OF INVENTION

This application relates to cell sampler units, cell sampler tools comprising the same and methods of cell sampling.

BACKGROUND OF INVENTION

Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the world. The increasing evidence of the correlation between HPV and anogenital cancers led to the development of HPV testing in both women and men. Cell sampling is a crucial step for HPV tests as the viral load of HPV in infected tissues is usually very low. To date, different kinds of samplers have been developed for the collection of cervical HPV samples in women. However, there is no similar sampler developed for men. In many clinical settings, sandpaper and medical swab are used together for sample collection. There are several drawbacks of this two-step collection method, such as causing discomfort and anxiety and more importantly the low successful collection rate. Therefore, there is an urgent need to develop an easy-to-use genital cell sampler for HPV DNA in men.

SUMMARY OF INVENTION

In certain embodiments, provided is cell sampler units, cell sampler tools comprising the same and methods of cell sampling. In certain embodiments, provided is a cell sampler with a novel microstructure for skin cell sampling such as genital HPV sampling in men. In certain embodiments, provided is a “Bio-Sandpaper” (BioS) technology that is similar to naturally-occurring microstructures, such as a dual-function microstructure mimicking micro to nano-scale structure setae in living organisms and pollen-trapping micro-patterned hairs on bee’s legs. Such biomimetic microstructure can remove and entrap skin cells easily. To achieve this, in some embodiments, the microstructure contains many micropillars, whose shape and size will be optimized to increase the surface area to volume ratio on the biocompatible polymer surface, resulting in a high friction for the removal of skin cells. In some embodiments, the micropillar spacing and patterning are optimized for efficient skin cell entrapment and release. In some embodiments, biocompatible polymers are tested to examine the consistency of microstructure fabrication in cast molding system, such that the provided sampler is feasible for future manufacturing scale-up. In some embodiments, cell sampler prototype is fabricated with a sampler head with microstructure and a handling stick. In some embodiments, provided cell sampler is easy-to-use, convenient and reliable skin cell sampling for HPV screening in men.
BRIEF DESCRIPTION OF FIGURES

FIG 1A is a photograph showing a mold design for a cell sampler unit with 42 x 35 micropillars, according to an example embodiment.

FIG. 1B is a schematic diagram showing dimensions of micropillars with 140 μm height and 140 μm width on a cell sampler unit, according to an example embodiment.

FIG. 1C is a photograph showing a master silicon wafer mold for manufacture of a cell sampler unit, according to an example embodiment.

FIG. 1D is a photograph showing a PDMS cell sampler unit in the size of 1x1.4 cm², according to an example embodiment.

FIG. 2A is a schematic diagram showing steps of fabrication of a cell sampler head unit from PDMS, according to an example embodiment.

FIG. 2B is a microscopic photograph showing the front view of a PDMS cell sampler head unit with 140 μm width of micropillars, under light microscope with magnification 100x, according to an example embodiment.

FIG. 2C is a microscopic photograph showing the side view of a PDMS cell sampler head unit with 140 μm height of micropillars, under light microscope with magnification 100x, according to an example embodiment.

FIG. 3A is a schematic diagram showing steps of fabrication of cell sampler heads from PCL, according to an example embodiment.

FIG. 3B is a microscopic photograph showing the front view of a PCL cell sampler head unit with 140 μm width of micropillars, under dissection microscope with magnification 80x, according to an example embodiment.

FIG. 3C is a microscopic photograph showing the side view of a PCL cell sampler unit with 108 μm height of micropillars, under light microscope with magnification 100x, according to an example embodiment.

FIG. 4A is a schematic diagram showing steps of fabrication of a cell sampler head from TPU, according to an example embodiment.

FIG. 4B is a microscopic photograph showing the front view of a TPU cell sampler head, under light microscope with magnification 100x, according to an example embodiment. The width of micropillars was 140 μm.

FIG. 4C is a microscopic photograph showing the side view of a TPU cell sampler head under light microscope with magnification 100x, according to an example embodiment. The height of micropillars was 140 μm.

FIG. 5A is a schematic diagram showing steps of analyzing PDMS cell sampler heads by scanning electron microscopy (SEM) , according to an example embodiment.

FIG. 5B is a SEM microscopic photograph showing the front view of a PDMS cell sampler head under SEM with magnification 100x, according to an example embodiment.. The density of the sampler was >1000 micropillars/cm².

FIG. 5C is a microscopic photograph showing the side view of PDMS cell sampler under SEM with magnification 100x, according to an example embodiment. The height and width of micropillars were 140 μm.

FIG. 6A are a schematic diagram (left) and a photograph (middle) of a silicon wafer mold of cell sampler unit with 42 x 35 square-micropillars, and a photograph (right) of a PDMS cell sampler heads of cell sampler unit with square-micropillars, respectively, according to an example embodiment.

FIG. 6B are microscopic photographs showing the front view (left) and side view (right) , of PDMS sampler with square-micropillars, under light microscope with magnification 100x, respectively, according to an example embodiment. The height and width of square-micropillars were 140 μm.

FIG. 6C is a schematic diagram (left) and a photograph (middle) of a silicon wafer mold of cell sampler unit with triangle-micropillars and a photograph (right) of a PDMS cell sampler unit with triangle-micropillars, according to an example embodiment.

