Versatile fabrication of size and shape-controllable nanofibrous

Oct 12, 2018 - Versatile fabrication of size and shape-controllable nanofibrous concave microwells for cell spheroid formation. Sang Min Park , Seong ...
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Biological and Medical Applications of Materials and Interfaces

Versatile fabrication of size and shape-controllable nanofibrous concave microwells for cell spheroid formation Sang Min Park, Seong Jin Lee, Jiwon Lim, Bum Chang Kim, Seon Jin Han, and Dong Sung Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15821 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Versatile fabrication of size and shape-controllable nanofibrous concave microwells for cell spheroid formation Sang Min Park, Seong Jin Lee, Jiwon Lim, Bum Chang Kim, Seon Jin Han, and Dong Sung Kim* Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Pohang, Gyeongbuk, 37673, South Korea KEYWORDS. Electrospinning; Microwell; Spheroid; Nanofiber; Electrolyte solution

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ABSTRACT

Though the microfabrication techniques for microwells enabled to guide physiologically relevant 3D cell spheroid formation, there have been substantial interests to more closely mimic nano/microtopographies of in vivo cellular microenvironment. Here, we developed a versatile fabrication process for nanofibrous concave microwells (NCMs) with a controllable size and shape. The key to the fabrication process was the use of an array of hemispherical convex electrolyte solution drops as the grounded collector for electrospinning, which greatly improved the degree of freedom of the size, shape and curvature of a NCM. A polymer substrate with through-holes was prepared for the electrolyte solution to come out through the hole and to naturally form a convex shape due to surface tension. Subsequent electrolyte-assisted electrospinning process enabled to achieve various arrays of NCMs of triangular, rectangular and circular shapes with size ranging from 1,000 µm down to 250 µm. As one example of biomedical applications, the formation of human hepatoma cell line (HepG2) spheroids was demonstrated on the NCMs. The results indicated that the NCM enabled uniform, size-controllable spheroid formation of HepG2 cells, resulting in 1.5 times higher secretion of albumin from HepG2 cells on the NCM on day 14 compared with those on a nanofibrous flat microwell (NFM) as a control.

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1. Introduction Three-dimensional (3D) cell spheroid cultures closely resemble the in vivo physiological microenvironment compared with the standard two-dimensional (2D) culture.1 In particular, 3D cell spheroid cultures provide a closely recapitulated microenvironment of in vivo tissue including 3D cell–cell interaction and physiological radial gradients of gas and nutrient,2 which makes 3D cell spheroids an excellent platform for tissue engineering,3 stem cell studies,4 cancer researches,5 and drug development.6-7 The common approaches for culturing a 3D cell spheroid in vitro are based on spinner flasks,8 hanging drops,9-12 and microwells.13-15 Although each method has its own advantages depending on the application, microwells have been highlighted due to their ability to produce well-organized 3D cell spheroids with good uniformity.13-15 Given recent advances in microfabrication techniques such as photo/soft-lithography,4,

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micromachining,5 and CO2 laser ablation,16 researchers can develop superior microwells with physiologically relevant size, shape, and curvature. These techniques are suitable for developing a precisely controlled 3D microscale environment, but it is generally challenging for them to achieve nanoscale topography. Given that cells in the human body are associated with complicated cell–cell and cell–nanostructured extracellular matrix (ECM) interaction, attempts have been made to implement nanoscale topographies in microwells to more closely recapitulate in vivo cellular microenvironment. Electrospinning, which is capable of producing an ECM mimetic nanofibrous environment, has attracted broad attention in biomedical applications including stem cell research17-19 and tissue regeneration.20-21 Electrospinning generally forms a 2D nonwoven nanofiber mat on a flat metal collector,22 and the 2D nanofiber mats have guided more favorable cellular responses than 2D flat surfaces.23-26 Interestingly, the 2D nanofiber mat has been known

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to guide the formation of 3D cell spheroids on the mat,27-28 but it is limited to controlling the size of the cell spheroids. Considering that the size of the 3D cell spheroid plays a critical role in its function,4,

