NanoSurface Cultureware User Manual

Download the NanoSurface Cultureware User Manual for protocol information and advice on how to achieve the best results with NanoSurface dishes and plates. Protocol guidelines are provided for several focus application areas. For additional information or advice with cell types not covered by this User Manual, please contact



+ 1. Guided Cell Migration on Microtextured Substrates with Variable Local Density and Anisotropy

Kim DH, Seo CH, Han K, Kwon KW, Levchenko A (1), Suh KY (2).

(1) Johns Hopkins University, (2) Seoul National University

Adv Funct Mater., 19(10): 1579–1586(2009)

Link To Paper

Abstract: This work reports the design of and experimentation with a topographically patterned cell culture substrate of variable local density and anisotropy as a facile and efficient platform to guide the organization and migration of cells in spatially desirable patterns. Using UV-assisted capillary force lithography, an optically transparent microstructured layer of a UV curable poly(urethane acrylate) resin is fabricated and employed as a cell-culture substrate after coating with fibronectin. With variable local pattern density and anisotropy present in a single cell-culture substrate, the differential polarization of cell morphology and movement in a single experiment is quantitatively characterized. It is found that cell shape and velocity are exquisitely sensitive to variation in the local anisotropy of the two-dimensional rectangular lattice arrays, with cell elongation and speed decreasing on symmetric lattice patterns. It is also found that cells could integrate orthogonal spatial cues when determining the direction of cell orientation and movement. Furthermore, cells preferentially migrate toward the topographically denser areas from sparser ones. Consistent with these results, it is demonstrated that systematic variation of local densities of rectangular lattice arrays enable a planar assembly of cells into a specified location. It is envisioned that lithographically defined substrates of variable local density and anisotropy not only provide a new route to tailoring the cell-material interface but could serve as a template for advanced tissue engineering.


Materials & Methods: Silicon wafers were spin-coated with photoresist (Shipley, Marlborough, MA) and then patterned via electron-beam lithography (JBX-9300FS, JEOL). After photoresist development (MF320, Shipley), exposed silicon was deep reactive ion etched (STS ICP Etcher) to form arrays of submicrometer-scale ridges with near-vertical sidewalls. The remaining photoresist on silicon wafers was removed using an ashing process (BMR ICP PR Asher) and then diced into silicon masters for subsequent replica molding. To cast topographic micro- and nanopattern arrays, PUA was used as a mold material from the silicon master as previously described [20]. Briefly, the UV-curable PUA was drop-dispensed onto a silicon master and then a flexible and transparent polyethylene terephthalate (PET) film was brought into contact with the dropped PUA liquid. Subsequently, it was exposed to UV light (λ = 200–400 nm) for 30 s through the transparent backplane (dose = 100 mJ cm−2). After UV curing, the mold was peeled off from the master and additionally cured overnight to terminate the remaining active acrylate groups on the surface prior to use as a first replica. The resulting PUA mold used in the experiment was a thin sheet with a thickness of ≈50 μm.

Microscopic Technique: Time-Lapse Microscopy

Cell Type(s): NIH 3T3

+ 2. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients

Kim DH, Han K, Gupta K, Kwon KW, Suh KY (1), Levchenko A (2)

(1) Seoul National University, (2) Johns Hopkins University

Biomaterials, 30(29):5433-44(2009)

Link To Paper

Abstract: In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell-substratum interactions. A single cell adhesion substratum with the constant ridge width (1 microm), and depth (400 nm) and variable groove widths (1-9.1 microm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term), suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of adhesive cell-cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell-matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell-material interface.

Keywords: Topography, Capillary force lithography, Grooves and ridges, Focal adhesions, Extracellular matrix, Cell migration, Fibroblast cells

Materials & Methods: Silicon wafers were spin-coated with a photoresist (Shipley, Marlborough, MA) and then patterned via electron-beam lithography (JBX-9300FS, JEOL). After photoresist development (MF320, Shipley), exposed silicon was deep reactive ion etched (STS ICP Etcher) to form arrays of sub-micron scale ridges with near-vertical sidewalls. The remaining photoresist on silicon wafers was removed using ashing process (BMR ICP PR Asher) and then diced into silicon masters for subsequent replica-molding. To fabricate topographic nanopattern arrays, PUA was used as a mold material from the silicon master as previously described [25]. Briefly, the UV-curable PUA was drop-dispensed onto a silicon master and then a flexible and transparent polyethylene terephthalate (PET) film was brought into contact with the dropped PUA liquid. Subsequently, it was exposed to UV light (λ= 200–400 nm) for 30 s through the transparent backplane (dose = 100 mJ cm−2). After UV curing, the mold was peeled off from the master and additionally cured overnight to terminate the remaining active acrylate groups on the surface prior to the use as a first replica. The resulting PUA mold used in the experiment was a thin sheet with a thickness of ∼50 µm.