FIG. 6D are microscopic photographs showing the front view (left) and side view (right) , respectively, of PDMS sampler with triangle-micropillars, under light microscope with magnification 100x, according to an example embodiment. The height and width of triangle-micropillars were 140 μm.

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

FIG. 8A is illustrative diagram showing friction generated by the microstructure to synthetic skin was measured in terms of a coefficient of friction (COF) tester, according to an example embodiment. In the diagram, different directions of movement for triangle micropillars were illustrated. The frictional force of the cell sampler head on the synthetic skin was measured with reference to ASTM D1894. The COF of different directions of movement for triangle micropillars was tested and compared.

FIG. 8B is a plot showing the static COF of different directions of movement for triangle micropillars, according to the example embodiment of FIG. 8A. FIG. 8C is a plot showing the dynamic COF of different directions of movement for triangle micropillars, according to the example embodiment of FIG. 8A.

FIG. 9A are microscopic photographs showing various heights (60μm, 100 μm and 140μm) of micropillars prepared to optimize the height of micropillars, according to an example embodiment. The frictional force of the cell sampler on the synthetic skin was measured with reference to ASTM D1894.

FIG. 9B is a plot showing the static COF of different heights of micropillars, according to the example embodiment of FIG. 9A. The frictional force of the cell sampler on the synthetic skin was measured with reference to ASTM D1894.

FIG. 9C is a plot showing the dynamic COF of different heights of micropillars, according to the example embodiment of FIG. 9A.

FIG. 10A are microscopic photographs under a light microscope with magnification 100x showing cell samplers with various widths (60μm, 100 μm and 140μm) of micropillars prepared to optimize the width of micropillars, according to an example embodiment. The frictional force of the cell sampler on the synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different widths were tested and compared.

FIG. 10B is a plot showing the static COF of different widths of micropillars, according to the example embodiment of FIG. 10A.

FIG. 10C is a plot showing the dynamic COF of different widths of micropillars, according to the example embodiment of FIG. 10A.

FIG. 11A are microscopic photographs under a light microscope with magnification 100x showing cell samplers with various densities of micropillars to optimize the density of micropillars, according to an example embodiment. The dimensions of cell samplers were viewed under a light microscope, magnification 100x. The frictional force of the cell sampler on the synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different densities were tested and compared.

FIG. 11B is a plot showing the static COF of different densities of micropillars, according to the example embodiment of FIG. 11A.

FIG. 11C is a plot showing the dynamic COF of different densities of micropillars, according to the example embodiment of FIG. 11A.

FIG. 12A are microscopic photographs under a light microscope with magnification 100x showing cell samplers with various patterns of micropillars to optimize the pattern of micropillars, according to an example embodiment. The dimensions of cell samplers were viewed under a light microscope, magnification 100x. The frictional force of the cell sampler on the synthetic skin was measured with reference to ASTM D1894. The COF of samplers with different patterns were tested and compared.

FIG. 12B is a plot showing the static COF of different patterns of micropillars, according to the example embodiment of FIG. 12A.

FIG. 12C is a plot showing the dynamic COF of different patterns of micropillars, according to the example embodiment of FIG. 12A.

FIG. 13A is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 1 time of swabbing; the photograph shows that skin cells/Big squares in the corner: (6+10+2+2) /4 = 5 Cells collected = 5x0.1x10⁴ = 5000 skin cells, according to an example embodiment. The cells on samplers were subjected to trypsin digestion and trypan blue staining. The cells were quantified with hemocytometer.

FIG. 13B is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 3 times of swabbing, the photograph shows that skin cells/Big squares in the corner: (10+10+6+6) /4 = 8 Cells collected = 8x0.1x10⁴ = 8000 skin cells, according to an example embodiment. The other experimental settings are the same as FIG. 13A.

FIG. 13C is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 5 times of swabbing, the photograph shows that skin cells/Big squares in the corner: (15+8+10+7) /4 = 10 Cells collected = 10x0.1x10⁴ = 10000 skin cells, according to an example embodiment. The other experimental settings are the same as FIG. 13A.

FIG. 13D is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 10 times of swabbing, the photograph shows that skin cells/Big squares in the corner: (11+10+9+9) /4 = 9.75 Cells collected = 9.75x0.1x10⁴ = 9750 skin cells, according to an example embodiment. The other experimental settings are the same as FIG. 13A.

FIG. 13E is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 15 times of swabbing, the photograph shows that skin cells/Big squares in the corner: (15+11+12+14) /4 = 13 Cells collected = 13x0.1x10⁴ = 13000 skin cells, according to an example embodiment. The other experimental settings are the same as FIG. 13A.

FIG. 13F is a microscopic photograph under a light microscope with magnification 100x showing evaluation of cell collection amount of PDMS samplers with 20 times of swabbing, the photograph shows that skin cells/Big squares in the corner: (16+14+18+15) /4 = 15.75 Cells collected = 15.75x0.1x10⁴ = 15750 skin cells, according to an example embodiment. The other experimental settings are the same as FIG. 13A.

FIG. 13G is a plot showing the cell amounts collected versus with numbers of swabbing of example embodiments of FIG. 13A to FIG. 13F, suggesting cells collection amount is directly proportional to the number of swabbing. Trypsinization with sonication also further improved the cell detachment efficiency comparing to trypsin alone, according to an example embodiment.

FIG. 13H is a photograph showing a sampler tool prototype fabricated by adding a polypropylene (PP) handle to the PDMS sampler head, according to an example embodiment.