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several studies have developed fabrication processes for a microwell-type

electrospun nanofiber mat to overcome such a problem.29-31 Constructing a physical barrier of polyethylene glycol (PEG) sidewalls on a 2D polystyrene (PS) nanofiber mat for a sizecontrolled PEG microwell produced hepatic multicellular spheroids with a precisely controlled size.29 However, the photolithography technique, which was utilized to build the PEG sidewalls, is limited to construct in vivo smooth, curved microenvironment that can effectively generate homogeneous spheroids.32 Template-assisted electrospinning with an array of stainless steel beads30, frozen water droplets31, and micropatterned photoresists33 could produce concaveshaped nanofibrous microwells, thereby forming embryonic stem cell spheroids, tumor cell spheroids, and artificial salivary gland, respectively. However, these methods have difficulty in controlling the size, shape and curvature of the nanofibrous concave microwells (NCMs) because imposing microscale smooth curvature to the metal-based collector required much time and cost, and frozen water droplets only produced circular microwells. A recent report suggested that the shape of the microwell influences the function of hepatic spheroids, given that hepatocytes in the human body form a distinct lobule that is typically hexagonal in cross-section.34 Similarly, many organs in vivo have their own distinctive microenvironment. Thus, it would be definitely valuable to develop a versatile method that can individually control the shape, size, and curvature of concave nanofibrous microwells. In this study, we developed a versatile fabrication process of an array of NCMs with controllable shape, size, and curvature via electrospinning. Recently, we reported a new type of electrospinning process that utilized an electrolyte solution as a grounded collector; this process

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is named electrolyte-assisted electrospinning (ELES).35 Here, we adopted the ELES process to produce NCMs and readily control their shape and size. An electrically connected array of electrolyte solution drops was introduced as a grounded collector for electrospun nanofibers to fabricate the array of NCMs. A polymer substrate with an array of through holes was fixed in a reservoir chamber such that an electrolyte solution was overfilled for the holes to form the electrically connected array of electrolyte solution drops. The convex shape and size of these drops were determined by the hole shape and size while their curvature was modulated by the internal pressure of the electrolyte solution in the chamber. The formation of uniform, sizecontrollable cell spheroids of HepG2 cells, human hepatoma cell line, was demonstrated on the NCMs. The metabolic functions of both HepG2 cell spheroids formed on the NCMs and nanofibrous flat microwells (NFMs), as a control, were compared by measuring albumin secretion levels per viable cells.

2. Materials and methods 2.1. Fabrication of nanofibrous concave microwells via ELES Figure 1 presents the schematic for the fabrication of NCMs. First, through holes were prepared in a 0.5 mm-thick poly(methyl methacrylate) (PMMA) substrate (Acryl Choika, Korea) by a micromachining machine (EGX-350, Roland, USA), and the micromachined PMMA substrate was fixed on a reservoir chamber connected to the ground. Four different types of circularshaped through holes with diameters of 250, 500, 750, and 1,000 µm with center-to-center distances of 1,000, 1,000, 1,500, and 2,000 µm, respectively, were fabricated, and also, two different shapes of equilateral rectangular- and triangular-shaped through holes with 1,000 µm sides and a center-to-center distance of 2,000 µm were produced. Second, the chamber was filled

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with 0.1 M potassium chloride (KCl) solution to form an electrically connected collector array of electrolyte solution drops. Third, the ELES process was conducted to obtain NCMs and NFMs. For the ELES process, we prepared 7.5% polycaprolactone (PCL, Mw = 80,000, Sigma–Aldrich) solution by dissolving PCL pellets in a mixture of methanol (Samcheon Chemicals, Korea) and chloroform (Sigma–Aldrich; 3:1 by volume) under magnetic stirring at 25 °C. The PCL solution was loaded in a 5 ml Hamilton syringe and fed at 0.5 ml h-1 by a syringe pump (KDS200, KD Scientific) through a 23-gauge metal needle, which was 20 cm apart from the collector. ELES was conducted by applying a high electrical potential of 19 kV between the metal needle and the KCl solution collector by a high voltage supplier (HV30, NanoNC, Korea) for 6 min at 40–50% relative humidity.