Microscopic Technique: Time-Lapse Microscopy

Cell Type(s): NIH 3T3

+ 3. Synergistically enhanced osteogenic differentiation of human mesenchymal stem cells by culture on nanostructured surfaces with induction media

You MH, Kwak MK, Kim DH, Kim K, Levchenko A, Kim DY (1), Suh KY (1)

(1) Seoul National University

Biomacromolecules, 12;11(7):1856-62(2010)

Link To Paper

Abstract: We have examined the effects of surface nanotopography on in vitro osteogenesis of human mesenchymal stem cells (hMSCs). UV-assisted capillary force lithography was employed to fabricate a scalable (4x5 cm), well-defined nanostructured substrate of a UV curable polyurethane polymer with dots (150, 400, 600 nm diameter) and lines (150, 400, 600 nm width). The influence of osteogenic differentiation of hMSCs was characterized at day 8 by alkaline phosphatase (ALP) assay, RT-PCR, and real-time PCR analysis. We found that hMSCs cultured on the nanostructured surfaces in osteogenic induction media showed significantly higher ALP activity compared to unpatterned PUA surface (control group). In particular, the hMSCs on the 400 nm dot pattern showed the highest level of ALP activity. Further investigation with real-time quantitative RT-PCR analysis demonstrated significantly higher expression of core binding factor 1 (Cbfa1), osteopontin (OP), and osteocalcin (OC) levels in hMSCs cultured on the 400 nm dot pattern in osteogenic induction media. These findings suggest that surface nanotopography can enhance osteogenic differentiation synergistically with biochemical induction substance.


Materials & Methods: A small amount (∼0.1 – 0.5 mL) of a UV curable PUA prepolymer was drop-dispensed on silicon master having positive patterns (features sticking out) and a supporting poly(ethylene terephthalate) (PET) film was carefully placed on top of the surface to make conformal contact. The PET film used in this study was surface modified with urethane groups to increase adhesion to the acrylate-containing monomer (Minuta Tech., Korea). The silicon masters had been prepared by photolithography or electron-beam lithography. To cure, the resin was exposed to UV (wavelength: 250 ∼ 400 nm) for 17 s at an intensity of 100 mW/cm2, and the cured 1st replica was peeled off from the master using a sharp tweezer. The 1st replica was additionally exposed to UV overnight to remove any uncured active groups on the surface. The 2nd replica was prepared by using a capillary molding process on glass coverslips with the over-cured 1st PUA as a mold, resulting in the same pattern as the silicon master. After curing, the 1st replica was removed from the surface using a sharp tweezers. The fabricated PUA nanopatterns were sterilized by rinsing with IPA (isopropyl alcohol) and D.I. water, and coated with 0.1 % gelatin for 1 hr at room temperature prior to cell culture. A schematic diagram of the fabrication procedure is illustrated in Figure 1 along with the self-replication characteristic of the PUA material.

Microscopic Technique: Atomic Force Microscopy

Cell Type(s): hMSCs

+ 4. Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs

Kim DH, Lipke EA, Kim P, Cheong R, Thompson S, Delannoy M, Suh KY (1), Tung L (2), Levchenko A (2)

(1) Seoul National University, (2) Johns Hopkins University

Proc Natl Acad Sci U S A, 107(2):565-70(2010)

Link To Paper

Abstract: Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell-cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell-material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

Keywords: action potential, cardiomyocytes, extracellular matrix, nanotopography, tissue engineering

Materials & Methods: Nanopatterned substrata of PEG hydrogels were fabricated by using UV-assisted capillary lithography-based nanomolding techniques as previously described (20). NRVMs were isolated as previously described (13) and then cultured on the nanopatterned PEG substratum to form the confluent monolayer. Optical mapping, contraction mapping, Western blot, SEM/TEM analysis, and immunostaining experiments were performed 6–7 days after plating. Quantitative analysis of conduction velocity and contraction was performed by using custom-written MATLAB scripts. Details are described in the SI Text.

Microscopic Technique: Scanning electron microscopy, Transmission electron microscopy

Cell Type(s): NRVMs

+ 5. Regulation of Brain Tumor Dispersal by NKCC1 Through a Novel Role in Focal Adhesion Regulation

T. Garzon-Muvdi, C. Aprhys, C. Smith, D.H. Kim, L. Kone, S.H. Farber, S. An, A. Levchenko (1), and A. Quinones-Hinojosa (1)

(1) Johns Hopkins University

PLoS Biology, vol. 10, pp. e1001320, 2012.

Link To Paper

Abstract: Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na+-K+-Cl− cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.


Materials & Methods: Migration of glioma cells was quantified using a novel directional migration assay using nano-ridges/grooves constructed of transparent poly(urethane acrylate) (PUA), and fabricated using UV-assisted capillary lithography (see Figure S3A–C) [94]. Nanopattern surfaces were coated with laminin (3 µg/cm2). Cell migration was quantified using time lapse microscopy (Video S3). Long-term observation was done on a motorized inverted microscope (Olympus IX81) equipped with a Cascade 512B II CCD camera and temperature and gas controlling environmental chamber. Phase-contrast and epi-fluorescent cell images were automatically recorded under 10× objective (NA = 0.30) using the Slidebook 4.1 (Intelligent Imaging Innovations, Denver, CO) for 15 h at 10–20-min intervals.