FIG. 14A. is an amplification plot of beta-globin, analyzed by qPCR, in Ca ski cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells was extracted by Realbest reagent provided by the sponsor.

FIG. 14B. is an amplification plot of beta-actin, analyzed by qPCR, in Ca ski cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells is extracted by Realbest reagent.

FIG. 14C. is an amplification plot of GAPDH, analyzed by qPCR, in Ca ski cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells is extracted by Realbest reagent.

FIG. 14D. is an amplification plot of beta-globin, analyzed by qPCR, in human forearm skin cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells is extracted by Realbest reagent.

FIG. 14E. is an amplification plot of beta-actin, analyzed by qPCR, in human forearm skin cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells is extracted by Realbest reagent.

FIG. 14F. is an amplification plot of GAPDH, analyzed by qPCR, in human forearm skin cells collected by cell samplers and detached by trypsinization with sonication, according to an example embodiment. The DNA of the cells is extracted by Realbest reagent.

FIG. 15A is an amplification plot of different concentrations of beta-actin standard template prepared and analyzed by qPCR, according to an example embodiment.

FIG. 15B is a melt curve of different concentrations of beta-actin standard template prepared and analyzed by qPCR, according to the example embodiment of FIG. 15A.

FIG. 15C is plot showing a standard value of Ct value with DNA copy in log scale of different concentrations of beta-actin standard template prepared and analyzed by qPCR, according to an example embodiment of FIG. 15A.

FIG. 15D is a plot showing a melt curve of human forearm skin cells sample of different concentrations of beta-actin standard template prepared and analyzed by qPCR, according to an example embodiment.

FIG. 16A are photographs and microscopic photographs showing Ca Ski cells utilized and seeded on glass slide, culture plate and synthetic skin, to simulate the cell collection with HPV DNA, according to an example embodiment. The cell samplers were swabbed on a glass slide and culture plate to collect Ca Ski cells. Ca ski cells was not able to cultivate on synthetic skin and all cells were suspended in culture medium. HPV-16 was analyzed by qPCR and beta-globin served as an internal control

FIG. 16B is an amplification plot showing HPV-16 and beta-globin, according to an example embodiment.

FIG. 16C is a melt curve of HPV-16, according to the example embodiment of FIG. 16B.

FIG. 16D is a melt curve of beta-globin, according to an example embodiment of FIG. 16B.

FIG. 17A is a photograph showing cell samplers with holder (left) and cell samplers (right) in accelerated stability test of the cell samplers performed at 55 ± 2℃ for 1 month, according to an example embodiment.

FIG. 17B is a plot showing the weight of cell samplers measured for 1 month, according to an example embodiment of FIG. 17A.

FIG. 17C are microscopic photographs showing front view of cell samplers with holder at Day 0 (Left) and Day 30 (right) , with width of micropillars 140 μm, under light microscope with magnification 100x, according to an example embodiment.

FIG. 17D are microscopic photographs showing front view of cell samplers at Day 0 (Left) and Day 30 (right) , with width of micropillars 140 μm, under light microscope with magnification 100x, according to an example embodiment.

FIG. 17E are microscopic photographs showing side view of micropillars of cell samplers at Day 0 (Left) and Day 30 (right) , with height of micropillars 140 μm, with holder, under light microscope with magnification 100x, according to an example embodiment.

FIG. 17F are microscopic photographs showing side view of micropillars of cell samplers at Day 0 (Left) and Day 30 (right) , with height of micropillars 140 μm, under light microscope with magnification 100x, according to an example embodiment.

FIG. 17G are microscopic photographs showing cell collection amounts of cell samplers evaluated, according to an example embodiment. The cells on samplers were subjected to trypsin digestion and trypan blue staining and the cells were quantified with hemocytometer under light microscope, magnification100x.

FIG. 17H is a graph showing the cell collecting ability of samplers was comparable between Month 0 and Month 1, according to an example embodiment.

FIG. 18A is a schematic diagram showing the dimensions of the rear side of an example sampler tool, according to an example embodiment.

FIG. 18B is a schematic diagram showing the dimensions of the front side of an example sampler tool with the grey area indicated the area comprising the example sampler unit, according to an example embodiment of FIG. 18A.

DETAILED DESCRIPTION

DEFINITIONS

As used herein and in the claims, the terms “comprising” (or any related form such as “comprise” and “comprises” ) , “including” (or any related forms such as “include” or “includes” ) , “containing” (or any related forms such as “contain” or “contains” ) , means including the following elements but not excluding others. It shall be understood that for every embodiment in which the term “comprising” (or any related form such as “comprise” and “comprises” ) , “including” (or any related forms such as “include” or “includes” ) , or “containing” (or any related forms such as “contain” or “contains” ) is used, this disclosure/application also includes alternate embodiments where the term “comprising” , “including, ” or “containing, ” is replaced with “consisting essentially of” or “consisting of” . These alternate embodiments that use “consisting of” or “consisting essentially of” are understood to be narrower embodiments of the “comprising” , “including, ” or “containing, ” embodiments.

For the sake of clarity, “comprising” , “including” , “containing” and “having” , and any related forms are open-ended terms which allows for additional elements or features beyond the named essential elements, whereas “consisting of” is a closed end term that is limited to the elements recited in the claim and excludes any element, step, or ingredient not specified in the claim.