2.2. Numerical simulation A numerical simulation on an electric field generated between a metal needle and an array of 0.1 M KCl solution convex drops or an metal collector as a control was conducted by COMSOL software (Version 5.0).36 The convex drops of 0.1 M KCl solution coming out from the through holes were surrounded by a 0.5-mm thick PMMA (εr = 3.6) substrate and a copper ground. The KCL solution was directly connected to the copper ground. An electric potential of 19 kV was applied between the metal needle and copper ground with a metal-needle-to-collector distance of 20 cm.

2.3. Characterization of nanofibrous concave microwells The nanostructural morphology of NCMs was examined by field-emission scanning electron microscopy (FE-SEM; SU6600, Hitachi, Japan). The 3D concave shape of the nanofibrous

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microwells was visualized by confocal laser scanning microscopy (LSM 510 Meta, Carl Zeiss, Germany) after immersing the nanofibrous microwells in a solution with a small amount of Rhodamine 6G for 30 min and drying them. The arrays of triangular, rectangular, and circular microwells with various sizes were captured by a digital camera (EOS 550D, Canon, Japan).

2.4. Cell cultures HepG2 cells were purchased from KCLB (Korean Cell Line Bank, Korea). HepG2 cells were cultured in high glucose Dulbecco’s minimal essential media (Hyclone) with 10 % horse serum (Hyclone), 5% FBS (Hyclone), and 0.5% penicillin/streptomycin (Gibco) before seeding in a humidified incubator at 37 °C and 5% CO2. The nanofibrous microwells were sterilized in 70% ethanol (Merk) for 30 min, rinsed twice with 1× PBS (Hyclone), and then radiated with UV for 3 h prior to cell seeding. For cell spheroid formation, HepG2 cells were seeded in the nanofibrous microwells (2.5 × 103 cells/mm2). The nanofibrous microwells were rinsed with 1× PBS to removed unattached cells after 30 min. The cells were cultured until forming spheroids in the nanofibrous microwells for 7 days.

2.5. Characterization of HepG2 cell spheroids After 3 days of cell culture to form cell spheroids, the cells were washed with 1× PBS and treated with a solution of calcein AM (green) and ethidium homodimer-1 (red) to distinguish live cells from dead ones using a LIVE/DEAD Viability kit (Molecular probes). The cells were then incubated for 45 min at room temperature, and the fluorescent images were acquired using a phase contrast inverted fluorescence microscope (Nikon TS100F). The quantitative data of the

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sizes of cell spheroids in the NCMs were obtained by Image J (http://rsbweb.nih.gov/ij/) with more than 20 cell spheroids for each nanofiber platform. HepG2 cell spheroids were fixed with 2.5% glutaraldehyde for 1 h, and then washed 2 times with 1× PBS. The fixed HepG2 cell spheroids were dehydrated with a series of graded ethanol (30%, 50%, 70%, 90%, and 100%). After dehydration, the HepG2 cell spheroids were dried in a freeze-dryer overnight. The morphology of the HepG2 cell spheroids on the nanofiberous microwells were examined by FE-SEM. Albumin secretion of HepG2 cells was evaluated by dividing the concentration of albumin in cell culture supernatant of HepG2 cell spheroids by the number of viable cells. Cell culture supernatant was collected after culturing for 7 and 14 days and preserved at -80 ℃. Albumin in cell culture supernatant was measured using a human albumin ELISA kit (Bethyl laboratories. Inc.) according to the manufacturer’s instructions. The number of viable cells was estimated at each time point using a Cell Counting Kit-8 (CCK, Dojindo, Japan) according to the manufacturer's instructions.