Microscopic Technique: Time-Lapse Microscopy

Cell Type(s):

+ 6. Quantitative Analysis of the Combined Effect of Substrate Rigidity and Topographic Guidance on Cell Morphology

JinSeok Park, Hong-Nam Kim, Deok-Ho Kim, Levchenko, A. (1), Kahp-Yang Suh (2)

(1) Johns Hopkins University, (2) Seoul National University,


Link To Paper

Abstract: Live cells are exquisitely sensitive to both the substratum rigidity and texture. To explore cell responses to both these types of inputs in a precisely controlled fashion, we analyzed the responses of Chinese hamster ovary (CHO) cells to nanotopographically defined substrata of different rigidities, ranging from 1.8 MPa to 1.1 GPa. Parallel arrays of nanogrooves (800-nm width, 800-nm space, and 800-nm depth) on polyurethane (PU)-based material surfaces were fabricated by UV-assisted capillary force lithography (CFL) over an area of 5 mm x 3 mm. We observed dramatic morphological responses of CHO cells, evident in their elongation and polarization along the nanogrooves direction. The cells were progressively more spread and elongated as the substratum rigidity increased, in an integrin beta 1 dependent manner. However, the degree of orientation was independent of substratum rigidity, suggesting that the cell shape is primarily determined by the topographical cues.

Keywords: Cell adhesion, Chinese Hamster Ovary (CHO) cells, microenvironment, nanofabrication, nanogrooves, substrate rigidity, topographical cues.

Materials & Methods: To prepare PUA molds, a small amount (0.1–0.5 mL) of polyurethane acrylate (PUA) prepolymer was drop-dispensed onto silicon master that had been prepared by photolithography. Then, a poly(ethylene terephthalate) (PET) film of Formula thickness was placed on the liquid prepolymer, followed by UV exposure Formula for a few tens of seconds. After the UV curing, the mold was peeled from the master, and fully cured by exposing UV for 10 h to render the surface inactive during subsequent pattern replication steps. Next, a similar amount of each PU material was drop-dispensed onto circular 25 mm cover glass and a PUA mold with preformed nanogrooves was placed carefully to make a uniform contact, leaving behind a replica of nanogrooves after UV exposure for a few tens of seconds followed by mold removal. Each prepolymer was obtained as follows: PU elastomer and intermediate PU (MINS 311 RM) were purchased from Minuta Tech. (Korea). Hard PU (NOA83 H) was purchased from Norland Optical Adhesive Inc. (NY, USA). The detailed information of soft and intermediate PU materials can be found elsewhere [38], [39]. To promote adhesion of the patterned layer, the glass coverslip was treated with an adhesion promoter (phosphoric acrylate or acrylic acid dissolved in propylene glycol monomethyl ether acetate (PGMEA), 10 vol%). Contact angles of water on various substrata were measured by a contact angle analyzer (DSA 100, Krüss, Germany). Data were averaged over at least 10 locations.

Microscopic Technique: Scanning Electron Microscopy, Atomic Force Microsope

Cell Type(s): CHO

+ 7. Matrix nanotopography as a regulator of cell function

Deok-Ho Kim (1), Paolo P. Provenzano, Chris L. Smith, and Andre Levchenko

(1) University of Washington

JCB, 197(3), 351-360(2012)

Link To Paper

Abstract: The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.


Materials & Methods: Refer to paper.

Microscopic Technique: Scanning Electron Microscopy

Cell Type(s): hMSCs, NIH 3T3 and CHO

+ 8. Nanopatterned cardiac cell patches promote stem cell niche formation and myocardial regeneration

Deok-Ho Kim (1),  Kshitiz,  Rachel R. Smith,  Pilnam Kim,  Eun Hyun Ahn,  Hong-Nam Kim,  Eduardo Marbán,  Kahp-Yang Suh and  Andre Levchenko

(1) University of Washington

Integr. Biol., 4, 1019-1033(2012)

Link To Paper

Abstract: Stem cell-based methods for myocardial regeneration suffer from considerable cell attrition. Artificial matrices reproducing mechanical and structural properties of the native tissue may facilitate survival, retention and functional integration of adult stem or progenitor cells, by conditioning the cells prior to, and during, transplantation. Here we combined autologous cardiosphere-derived cells (CDCs) with nanotopographically defined hydrogels mimicking the native myocardial matrix, to form in vitro cardiac stem cell niches, and control cell function and fate. These platforms were used to produce cardiac patches that could be transplanted at the site of infarct. In culture, highly anisotropic, but not more randomized nanotopographic, control augmented cell adhesion, migration, and proliferation. It also dramatically enhanced early, and, in the presence of mature cardiomyocytes, late cardiomyogenesis. Nanotopography sensing and transcriptional response was mediated via p190RhoGAP. In a rat infarction model, engraftment of nanofabricated scaffolds with CDCs enhanced retention and growth of transplanted cells, and their integration with the host tissue. The infarcted ventricle wall increased in thickness, with higher cell viability and better collagen organization. These results suggest that nanostructured polymeric materials that closely mimic the extracellular matrix structure on which cardiac cells reside in vivo can be both very effective tools in investigating the mechanisms of cardiac differentiation and the basis for cardiac tissue engineering, thus facilitating stem cell-based therapy in the heart.