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. Where a range is referred in the specification, the range is understood to include each discrete point within the range. For example, 1-7 means 1, 2, 3, 4, 5, 6, and 7.

As used herein, the term "about" is understood as within a range of normal tolerance in the art and not more than ±10%of a 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 specific value also includes the specific value, for example, about 50 includes 50.

It is to be understood that terms such as “top” , “bottom” , “middle” , “side” , “length” , “inner” , “outer” , “interior” , “exterior” , “outside” , “vertical” , “horizontal” and the like as may be used herein, merely describe points of reference and do not limit the present invention to any particular orientation or configuration. Further, terms such as “first, ” “second, ” “third, ” etc., merely identify one of a number of portions, components and/or points of reference as disclosed herein, and likewise do not limit the present invention to any particular configuration or orientation.

Although the description referred to particular embodiments, the disclosure should not be construed as limited to the embodiments set forth herein.
NUMBERED EMBODIMENT

Embodiment 1. A cell sampling unit, comprising: a plurality of micropillars for contacting a sample, e.g. skin or other surfaces, and obtaining cells from surface of the sample, e.g. skin or other surfaces, by friction; and a base layer comprising a front side for supporting the plurality of micropillars and the cells obtained, wherein one end of each micropillar is fixed to or extending from the front side of the base layer and the opposing end is a free end, the free end comprising a contacting surface having friction; wherein each micropillar has a diameter of about 60-140 μm and a height of about 60-140 μm; wherein the plurality of micropillars are configured in an array arrangement; the micropillars are distributed with a density of between: about 1000-1400 micropillars/cm².

Embodiment 2. The cell sampling unit of embodiment 1, wherein each micropillar has a height of 60 μm, 100 μm or 140 μm.

Embodiment 3. The cell sampling unit of any one of the preceding embodiments, wherein the sample, e.g. skin or other surfaces, is derived from human skin such as skin from male genital organ for male human papillomavirus (HPV) detection.

Embodiment 4. The cell sampling unit of any one of the preceding embodiments, wherein the cell sampling unit has an overall length of about 1-2cm and a width of about 1-3cm.

Embodiment 5. The cell sampling unit of any one of the preceding embodiments, wherein the each micropillar is cylindrical, triangular prism, square prism, hexagonal prism or pentagonal prism.

Embodiment 6. The cell sampling unit of any one of the preceding embodiments, wherein each micropillar and/or the base layer is made of a biocompatible polymeric material such as polydimethylsiloxane (PDMS) , thermoplastic polyurethane (TPU) and/or polycaprolactone (PCL) .

Embodiment 7. A cell sampling unit for human papillomavirus (HPV) detection in male, comprising: a plurality of micropillars for contacting a sample and obtaining cells on surface of the sample by friction; and a base layer comprising a front side for supporting the plurality of micropillars and the cells obtained, wherein one end of each micropillar is fixed to or extending from the front side of the base layer and the opposing end is a free end, the free end comprising a contacting surface having friction; wherein each micropillar is cylindrical in shape, having a diameter of about 60-140 μm and a height of about 100-140 μm; wherein the plurality of micropillars are configured in an array arrangement; the micropillars are distributed with a density of about 1000 micropillars/cm²; and wherein each micropillar and/or the base layer is made of polydimethylsiloxane (PDMS) .

Embodiment 8. The cell sampling unit for human papillomavirus (HPV) detection in male of embodiment 7, wherein each micropillar has a height of 60 μm, 100 μm or 140 μm.

Embodiment 9. A cell sampling tool, comprising: the cell sampling unit as claimed in any one of embodiments 1-8; and an effector; wherein the base layer of the cell sampling unit is attached to or extending from the effector such that the cell sampling unit rubs against the sample to obtain cells, under an external force exerted on the effector.

Embodiment 10. A method of cell sampling, comprising the steps of: rubbing against a sample to obtain cells therefrom using the cell sampling unit as claimed in any one of embodiments 1-8 or the cell sampling tool in embodiment 9.

Embodiment 11. The method of embodiment 10, wherein not less than 5,000 cells are obtained on the cell sampling unit.

Embodiment 12. The method of embodiments 10 or 11, wherein more than 25,000 cells are obtained.

Embodiment 13. The method of any one of embodiments 10 -12, wherein the rubbing against a sample comprises the steps of: disposing the cell sampling unit or the cell sampling tool on a sample surface, e.g. skin or other surfaces, the free end facing the sample surface, e.g. skin or other surfaces, and rubbing the sample back and forth under an external force to collect the cells.

Embodiment 14. The method of any one of embodiments 10-13, wherein the sample is human skin, such as male genital organ skin for male human papillomavirus (HPV) detection, or skin, surfaces of other animals or human body.

Embodiment 15. The method of any one of embodiments 10-14, further comprising the step of: immersing the cell sampling unit or the cell sampling tool in an extraction solution to detach the cells from the sampling unit, and facilitating detachment of the cells from the sampler under an external force; and separating the detached cells from the extraction solution by centrifugal force to remove supernatant, such that cell pellet that can be used for DNA extraction is obtained.