3. Results and discussion 3.1. Fabrication of nanofibrous concave microwells by ELES Our group recently suggested an electrolyte solution in the electrospinning process as a grounded collector to produce a complicated architecture of electrospun nanofibers.35, 37 The fluidic nature of electrolyte solution enabled facile fabrication and integration of a free-standing nanofiber membrane with various polymer platforms including a microfluidic device35 and well insert.38 By taking advantages of its fluidic nature, we newly applied the electrically connected convex

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menisci of electrolyte solution as a grounded collector on the PMMA substrate followed by fabricating 3D curved PCL nanofiber membranes by ELES to realize the NCMs. Figure 1 shows the sequential steps to fabricate NCMs based on the ELES process on the array of convex electrolyte solution drops. First, an array of through holes with various orifice shapes was produced in a PMMA substrate by a micromachining process (Figure 1a). In this study, we utilized CNC micromachining because of its moderate resolution of 100 µm. Other cost-effective or precise microfabrication techniques, such as laser micromachining and soft lithography, could also be adopted to generate the through holes in the substrate. Subsequently, the micromachined PMMA substrate was tightly fixed on the top of a reservoir chamber and sealed by a mechanical clamping system (Figure S1). When the chamber was filled with the electrolyte solution to overflow, the overflowed electrolyte solution came out from the through holes in the top surface of substrate and naturally formed an array of convex drops on the substrate due to surface tension as shown in Figure 1b. Finally, ELES was performed by applying the electrical potential of 19 kV between the metal needle and electrolyte solution to produce nanofibrous microwells directly on the convex menisci of the electrolyte solution drops. After depositing PCL nanofibers on both the electrolyte solution and the substrate, the electrolyte solution was simply removed by disassembling the PMMA substrate with the fabricated nanofibrous microwells from the chamber. When we observed the bottom surface of the substrate by turning it upside down, we could confirm the concave shape of the nanofiber microwells (Figure 1c). Thus, we utilized the inverted PMMA substrate with the NCMs for cell spheroid formation. The curvature of the convex electrolyte solution drops was controlled by the internal pressure of the chamber, which was manipulated by a height-adjustable bath filled with electrolyte solution connected to the chamber as shown in Figure 2. The change in segment angle

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of the convex electrolyte solution drop from 30 to 90° in accordance with the alteration in the bath height is presented in Figure 2a–c. The segment angle between the free surface of convex electrolyte solution drop and the substrate formed by the Young–Laplace pressure in response to the change in height of the bath could be approximately estimated as follows:



 = 



,

(1)

where  is the segment angle between the free surface of convex electrolyte solution drop and the top surface of substrate;  is the density of the electrolyte solution;  is the gravitational acceleration;  is the diameter of the through-hole; ℎ is the height difference between the free surface of electrolyte solution in the bath and substrate; and  is the surface tension of the electrolyte solution. The convex electrolyte solution drop could be approximated to a segment of the hemisphere as shown in Figure 2d. This approximation would be valid when the Bond number, which is the ratio of the gravity to the surface tension, has a low value (Bo ≪ 1). For the case of a 500-µm microwell, the Bond number is 3.5 × 10-5, and thus, the approximation of the segment of the hemisphere is valid. Thus, the radius of curvature (!) could be presented as follow: ! = /(2  ()),

(2)

And, the relationship between the angle () and the radius () of the drop could be presented as follow: (&) = '(! ( − (& + ! + )( ),

(3)

where  is the radius of the convex electrolyte solution drop, and & is the height above the substrate. Equation (2) suggested that the lower the angle is, the curvature of the electrolyte solution drop increases as shown in Figure 2a–c. When the angle become zero, the curvature of the electrolyte solution drop become infinite, which implies the flat surface of the electrolyte

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solution collector. Thus, by adjusting the angle to zero, we achieved the array of flat electrolyte solution collectors for the fabrication of NFMs, as a control for the NCMs. And, the shape of the electrolyte solution drop would be estimated by the Equation (3), which provided the design guideline for the NCMs. Figures S2 shows typical confocal images of the circular NCM with a 3D curved nanofiber membrane. These confocal images confirmed that the fabricated nanofibrous microwell exhibited a circular concave shape, which could confine cells to form an aggregate. Many researchers attempted to develop a fabrication method for concave microwells as an alternative to conventional cylindrical microwells which lack 3D physiological curvature of in vivo microenvironment (e.g. embryonic body).4-5,

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Our fabrication method based on the