Materials & Methods: The ultraviolet (UV)-curable poly(urethane acrylate) (PUA) mold material consists of a functionalized precursor with an acrylate group for cross-linking, a monomeric modulator, a photoinitiator and a radiation-curable releasing agent for surface activity.42 To fabricate a sheet-type mold, the liquid mixture was drop-dispensed onto a silicon master pattern and then a flexible, transparent polyethylene terephthalate (PET) film was brought into contact with the liquid mixture. Subsequently, it was exposed to UV light (l = 200–400 nm) for 20 sec. through the transparent backplane (dose = 100 mJ cm2). After UV curing, the mold was peeled off from the master pattern and additionally cured overnight to terminate the remaining active acrylate groups on the surface. The resulting PUA mold used in the experiment was a thin sheet with a thickness of B50 mm. The anisotropic nano-fabricated substratum (ANFS) made of PEG hydrogel was fabricated on a glass cover slip using UV-assisted capillary force lithography (CFL).19 Prior to application of the PUA mold, the glass substratum was thoroughly rinsed with ethanol to remove excess organic molecules and dried in a stream of nitrogen. The glass coverslips were first rinsed with isopropyl alcohol (IPA) in an ultrasonic bath for 30 min. and dried in a stream of nitrogen. To increase the adhesion at the PEG-DA nanostructure/glass interface and prevent the nanopatterned PEG hydrogels from peeling off, an adhesion promoter (volume ratio of phosphoric acrylate: propylene glycol monomethyl ether acetate = 1 : 10) was first spin-coated onto the glass coverslips to form a thin layer(B200nm) prior to dispensing of the precursor solution. Then a small amount (B0.1–0.5 mL) of PEG-DA (M.W. = 575) precursor was drop-dispensed onto the glass coverslips, and a PUA mold was directly placed onto the surface. The PEG-DA precursor was spontaneously drawn into the cavity of the mold by means of capillary action and was cured by exposure to UV for B30 s. The elastic modulus of the cured PEG nanostructure was B70 MPa, providing a compliant environment for cell cultures (cf. B90 GPa for glass). After curing, the mold was peeled from the substratum using a sharp tweezers.

Microscopic Technique: Scanning Electron Microscope, Transmission Electron Microscope

Cell Type(s): NRVMs

+ 9. Nanotopography-guided tissue engineering and regenerative medicine

Hong Nam Kim, Alex Jiao, Nathaniel S. Hwang, Min Sung Kim, Do Hyun Kang, Deok-Ho Kim, Kahp-Yang Suh (1)

(1) Seoul National University

Advanced Drug Delivery Reviews ,65(4), 536–558(2013)

Link To Paper

Abstract: Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end.

Keywords: Nanotopography, Tissue engineering, Regenerative medicine, Biomaterials, Cell–material interface

Materials & Methods: The controlled microenvironment is intended for elucidating or guiding the expression of cellular functions. To address this controllability, a great number of fabrication methods have been introduced in the last few decades. Since many reviews are currently available for numerous fabrication methods [42], [43], [44], [45], [46], [47], [48] and [49], we will focus on the methods that deal with synthetic polymers and their related fabrication techniques. Some fabrication methods described in this section are still valid for the structuring of natural polymers.

Microscopic Technique: Atomic Force Microscopy, Scanning Electron Microscope

Cell Type(s): Endothelial cells

+ 10. Synergistic Effects of Matrix Nanotopography and Stiffness on Vascular Smooth Muscle Cell Function

Somali Chaterji, PhD, Peter Kim, BS, Seung H. Choe, Jonathan H. Tsui, MS, Christoffer H. Lam, Derek S. Ho, BS, Aaron B. Baker, PhD, and Deok-Ho Kim, PhD (1)