Embodiment 16. The method of any one of embodiments 10-15, further comprising the step of: rubbing the sample such as human skin 1-20 times to obtain 5,000-50,000 cells or more.
EXAMPLES
Methodology, Sampler Head Optimization and Results
1. Fabricate an example cell sampler head with biomimetic microstructure

In the following examples, the cell sampler unit can be named as cell sampler head, sampling head or sampler head. A silicon wafer mold was selected as the master mold of the example cell sampler head. Such mold has a smooth surface, high melting point, and a precise dimension from tens to hundreds of microns, which is suitable for the fabrication of the cell sampler head with different biocompatible polymers. As shown in Figure 1, a master negative mold was made by silicon wafer with microstructure (at least 1400 micropillars/1.4 cm²) . The cell sampler head was cast by PDMS. The dimensions of the microstructure of the cell sampler head made by PDMS were certified by the vendor using the white light interferometer. The height and width of the cell sampler head made by the vendor were about 140 μm.
2. Evaluate the consistency of microstructure fabrication with biocompatible
polymers for the example cell sampler head.

In this example, three example biocompatible polymers, including TPU, PDMS, and PCL were tested as the example cell sampler head. To produce a PDMS cell sampler head, Silicone elastomer kit was used (Figure 2A) . 10 parts of the silicon base were mixed well with 1 part of the curing agent. The bubbles in the solution were removed by desiccator for about 30 min. Then the clear solution was cast on the silicon wafer master mold. The mold with PDMS was placed in the desiccator to remove the bubbles on the micropillars hole for 18 h, as a result, all the PDMS could fill up the micropillar hole. Then the mold was placed in the oven at 65℃ for 2 h and cut to the size of 1x1.4 cm². The height and width of micropillars were measured by a light microscope. Both height and width were about 140 μm (Figures 2B and 2C) .

For the PCL cell sampler head, the PCL solid was placed on the master mold and incubated in at 80℃ vacuum oven for 18 h (Figure 3A) . The sampler head was cooled down to room temperature. PCL sampler head was carefully removed by scalpel and cut to the size of about 1x1.4 cm². The height and width of micropillars were measured by a light microscope. The width of micropillars was about 140 μm (Figure 3B) and the height was about 108 μm (Figure 3C) .

To fabricate the TPU cell sampler head, TPU pellets were placed on the master mold and incubated at a 200℃ vacuum oven for 18 h (Figure 4A) . The sampler head was cooled down to room temperature. The TPU sampler head was carefully removed with kitchen scissors and cut to the size of 1x1.4 cm². The height and width of micropillars were measured by a light microscope. Both height and width of the micropillars reached 140 μm (Figure 4B and 4C) .

The height and width of the micropillar made from PDMS could both reach 140 μm. The PDMS cell sampler head was soft and elastic which is comfortable to swab on the forearm. It is the best among the 3 tested materials. The height of the PCL micropillar did not reach 140μm even incubated in a vacuum oven. It may be due to higher viscosity in the liquid state compared to PDMS and TPU. Besides, the PCL sampler head was brittle and hard to swab on the forearm. It would not be considered material for the sampler head. Although the sampler head made of TPU could satisfy the target dimensions of the sampling head, TPU was difficult to remove from the mold. TPU is a little hard to swab on the forearm. The production of a TPU sampler head also requires high temperatures. Compared with PDMS, TPU is the second-priority material for the sampler head. Results showed that PDMS was comparatively better option for the material of the cell sampler head due to the texture and ease of production.
3. Characterize the microstructure of the cell sampler head by scanning
electron microscopy (SEM) .

As indicated in Figure 5A, the example PDMS cell sampler head was cut and fixed on the metal stand. After gold coating, the microstructure was characterized with SEM, and ten regions of interest (ROI) for each sample were examined. The height and width of micropillars were 140 μm (Figure 5B and Figure 5C) and details of each measurement were listed in Table 1. The density was also greater than 1000 pillars/cm².

Table 1. The microstructure was characterized with scanning electron microscopy and ten ROI for
each sample was examined. All ROI of height and width were about 140 μm.
4. Optimize the shape, dimension, density and pattern of micropillars on the
cell sampler head and measure coefficient of friction on skin model

a) Optimize the shape of micropillars

In addition to the round pillars, square and rectangular pillars were prepared. The square and triangle master mold was produced (Figures 6A and 6C) . The height and width of the micropillars were 140 μm and the design was at least 1400micropillars/1.4 cm² (Figure 6B and 6D) .

The friction generated by the microstructure to synthetic skin was measured in terms of coefficient of friction (COF) by a COF Tester (Labthink MXD-02) . An 8N synthetic skin was used to assimilate the human forearm, and the synthetic skin was immobilized on the slide plane of the tester. The PDMS sampler head was fixed to the center of the sled with double-sided tape, and the weight of the sled with sampler heads was recorded. The testing speed was set at 150 mm/min. As shown in Figure 7, the frictional force of the cell sampler head on the synthetic skin was measured with reference to ASTM D1894. The COF of different shapes of micropillars was tested and compared. In general, the frictional force of sampler heads on the human forearm skin was about 0.2-0.5 N. The frictional force was in the following order: triangle-micropillars > circle-micropillars > square-micropillars > flat surface (without micropillars) .