ELES allowed to form an ECM mimetic nanofibrous environment through the NCM, recapitulating not only the nanofibrous environment but also the contour of the in vivo microenvironment. Notably, the shape, size, and interspacing of the array of NCMs could be individually controlled by changing the orifice shape and configuration of the through holes in the polymer substrate. Figure 3 shows the various arrays of PCL NCMs that were fabricated by ELES using electrically connected convex menisci of 0.1 M KCl solution collectors. We demonstrated that the size of the microwell could vary from 1,000 µm down to 250 µm (Figure 3a). We expected that even smaller or larger microwells compared with the present demonstration could be produced based on precisely prepared through holes in the polymer substrate. Furthermore, given that the electrolyte solution drops were internally connected, when we positioned an array of the same-sized through holes in the same height, an array of electrolyte solution drops in the through holes would have the same internal pressure. Thus, we can simultaneously control the curvature of the large quantity of electrolyte solution drops by adjusting the internal pressure. With

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subsequent ELES process, these electrolyte solution drops allowed to produce a number of microwells with the same shape (Figure S3), which would be utilized for high throughput formation of cell spheroids. Figure 3b shows the fabricated arrays of NCMs with different shapes of microwell including circle, square, and triangle. Given that the shape of the microwell could influence the 3D morphology and function of cell spheroids, such as hepatocytes, the controllability in the shape and configuration of the NCMs would be powerful to recapitulate in vivo cellular microenvironments.

3.2. Mechanism of fabrication of nanofibrous concave microwells Figure 4a shows a computed drawing of electric field vectors between the metal needle and the collector system. In this study, we prepared two types of collector systems of an array of circular convex drops of 0.1 M KCl solution and a metal plate. We have previously demonstrated that the electrolyte solution was capable of concentrating the electric field with the same magnitude as a metal collector during electrospinning.36 Likewise, the electric field vectors above the through holes with the KCl solution drops pointed directly toward the surface of KCl solution as shown in Figure 4b. The focused electric field toward the KCl solution drops attracted as-spun nanofibers to the surface of KCL solution. To calculate the electric field in the absence of an electrolyte solution collector, the copper ground was placed below the PMMA substrate while the electrolyte solution was removed. When a metal collector was placed on the bottom of a PMMA substrate without electrolyte solution, the electric field was not concentrated toward the through holes, but toward the PMMA substrate as shown in Figure 4c. This phenomenon was attributed to a higher dielectric constant of the PMMA substrate compared to air filled in the

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through hole, given that, for the case of dielectric materials, the material with a higher dielectric constant was known to better concentrate the electric field.39 Figure 5a shows SEM images of a NCM produced by ELES process. On the top surface of the PMMA substrate, a convex shape of a nanofibrous microwell was shown at the center, which replicated the curvature of a convex electrolyte solution drop, and aligned nanofibers toward the outside of the nanofibrous microwells were also observed (Figure 5a-(i)). As shown in the typical SEM images of the array of circular NCMs (Figure S4), these aligned nanofibers were positioned between nanofibrous microwells, creating the interconnections between them. However, these aligned nanofibers did not influence the formation of cell spheroid, and thus, we presented the detailed explanation of the formation of these aligned nanofibers in the Supporting Information. Figure 5a-(ii), which is an upside-down image of Figure 5a-(i), shows the bottom surface of the PMMA substrate with a NCM. Randomly-oriented nanofibers of the NCM were observed inside the through hole of the PMMA substrate, which was to be used in cell culture, and in contrast, no nanofiber was observed on the bottom surface of PMMA substrate (Figure 5a(ii), (iii)). For the case of a metal collector without an electrolyte solution, electrospinning was conducted under the same conditions as ELES except for an electrospinning time of 30 min. Figure 5b shows the nanofibers formed on the PMMA substrate with the through-holes by electrospinning with a metal collector without electrolyte solution. As the numerical simulation demonstrated that the electric field was focused on the top surface of PMMA substrate, not within the holes, most of as-spun nanofibers were attracted toward the PMMA substrate, while a small number of nanofibers were located at the through holes (Figure 5b-(i–iii)). Though electrospinning with the metal collector without electrolyte solution was performed for 30 min, which was 5 times more than the present ELES process, the number of deposited nanofibers at