(1) University of Washington

Tissue Engineering Part A, 20(15-16)2014

Link To Paper

Abstract: Vascular smooth muscle cells (vSMCs) retain the ability to undergo modulation in their phenotypic continuum, ranging from a mature contractile state to a proliferative, secretory state. vSMC differentiation is modulated by a complex array of microenvironmental cues, which include the biochemical milieu of the cells and the architecture and stiffness of the extracellular matrix. In this study, we demonstrate that by using UV-assisted capillary force lithography (CFL) to engineer a polyurethane substratum of defined nanotopography and stiffness, we can facilitate the differentiation of cultured vSMCs, reduce their inflammatory signature, and potentially promote the optimal functioning of the vSMC contractile and cytoskeletal machinery. Specifically, we found that the combination of medial tissue-like stiffness (11 MPa) and anisotropic nanotopography (ridge widthgroove widthridge height of 800800600 nm) resulted in significant upregulation of calponin, desmin, and smoothelin, in addition to the downregulation of intercellular adhesion molecule-1, tissue factor, interleukin-6, and monocyte chemoattractant protein-1. Further, our results allude to the mechanistic role of the RhoA/ROCK pathway and caveolin-1 in altered cellular mechanotransduction pathways via differential matrix nanotopography and stiffness. Notably, the nanopatterning of the stiffer substrata (1.1 GPa) resulted in the significant upregulation of RhoA, ROCK1, and ROCK2. This indicates that nanopatterning an 800800600 nm pattern on a stiff substratum may trigger the mechanical plasticity of vSMCs resulting in a hypercontractile vSMC phenotype, as observed in diabetes or hypertension. Given that matrix stiffness is an independent risk factor for cardiovascular disease and that CFL can create different matrix nanotopographic patterns with high pattern fidelity, we are poised to create a combinatorial library of arterial test beds, whether they are healthy, diseased, injured, or aged. Such high-throughput testing environments will pave the way for the evolution of the next generation of vascular scaffolds that can effectively crosstalk with the scaffold microenvironment and result in improved clinical outcomes.


Materials & Methods: Silicon wafers with 800_800nm nanogroove features were fabricated by a micro-stamping method as described in Kim et al.17 Briefly, this involved spin-coating the silicon wafers with a photoresist (Shipley), patterning via electronbeam lithography (JBX-9300FS, JEOL), photoresist development (MF320, Shipley) and deep reactive ion etching of exposed silicon, removal of the remaining photoresist, and finally, dicing into silicon masters for subsequent replica molding. These etched features on the silicon masters were then transferred to poly(urethane acrylate) (PUA) molds (*50mm thickness) on polyester film for the fabrication of nanopatterned PUA substrata by a UV-assisted nanomolding method used previously17,18 (Fig. 1A).

Microscopic Technique: Atomic Force Microscopy, Scanning Electron Microscope

Cell Type(s): vSMC

+ 11. Thermoresponsive Nanofabricated Substratum for the Engineering of Three-Dimensional Tissues with Layer-by-Layer Architectural Control

Alex Jiao, Nicole E. Trosper, Hee Seok Yang, Jinsung Kim, Jonathan H. Tsui, Samuel D. Frankel, Charles E. Murry, and Deok-Ho Kim (1)

(1) University of Washington

ACS Nano, 8 (5), 4430–4439(2014)

Link To Paper

Abstract: Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Keywords: tissue engineering; thermoresponsive; three-dimensional

Materials & Methods: AUV-curablepoly(urethane acrylate) (PUA, Minutatek, Korea) mold was fabricated using capillary force lithography as published previously32,33,35 and was used as the nanopatterned template for the PUA-PGMA substratum (Figure 1a). To allow for epoxy functionalization of the fabricated nanopatterned substratum, 1% GMA weight/ volume monomer(Sigma-Aldrich) was added to the liquid PUA precursor (Norland Optical Adhesive), sonicated for 1 h, and then hand mixed for 10min. Solution was then degassed under a vacuum for 1 h to remove air bubbles. A glass coverslip (Ø18 mm, Fisher) was cleaned using isopropyl alcohol and brush coated with an adhesion promoter (Glass Primer, Minuta Tech) to allow for attachment of the polymer to the glass surface and air-dried. Twenty microliters of PUA-PGMA prepolymer was added to the coverslip and covered with the PUA template consisting of 800 nm wide and 500 nm deep parallel grooves and ridges. The PUA-PGMA prepolymer was spontaneously drawn into the nanofeatures of the PUA template via capillary force. The template-prepolymer-glasswascuredunder365nm UV light to initiate photopolymerization for 5 min. After polymerization, the PUA template was peeled off from the PUA-PGMA substratum using forceps and the substratum was UV-cured overnight to finalize polymerization.

Microscopic Technique: Scanning Electron Microscope

Cell Type(s): C2C12

+ 12. Fabrication of poly(ethylene glycol): gelatin methacrylate composite nanostructures with tunable stiffness and degradation for vascular tissue engineering

Peter Kim, Alex Yuan, Ki-Hwan Nam, Alex Jiao and Deok-Ho Kim (1)