COF of different shapes of cell sampler heads was calculated by the software “MXD-02 coefficient of friction tester” . Static friction time was defined as 0-10 sec, while dynamic COF was calculated based on 10-60 sec as indicated in Figure 7C. The static and dynamic COF of sampler heads were shown in Table 2. The percentage increase of COF was calculated as [ (COF_micropillars -COF_{flat surface}) /COF_{flat surface} *100%] . The static and dynamic COF of circle-micropillars increased by 57.9%and 32.8%, respectively, compared with a flat surface without the microstructure. The static and dynamic COF of triangle micropillars both increased by more than 50%, whereas the static and dynamic COF of square micropillars increased by less than 20%. From the COF results, circle and triangle–micropillars were comparatively better options for utilization in sampler heads.

Regarding the triangle micropillars, different directions of movement may affect the COF as well as the cell's collection ability. The COF of triangle micropillars in side movement, forward movement, and backward movement was tested with the COF tester on the synthetic skin (Figure 8A) . The frictional force of the cell sampler head on the synthetic skin was measured with reference to ASTM D1894. The COF of different directions of movement varied from 0.199 to 0.238, and backward movement showed the highest static COF (Figure 8B) . The values of dynamics COF varied from 0.198 -0.289, whereas forward movement showed the highest value (Figure 8C) .
Static coefficient of friction

Dynamic coefficient of friction

Table 2. Coefficient of Friction (COF) of different shapes of cell sampler heads was measured by
COF tester with reference to ASTM D1894. The percentage increase of COF was defined as [ (COF_micropillars–COF_{flat surface}) /COF_{flat surface} *100%] .
b) Optimize the dimension of micropillars

To optimize the dimension of micropillars, 60μm, 100μm, and 140μm height of micropillars were prepared (Figure 9A) . 140μm-height micropillars showed the highest value of static COF (Figure 9B) . 100μm-height and 140μm-height micropillars showed the highest dynamic COF (Figure 9C) . Furthermore, 60μm, 100μm, and 140μm width of micropillars were prepared (Figure 10A) . 140μm-width micropillars showed the highest value of static COF (Figure 10B) , whereas 100μm-width micropillars showed the highest dynamic COF (Figure 10C) .
c) Optimize the density of micropillars

For the density of cell sampler heads, 1000, 1200, and 1400 micropillars/cm² were prepared (Figure 11A) . 1000 micropillars/cm² showed the highest static COF and dynamic COF and the density of micropillars were negatively related to the dynamic COF (Figures 11B and 11C) .
d) Optimize the pattern of micropillars

For the patterning, cell sampler heads with array and cross pattern of micropillars were prepared (Figure 12A) . The array pattern showed higher static COF and dynamic COF than the cross pattern (Figure 12B and 12C) .

To summarize, the cell samplers 1, 3 and 4 both exerted high value of dynamics COF on synthetic skin (Table 3) .

Table 3. Dynamic Coefficient of Friction of Samplers with different dimensions. n=3.
5. Evaluate the skin cell collection ability of the cell sampler prototype from
human forearm.

The sampler tool prototype was fabricated by adding a polypropylene (PP) handle to the sampler head (Figure 13H) . Concisely, the master silicon mold was placed in a soft silicon mold. The PP handle was inserted in the soft mold and PDMS was poured into the mold. The mold was degassed in a desiccator and incubated at 65℃ as mentioned in section 2. The sampler head was then cut to the size of 1x1.4 cm².

It was used to swab the forearm 1, 3, 5, 10, 15 and 20 times. The sampler head was placed in a 50 ml tube and 2 ml trypsin was added to immerse the sampling head completely, followed by incubation at 37℃ for 3 min. Then the sampler head was removed from the solution, and the solution was centrifuged at 3000 rcf for 5 min. The supernatant was carefully removed using a pipette. Upon adding 50 μl Tris-buffered saline (TBS) to the bottom of the tube, 50μl trypan blue was then added. 10μl solution was transferred on a hemocytometer and the number of cells was counted under a light microscope. The average cells on the four big squares in the corner were measured (Figures 13A-F) . Our data demonstrated that about 5000-15000 cells were collected with different numbers of swabbing. The number of cells collected was directly proportional to the number of swabbing (Figure 13G) .

To further improve cell detachment, trypsinization with sonication was performed. Upon collecting skin cells. The sampler head was placed in a 15 ml tube and 2.5 ml trypsin was added to immerse the sampling head completely, followed by incubation and sonication in the temperature-controlled sonicator at 37℃ for 10 min. Then the sampler head was removed from the solution, and about 1.4 ml sample solution was transferred to a 1.5 ml tube. The sample solution was centrifuged at 20000 rcf for 5 min. The supernatant was carefully removed using a pipette, and the remaining solution in the 15 ml tube (about 1.1 ml) was transferred to the 1.5 ml tube. The sample solution was centrifuged at 20000 rcf for 5 min again. After carefully removing the supernatant, 50 μl TBS was added to the pellet. Then 50μl trypan blue was added to a 1.5 ml tube. 10 μl solution was transferred on a hemocytometer and the number of cells was counted under a light microscope. About 25000-48000 cells were collected with different numbers of swabbing. Thus, trypsinization with sonication was utilized as the cell detachment method.

The cell collection ability of sampler heads 1, 3, and 4 was examined. The cell collection amount was comparable between sampler heads 1, 3, and 4. Sampler head 1 was in use, and therefore selected sampler head 1 for further development.
6. Evaluate the quantity of genomic DNA extracted from collected skin cells by
the cell sampler prototype.