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the through hole was much fewer compared with the ELES due to its low capability to concentrate the electric field. These results implied that the ELES process could dramatically increase the productivity to fabricate the nanofibrous microwells. Like the electrolyte solution, various templates, such as frozen water droplets31, micropatterned photoresists33, and stainless steel beads30, were also utilized to produce the NCMs. However, the frozen water droplets and micropatterned photoresists less effectively attracted the electric field because they are not electrically conductive, and thus, the deposition efficiency of electrospun nanofibers on the template was greatly reduced compared to an electroconductive template such as electrolyte solution or stainless steel beads. In view of controlling the size, shape and curvature of the NCMs, the utilization of the electroconductive stainless steel beads as a collector also has difficulty in modulating the dimension of the template. In this regard, the electrolyte solution would be an effective template to form the NCMs through electrospinning.

3.3. Formation of HepG2 cell spheroids on nanofiber concave microwells The main purpose of utilizing NCMs was the formation of 3D cell spheroids by physically confining cells within the nanofibrous microwells mimicking in vivo microenvironment. Hepatocyte spheroids have great potential in various biomedical applications including liver regeneration and development of artificial liver, and in vitro model for physiological study or drug assessment.32, 40 Here, we demonstrated the potential of the NCMs to promote the formation of uniform, size-controllable HepG2 cell spheroids with improved metabolic function. Figure 6a shows a typical confocal image of a HepG2 cell spheroid formed on the circular NCM of 500-um diameter, indicating successful confinement of the cells within the nanofibrous microwell to make an aggregate. To investigate the influence of the microwell size of the NCM on the

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formation of HepG2 cell spheroids, we fabricated four types of NCMs with different diameters of 250, 500, 750, and 1,000 µm. Figure 6b shows various sizes of cell spheroids formed on the four samples, suggesting that the cells were successfully confined within the NCMs and the size of HepG2 cell spheroids could be modulated by the size of NCM. We also measured the sizes of cell spheroids generated on the four samples and found that the HepG2 cell spheroids with a uniform size distribution were formed on the all NCMs (Figure 6c). Furthermore, we confirmed that the NCMs improved the formation of uniform-sized tumor spheroids composed of PC12 rat pheochromocytoma cells (Figure S6). We observed the formation of PC12 cell spheroids on a 2D flat nanofiber mat, but the sizes of the PC12 cell spheroids varied considerably. On the NCMs, uniform-sized PC12 cells spheroids were formed within the individual microwells. Such promoted formation of the uniform cell spheroids could be explained by the confinement of cells in the NCMs which was achieved by not only the structure of nanofiber membrane of the NCM but also the through-hole structure of the NCM in the PMMA substrate that supported the nanofiber membrane (Figure 5a-(ii)). The concaveness of the nanofiber membrane made cells difficult to escape from the NCMs. In addition, the through holes of 500-µm height in the PMMA substrate also played a critical role as a physical barrier to prevent the cell migration out from the NCMs. We further investigated the metabolic function of the HepG2 cell spheroids formed on the NCMs. As a control for the NCMs, NFMs that replaced the concave nanofiber membrane of the NCM to a flat nanofibrous membrane was prepared. To precisely compare the albumin secretion per viable cells in the NCMs and NFMs, we prepared 1-mm diameter NCMs and NFMs, and a 1 µl suspension with HepG2 cells (2.0 × 103 cells/well) was manually added. Figure 7a schematically illustrates the cell aggregation within the NCMs and NFMs. The

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curvature of the concave membrane would facilitate HepG2 cells to be collected at the center of the microwell (Figure 7a-(i)), whereas the HepG2 on the flat membrane would prefer to be seeded across the whole surface of the microwell (Figure 7a-(ii)). The cell culture results in Figure 7b exhibited that the HepG2 cells on the NCM were aggregated at the center of the microwell to form a well-organized spheroid, while the HepG2 cells on the NFM were spread out over the area of the microwell, which reduced the chance of forming a cell spheroid. Furthermore, the metabolic functions of both HepG2 cell spheroids formed on the NCMs and NFMs were evaluated, given that the formation of HepG2 cell spheroid is known to enhance the secretion of albumin.41 Notably, the measurement of the albumin levels from the HepG2 cells indicated that the HepG2 cells on the NCMs secreted about 1.5 times higher amounts of albumin compared with those on the NFMs (Figure 7c). These results indirectly showed a positive effect of the NCM on HepG2 cell spheroid formation, demonstrating that the NCMs more effectively generated cell spheroids compared with the NFMs. The application of the nanofibrous microwells is not restricted to generating cell spheroids. Xie et al. demonstrated that interconnected nanofibrous microwells can produce neural networks30. In addition, Ma et al. showed that square-arrayed microwells can enhance cell migration, thereby accelerating skin regeneration.42 Given that our fabrication technique is versatile to fabricate nanofibrous microwells with controllable size, shape and curvature, it would also broaden the potential of the nanofibrous microwells in the biomedical fields.