(1) University of Washington

Biofabrication, vol.6, 024112, 2014

Link To Paper

Abstract: Although synthetic polymers are desirable in tissue engineering applications for the reproducibility and tunability of their properties, synthetic small diameter vascular grafts lack the capability to endothelialize in vivo. Thus, synthetically fabricated biodegradable tissue scaffolds that reproduce important aspects of the extracellular environment are required to meet the urgent need for improved vascular grafting materials. In this study, we have successfully fabricated well-defined nanopatterned cell culture substrates made of a biodegradable composite hydrogel consisting of poly(ethylene glycol) dimethacrylate (PEGDMA) and gelatin methacrylate (GelMA) by using UV-assisted capillary force lithography. The elasticity and degradation rate of the composite PEG–GelMA nanostructures were tuned by varying the ratios of PEGDMA and GelMA. Human umbilical vein endothelial cells (HUVECs) cultured on nanopatterned PEG–GelMA substrates exhibited enhanced cell attachment compared with those cultured on unpatterned PEG–GelMA substrates. Additionally, HUVECs cultured on nanopatterned PEG-GelM substrates displayed well-aligned, elongated morphology similar to that of native vascular endothelial cells and demonstrated rapid and directionally persistent migration. The ability to alter both substrate stiffness and degradation rate and culture endothelial cells with increased elongation and alignment is a promising next step in recapitulating the properties of native human vascular tissue for tissue engineering applications.


Materials & Methods: Lyophilized GelMA and PEGDMA (PEGDMA MW 1000 Da; Polyscience) were dissolved in DPBS at 80 °C with a UV crosslinker at a ratio of 5 µL crosslinker per 1 mL of solution. The UV crosslinker was prepared by dissolving 2,2-dimethoxy-2-phenylacetophenone (Aldrich) into 1-vinyl-2-pyrrolidinone (Aldrich) at 30% (w/v). The UV crosslinker was stored protected from light to prevent photochemical reaction. PEGDMA was used at 5%, 20% (w/v) concentrations and GelMA concentration was varied among 0%, 5%, 10% and 20% (w/v). The solution was stored at 40 °C to prevent gelation and used within seven days. A silicon wafer with ridge and groove width of 800 nm and height of 600 nm nanopatterned features was fabricated via ion etching as described in [30]. The features were transferred to a PU mold on a polyester film prior to the fabrication of PEG nanopatterns on the glass by a UV-assisted nanomolding method used previously [34]. To prepare the glass coverslips for nanopatterning, glass coverslips were first rinsed in isopropyl alcohol in a sonicated bath at 35 °C for 20 min and dried under a stream of compressed air. The coverslips were then oxygen-plasma treated for 5 min. An adhesion promoter (Glass Primer, Minuta Tech) was then applied to the 35 mm glass coverslips by spin-coating at 2000 rpm for 20 s. Once spin-coated, the coverslips were baked at 65 °C for 20 min and then treated with UV light (365 nm) for 60 s. Treated glass coverslips were stored up to seven days in a desiccator before usage. PEGDMA and GelMA solution was drop dispensed on the coverslips and PUA master was placed over. The pattern was UV cured (365 nm) for 5 min, resulting in a polymer of 35 mm in diameter, ~100 µm in thickness on top of the coverglass. After polymerization, the nanopatterned PUA master mold was removed. The sample was kept under UV for 12 h to complete the UV curing. Unpatterned substrates were fabricated from an unpatterned polyester film instead of a PUA copy of the nanopatterned substrates. Fidelity of the nanopattern was confirmed by scanning electron microscope (SEM) (JEOL) at 15 kV, 4000× magnification after sputter coating gold on the surface.

Microscopic Technique: Scanning Electron Microscope, Atomic Force Microscopy

Cell Type(s): HUVECs

+ 13. Nanopatterned muscle cell patches for enhanced myogenesis and dystrophin expression in a mouse model of muscular dystrophy

Hee Seok Yanga, Nicholas Ieronimakisc, Jonathan H. Tsuia, Hong Nam Kime, Kahp-Yang Suhe, Morayma Reyesc, Deok-Ho Kim (1)

(1) University of Washington

Biomaterials, 35(5), Pages 1478–1486(2014)

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Abstract: Skeletal muscle is a highly organized tissue in which the extracellular matrix (ECM) is composed of highly-aligned cables of collagen with nanoscale feature sizes, and provides structural and functional support to muscle fibers. As such, the transplantation of disorganized tissues or the direct injection of cells into muscles for regenerative therapy often results in suboptimal functional improvement due to a failure to integrate with native tissue properly. Here, we present a simple method in which biodegradable, biomimetic substrates with precisely controlled nanotopography were fabricated using solvent-assisted capillary force lithography (CFL) and were able to induce the proper development and differentiation of primary mononucleated cells to form mature muscle patches. Cells cultured on these nanopatterned substrates were highly-aligned and elongated, and formed more mature myotubes as evidenced by up-regulated expression of the myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). When transplanted into mdx mice models for Duchenne muscular dystrophy (DMD), the proposed muscle patches led to the formation of a significantly greater number of dystrophin-positive muscle fibers, indicating that dystrophin replacement and myogenesis is achievable in vivo with this approach. These results demonstrate the feasibility of utilizing biomimetic substrates not only as platforms for studying the influences of the ECM on skeletal muscle function and maturation, but also to create transplantable muscle cell patches for the treatment of chronic and acute muscle diseases or injuries.