Genomic DNA was extracted from cells collected from the cell sampler by a commercially available kit (Qiagen) . The sampler was held and swabbed on the forearm 20 times. The sampler head was placed in a 50 ml tube and 2 ml trypsin was added to the sampler head. The tube was incubated at 37℃ for 3 min. Then the sampler head was 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 PBS, 20 μl proteinase K, and 200 μl Buffer AL were added to the tube. The solution was mixed well and incubated at 56℃ for 10 min with shaking at 300 rpm. The solution was allowed to cool down to room temperature and 200 μl ethanol was added to the solution. The solution was transferred to the 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 in the collection tube. Then the spin column was placed in a new 2 ml collection tube. 500 μl buffer AW1 was added to the spin column and centrifuged at 6000 rcf for 1 min. The flow-through was discarded in the collection tube. Upon placing the spin column in a new 2 ml collection tube, 500 μl buffer AW2 was added to the spin column. The column was centrifuged at 20000 rcf 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 μl buffer AE to the center of the spin column membrane. The column was incubated at room temperature (15-25℃) for 1 min. The spin column was centrifuged at 6000 rcf for 1 min.

Quantification of the DNA was determined by UV absorbance at 260 nm by a NanoDrop (Implen NP80) . In brief, 1 μl buffer AE was pipetted to the NanoDrop and calibrated as blank. Then 1μl filtrate was pipetted to the NanoDrop for the DNA measurement at a concentration of ng/ml. As indicated in Table 4, the extracted DNA amount was above 4 ng/μl per cell sampler head when trypsin was used for cell detachment from cell sampler heads, whereas the extracted DNA amount was increased to 5.67 ng/μl when trypsin with sonication was used for cell detachment.

Table 4. The sampler head swabbed on human forearm skin and the skin cells detached by trypsin
or trypsin with sonication. Genomic DNA was extracted from cells collected from the cell sampler head by a commercially available kit (Qiagen) . The DNA concentration of the extracted filtrate was quantified at UV absorbance of 260 nm.
7. Evaluate the gene copy number of house-keeping gene of the extracted
DNA by Real-time quantitative PCR

Following the genomic DNA extraction by Realbest reagent (provided by the sponsor) , the copies of a housekeeping gene present in the sample were obtained by real-time quantitative PCR (qPCR) . In brief, the cells collected were detached by trypsin with sonication. Then the collected cell pellet was re-suspended with 50 μl Realbest reagent and incubated at 98℃for 30 min with shaking at 300 rpm. The sample solution was centrifuged at 8000 rpm for 5 min. 40 μl supernatant was transferred to a new tube. Extracted DNA was mixed with the qPCR master mix and primers for the housekeeping gene. Then, the sample was processed in a qPCR thermocycler. A plasmid containing the housekeeping gene was used for the establishment of the standard curve. The gene copy number of the housekeeping gene in the extracted DNA sample was calculated from the generated standard curve.

Originally, beta-globin was proposed as the housekeeping gene. However, beta-globin was detected in Ca Ski cells but was not detected 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) . Beta-actin has the highest CT value than GAPDH and thus was selected as the housekeeping gene as the internal control of human skin cells.

To calculate the gene copy number of the housekeeping gene of the cells collected from human forearm skin. A beta-actin standard template was used for the establishment of a standard curve (Figures 15A and 15C) . The melt curve confirms the specificity of the beta-actin standard (Figure 15B) .

Upon swabbing on the human forearm 20 times by samplers, 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/μl.

Table 5. Gene copy number of human beta-actin collected from human forearm and analyzed
by qPCR.
8. Utilize Cell line with HPV DNA as simulation of cell collection with HPV DNA

Since it is not possible to obtain HPV-infected cells from healthy volunteers, a surrogate approach was employed. A human carcinoma cell line previously infected with HPV Ca Ski cells, was utilized. Cells were seeded on a glass slide, culture plate, and synthetic skin at a density of 5 x 10⁴ cells/mm², similar to the epidermal cell density of the human forearm (Figure 16A) . Ca Ski cells were not able to cultivate on synthetic skin and all cells were suspended in a culture medium. Samplers were swabbed on the glass slide and culture plate 20 times. The cells were detached by trypsinization with sonication. The DNA was extracted with a Qiagen DNA extraction kit. HPV-16 was analysed by qPCR and beta-globin served as an internal control. Ca Ski cells alone served as a positive control. On the amplification graph, both CT value o f HPV-16 and beta-globin were under 40 indicating the present of HPV DNA (Figure 16B) . The melt curve also confirms the specificity of the targeted HPV DNA (Figure 16C) .

Table 6. A HPV-16 positive cell line, Ca Ski cells, was seeded on a glass slide and culture plate to
utilize as simulations of cell collection with HPV DNA. HPV-16 was analyzed by qPCR and beta-globin served as an internal control.
9. Accelerated stability test of the cell sampler at 55 ± 2℃ for 1 month and
evaluate its cell collecting ability.