4. Conclusion In this study, we present a versatile fabrication process for the nanofibrous concave microwells (NCMs) with controllable structural cues via electrolyte-assisted electrospinning on an array of

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hemispherical convex electrolyte solution collectors. We demonstrated the versatility of this process by fabricating the NCMs with various shapes and sizes. Uniform, size-controllable HepG2 cell spheroids could be successfully formed in the NCMs, showing an improved metabolic function. Given that our fabrication process would provide a powerful tool to closely mimic in vivo microenvironment, it paves the way for developing an effective platform in the biomedical fields, which is not possible with conventional culture plates.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Schematic of a mechanical clamping system and a height adjustable system; confocal images of nanofibrous concave microwell; nanofibrous concave microwells on the PMMA substrate; SEM images of the array of circular nanofibrous concave microwells; and formation of PC12 cell spheroids on nanofibrous concave microwells (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (D.S.K.).

ACKNOWLEDGMENT

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This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP). (No. 2017R1A2A1A05001090, 2011-0030075)

Notes The authors declare no competing financial interest.

ABBREVIATIONS ELES, electrolyte-assisted electrospinning; PMMA, poly(methyl methacrylate); NCM, nanofibrous concave microwell; NFM, nanofibrous flat microwell

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Figure 1. Schematic for the fabrication process of NCMs based on ELES, which is a sequential process of preparing a PMMA substrate with through holes, placing it on a chamber (a), performing ELES on an array of electrolyte solution in a 3D convex shape (b), and obtaining NCMs by removing electrolyte solution (c).

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Figure 2. The convex electrolyte solution drop with different contact angles of 30 (a), 60 (b), and 90° (c). (d) The schematic of the convex electrolyte solution drop on a polymer substrate. All scale bars are 500 µm.

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Figure 3. Photographic images of the polymer substrate with micro-holes and NCMs with various sizes of 250, 500, and 750 µm (a) and shapes including circular, rectangular and triangular (b). All scale bars are 1 mm.

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Figure 4. (a) Numerical simulation of electric field vectors between the metal collector and array of electrolyte solution in a convex shape with the electrical potential of 19 kV. A magnified image of the collector system with an electrolyte solution (b) and with a metal (c) presenting electric field vectors.

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Figure 5. SEM images of nanofibrous microwells produced by electrospinning with an electrolyte solution collector (a) and a metal collector (b) (Top view: (i), Bottom view: (ii), and magnified view: (iii)). Scale bars are 200 µm (a-(i, ii), b-(i, ii)) and 10 µm (a-(iii), b-(iii)).

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Figure 6. (a) Confocal image of a HepG2 cell spheroid (b, c) Live/dead images (b) and size distribution analysis (c) of HepG2 cell spheroid culture on 250, 500, 750, and 1000 µm-diameter NCMs. Statistical significance: ***p < 0.001. Scale bars are 100 µm (a) and 500 µm (b).

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Figure 7. (a) Schematic illustration of HepG2 cell spheroid on NCMs (a-i) and NFMs (a-ii) (b) SEM images of HepG2 cell spheroid on NCMs and NFMs (c) Albumin secretion per viable cells at day 7 and 14 on NCM and NFM. The albumin secretion per viable cell was calculated relative to that on the NCM. Statistical significance: ***p < 0.001. All scale bars are 100 µm.

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BRIEFS (Table of Contents)

We presented versatile fabrication process of nanofibrous concave microwells for HepG2 cell spheroid formation with improved metabolic function.

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