Keywords: Muscle tissue engineering, Nanotopography, Myogenesis, Poly(lactic-co-glycolic acid), Muscular dystrophy

Materials & Methods: Polyurethane acrylate (PUA, MINS 301 RM, Minuta Technology), and polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) were used as the mold and solvent-absorbing sheet materials, respectively. PUA molds were fabricated by first dispensing PUA precursor onto a patterned silicon wafer master which had been made using standard photolithography techniques, and lightly pressing a polyethylene terephthalate (PET, Skyrol®, SKC) film (thickness = 75 μm) against the PUA. The PUA precursor spontaneously filled the cavities of the master mold by means of capillary action and was cured by exposure to UV light (λ = 250–400 nm) for approximately 30 s (dose = 100 mJ cm−2). After this initial curing, the PUA mold was peeled off from the substrate and further exposed to UV light overnight for complete curing. The PDMS was made with a precursor:curing agent mixing ratio of 10:1 and cured at 60 °C for 10 h. The cured PDMS sheets were manually removed and cut prior to use.

To fabricate the PLGA substrates, a cover glass (ø 25 mm, Fisher) was washed with isopropyl alcohol for 30 min in a water sonicator and dried in nitrogen stream. PLGA (MW: 50,000–75,000, 50:50 lactide:glycolide ratio, Sigma–Aldrich) dissolved in chloroform (100 μl, 15% w/v) was drop-dispensed onto the cover glass. A flat PDMS sheet with an RMS roughness of ∼1.21 nm (Fig. S1) was placed onto the dispensed PLGA solution, and slight pressure (∼10 kPa) was applied evenly on the blanket for 5 min to absorb solvent and to obtain a flat PLGA layer. The cover glass was then placed on a hot plate preheated to 120 °C for 5 min to remove residual solvent and to increase adhesion between the PLGA and the cover glass. Then, a nanopatterned PUA mold was placed onto PLGA-coated glass and the PLGA was embossed with constant pressure (∼100 kPa) on the hot plate for 15 min. After this molding process, the sample was cooled to room temperature, and the PUA mold was carefully peeled off from the PLGA-coated glass, leaving behind a nanopatterned PLGA substrate. To prepare the flat substrates, the patterning steps were simply excluded. Both patterned and flat PLGA substrates were stored in a desiccator to remove residual solvent before use.

Microscopic Technique: Atomic Force Microscopy, Scanning Electron Microscope

Cell Type(s): Mouse Muscle cells

+ 14. Spatial control of adult stem cell fate using nanotopographic cues

Eun Hyun Ahn, Younghoon Kim, Kshitizc, Steven S. An, Junaid Afzal, Suengwon Lee, Moonkyu Kwak, Kahp-Yang Suh, Deok-Ho Kim (1), Andre Levchenko (2)

(1) University of Washington, (2) Johns Hopkins University

Biomaterials, 35(8), Pages 2401–2410(2014)

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Abstract: Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.

Keywords: Human mesenchymal stem cells, Differentiation, Nanotopography, Osteogenesis, Adipogenesis, Capillary force lithography

Materials & Methods: Fabrication of nanostructured posts composed of polyurethane acrylate (PUA) using UV-assisted CFL Nanostructured PUA surfaces with various post-to-post distances (1.2, 2.4, 3.6, and 5.6 μm) were fabricated as described previously [24].

Microscopic Technique: Optical and immunofluorescence confocal microscopy

Cell Type(s): hMSC

+ 15. Enhanced Chondrogenic Differentiation of Dental Pulp Stem Cells Using Nanopatterned PEG-GelMA-HA Hydrogels

Nemeth Cameron L. (1), Janebodin Kajohnkiart (1), Yuan Alex E., Dennis James E., Reyes Morayma, and Kim Deok-Ho.

(1) University of Washington

Tissue Engineering Part A, 20(21-22): 2817-2829(2014)

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Abstract: We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial–mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs.


Materials & Methods: Glass coverslips (BioScience Tools) were cleaned in a piranha solution consisting of a 3:1 ratio of 100% sulfuric acid (Sigma-Aldrich) and 30% aqueous hydrogen peroxide (SigmaAldrich) for 30min to remove organic material and provide additional hydroxyl groups before silane treatment. Then, coverslips were thoroughly cleaned using deionized water and dried under an air stream before being submerged in 2mM 3-(trimethoxysilyl) propyl methacrylate (Sigma-Aldrich) in anhydrous toluene (Sigma-Aldrich) for 60min. The glass coverslips were rinsed in toluene again and dried under an air stream. The cleaned and silane-treated coverslips were stored under vacuum inside a desiccator until used. UV curable nanopatterned polyurethane acrylate (PUA) (Minuta Tech) molds were prepared for fabrication. Characterization and synthesis were previously described.5 The PUA mold consisted of a pattern of ridge·groove·height dimensions of 800·800·500nm. Anisotropically nanopatterned PEG-GelMA-HA hydrogels were fabricated on the pretreated glass coverslips using UV-assisted CFL. A PUA mold was rinsed with 100% ethyl alcohol to remove organic contaminants and was carefully placed onto the surface. A small amount (*10mL) of PEG-GelMA-HA precursor solution was pipetted onto a single glass coverslip. The solution was drawn into the nanogrooves of the pattern through capillary action and cured by exposure to UV light (l=365nm) for 5min. After curing, the PUA mold was peeled off leaving a nanopatterned PEG-GelMA-HA hydrogel scaffold.