The stability of the cell sampler prototype will be evaluated under accelerated aging conditions. Accelerated aging condition at 55℃, above an ambient temperature of 25℃, equals an aging factor of 23 = 8. The cell sampler prototypes (with holder/ without a holder, Figure 17A) were stored in a temperature-controlled chamber at 55 ± 2℃, atmospheric humidity for 1 month (31 days) and the cell collection ability was evaluated as described in section 5 (trypsinization with sonication) . The weight of cell samplers was measured for 1 month, and the weight of all samplers was stable throughout a whole month (Figure 17B) . The dimensions of micropillars on the samplers were monitored. Changes in the width and height of samplers were not observed after one month in all samplers (Figures 17C, 17D, 17E, and 17F) . The width and height of all samplers (Figures 17C and 17D) remained at about 140μm. The cell collection amount of cell samples was evaluated. The cells on samplers were subjected to trypsin digestion and trypan blue staining. The cells were quantified with a hemocytometer under a light microscope. From Figure 17H, a significant difference in cell collecting ability of samplers was not observed between Month 0 and Month 1.
10. Example sampling tools

Figure 13C showed an example sampling tool comprising a handle and a cell sampling unit. The sampler tool prototype was fabricated by attaching a polypropylene (PP) handle to the PDMS sampler head.

Figures 18A and 18B showed another example sampler tool containing any one of the example cell sampler units as described herein. In this example, the example cell sampler unit is formed with the effector as one integral piece. The grey area indicated the area comprising the example sampler unit, which is disposed on the font side of the effector. The effector has two curved portions facilitating a user to hold the effector to exert an external force onto the effector, thereby onto the sampler unit.
Conclusion

In summary, the provided example of biocompatible polymer-based cell sampler units and the cell samplers that can be used to collect cells efficiently and painlessly for HPV tests in men was developed. In some embodiments, the provided cell samplers are small and elastic. In some embodiments, cell sampler head was in size of about 1x1.4 cm². In some embodiments, the sampling surface contains a regular array of >1000 micropillars per cm², each <150 μm in height and width. Due to the novel design, the cell sampler is able to collect a sufficient number of cells for the downstream analysis of HPV. Upon cell collection, the cell sampler will be subjected to enzymatic digestion for cell detachment. The detached cells will be utilized for DNA extraction and further qPCR analysis.

The provided example cell sampler acts as an easy-to-use, convenient, and reliable sampling device, which enables self-sampling or remote sampling. With the designated size and microstructure, the sampler not only can apply to HPV detection but also for other diagnostic tests, such as skin diseases.

Claims

A cell sampling unit, comprising:

a plurality of micropillars for contacting a sample and obtaining cells from surface of the sample by friction; and

a base layer comprising a front side for supporting the plurality of micropillars and the cells obtained,

wherein one end of each micropillar is fixed to or extending from the front side of the base layer and the opposing end is a free end, the free end comprising a contacting surface having friction;

wherein each micropillar has a diameter of about 60-140 μm and a height of about 60-140 μm;

wherein the plurality of micropillars are configured in an array arrangement; the micropillars are distributed with a density of between: about 1000-1400 micropillars/cm².
The cell sampling unit of claim 1, wherein the sample is derived from human skin such as skin from male genital organ for male human papillomavirus (HPV) detection.
The cell sampling unit of claim 1, wherein the cell sampling unit has an overall length of about 1-2cm and a width of about 1-3cm.
The cell sampling unit of claim 1, wherein the each micropillar is cylindrical, triangular prism, square prism, hexagonal prism or pentagonal prism.
The cell sampling unit of claim 1, wherein each micropillar and/or the base layer is made of a biocompatible polymeric material such as polydimethylsiloxane (PDMS) , thermoplastic polyurethane (TPU) and/or polycaprolactone (PCL) .
A cell sampling unit for human papillomavirus (HPV) detection in male, comprising:

a plurality of micropillars for contacting a sample and obtaining cells on surface of the sample by friction; and

a base layer comprising a front side for supporting the plurality of micropillars and the cells obtained,

wherein one end of each micropillar is fixed to or extending from the front side of the base layer and the opposing end is a free end, the free end comprising a contacting surface having friction;

wherein each micropillar is cylindrical in shape, having a diameter of about 60-140 μm and a height of about 100-140 μm;

wherein the plurality of micropillars are configured in an array arrangement; the micropillars are distributed with a density of about 1000 micropillars/cm²; and

wherein each micropillar and/or the base layer is made of polydimethylsiloxane (PDMS) .
A cell sampling tool, comprising:

the cell sampling unit as claimed in any one of claims 1-6; and

an effector;

wherein the base layer of the cell sampling unit is attached to or extending from the effector such that the cell sampling unit rubs against the sample to obtain cells, under an external force exerted on the effector.
A method of cell sampling, comprising the steps of: rubbing against a sample to obtain cells therefrom using the cell sampling unit as claimed in any one of claims 1-6 or the cell sampling tool as claimed in claim 7.
The method of claim 8, wherein the rubbing against a sample comprises the steps of: disposing the cell sampling unit or the cell sampling tool on a sample surface, the free end facing the sample surface, and rubbing the sample back and forth under an external force to collect the cells.
The method of claim 8 or claim 9, wherein the sample is human skin, such as male genital organ skin for male human papillomavirus (HPV) detection.
The method of any one of claims 8 -10, further comprising the step of:

immersing the cell sampling unit or the cell sampling tool in an extraction solution to detach the cells from the sampling unit, and facilitating detachment of the cells from the sampler under an external force; and

separating the detached cells from the extraction solution by centrifugal force to remove supernatant, such that a precipitate that can be used for DNA extraction is obtained.
The method of any one of claims 8 -10, further comprising the step of: rubbing the sample such as human skin 1-20 times to obtain 5,000-50,000 cells or more.