Microscopic Technique: Scanning Electron Microscope

Cell Type(s): DPSC

+ 16. A nanotopography approach for studying the structure-function relationships of cells and tissues

Kshitiz, Junaid Afzal, Sang-Yeob Kim, Deok-Ho Kim (1)

(1) University of Washington

Cell Adhesion and Migration, 8(4), Pages 1–8(2015)

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Abstract: Most cells in the body secrete, or are in intimate contact with extracellular matrix (ECM), which provides structure to tissues and regulates various cellular phenotypes. Cells are well known to respond to biochemical signals from the ECM, but recent evidence has highlighted the mechanical properties of the matrix, including matrix elasticity and nanotopography, as fundamental instructive cues regulating signal transduction pathways and gene transcription. Recent observations also highlight the importance of matrix nanotopography as a regulator of cellular functions, but lack of facile experimental platforms has resulted in a continued negligence of this important microenvironmental cue in tissue culture experimentation. In this review, we present our opinion on the importance of nanotopography as a biological cue, contexts in which it plays a primary role influencing cell behavior, and detail advanced techniques to incorporate nanotopography into the design of experiments, or in cell culture environments. In addition, we highlight signal transduction pathways that are involved in conveying the extracellular matrix nanotopography information within the cells to influence cell behavior.

Keywords: nanotopography, extracellular matrix, structure-function, tissue engineering, cell biology

Materials & Methods: We recapitulated the highly anisotropic collagen fiber bundles seen in heart muscle (Fig. 3A) using capillary force lithography (CFL) to form a scalable polymeric nanogrooved pattern (Fig. 3B–C). [20] Engineering techniques used to study topographical cues can directly provide guidance cues on which cells can track. Metastatic cancer cells can migrate, as well as remodel the collagen fibrils (Fig. 3D), [21] while even immune T cells respond to the underlying nanotopography in combination with biochemical ligand based stimulation (Fig. 3E). 6 Nanotopographical features also structurally and functionally mature cardiac progenitors, [29] and skeletal muscle satellite cells. [30] We also found that adult mesenchymal cells could be directed toward adipogenesis or osteogenesis depending on the features of nanotopographical cues, facilitating creation of a mosaic tissue with spatial control of cell lineage. [28]

Microscopic Technique: Scanning Electron Microscope

Cell Type(s): T cells

+ 17. Combined Effects of Substrate Topography and Stiffness on Endothelial Cytokine and Chemokine Secretion

Hyeona Jeon, Jonathan H. Tsui, Sue Im Jang, Justin H. Lee, Soojin Park, Kevin Mun, Yong Chool Boo, and Deok-Ho Kim (1)

(1) University of Washington

ACS Appl. Mater. Interfaces, 7 (8), pp 4525–4532(2015)

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Abstract: Endothelial physiology is regulated not only by humoral factors, but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nanopatterns tested, the 1:1 and 1:2 type patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1β, IL-3, and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling.

Keywords: nanotopography; substrate stiffness; endothelial cells; cytokines; chemokines

Materials & Methods: Nanopatterned substrates were fabricated from UV-curable polymers by utilizing capillary force lithography (Figure 1A). This technique allows for the simple and reproducible fabrication of a variety of molded nanopatterned substrates with excellent pattern fidelity regardless of the polymer used, and this was confirmed using using SEM and AFM (Figure 1B). It is well-known that cell reorganization, motility, and adhesion is greatly affected by substrate surface wettability.23−26 Therefore, to ensure that changes in endothelial cell morphology and cytokine/ chemokine secretion would be due to differences in substrate stiffnesses and topographies, rather than surface chemistries, surface wettability measurements on both unpatterned and patterned substrates were taken, and the results indicate that there were no signi ficant differences due to polymer composition (Figure 2). The specific nanopattern dimensions and structures used for this study were based on the native extracellular matrix (ECM) structure of vascular tissue,27 thus providing a biomimetic platform for studying the effects of the ECM on endothelial cell morphology and activity. As changes in the structure and stiffness of vascular ECM can occur as a result of disease,28,29 our platform could be used in future studies that utilize disease-in-a-dish models to better understand how these pathologies can affect cell behavior, and how this leads to the disease progression and complications seen in patients.

Microscopic Technique: Confocal Fuorescent Microscope

Cell Type(s): Human endothelial cells