Single-cell manipulation by two-dimensional micropatterning

Cells are highly sensitive to their geometrical and mechanical microenvironment that directly regulate cell shape,cytoskeleton and organelle,as well as the nucleus morphology and genetic expression.The emerging two-dimensional micropatterning techniques offer powerful tools to construct controllable and well-organized microenvironment for single-cell level investigations with qualitative analysis,cellular standardization,and in vivo environment mimicking.Here,we provide an overview of the basic principle and characteristics of the two most widely-used micropatterning techniques,including photolithographic micropatterning and soft lithography micropatterning.Moreover,we summarize the application of micropatterning technique in controlling cytoskeleton,cell migration,nucleus and gene expression,as well as intercellular communication.

Regulation of actin cytoskeleton via photolithographic micropatterning

Actin cytoskeleton plays crucial roles in various cellular functions.Extracellular matrix(ECM)can modulate cell morphology by remodeling the internal cytoskeleton.To define how geometry of ECM regulates the organization of actin cytoskeleton,we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes.It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern,but were absent from the opening edge of V-shaped pattern,indicating that the organization of actin cytoskeleton was dependent on the mechanical environment.Furthermore,a secondary actin ring was observed on 50μm circular pattern while did not appear on 30μm and 40μm pattern,showing a size-dependent organization of actin cytoskeleton.Finally,osteoblasts,MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern,suggesting a cell-type specificity in arrangement of actin cytoskeleton.Together,our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.

Effects of femtosecond laser micropatterning on the surface properties and cellular response of biomedical tantalum-blended composites

Poly(ε-caprolactone)(PCL)holds unique bioresorbability and competent biomechanical properties for tissueengineering application.However,PCL is hydrophobic intrinsically and poor in cell-biomaterial interaction.In this study,we prepared a composite based on PCL and bioactive tantalum(Ta)to understand the effects of direct laser micropatterning on composite surface properties.The PCL/Ta composite after preparation was surface-patterned by femtosecond laser and characterized with surface morphology,crystal structure,chemical composition,wettability and cellular response of fibroblast.It was found that laser micropatterning enlarged the difference of wetting properties(~15°)on PCL and PCL/Ta surfaces.The wetting changes was dependent on both material composition and lasermachined geometry.The blending of Ta enhanced surface wettability with prolonged contact time on the laser-machined line and rectangle microarrays.In vitro culture results showed beneficial effects of laser micropatterning on cell morphology of the fibroblasts.On the PCL/Ta surfaces with line and rectangle microarrays,the cells were more likely to bridge the sidewalls of the microgrooves,showing adaptive 3D morphologies to the micro/nano topographies on the sidewalls.These findings are envisaged to facilitate surface design and micropattern optimization for favorable tuning the cell response to biomedical PCL/Ta composites.

Additive manufacturing of micropatterned functional surfaces:a review

Over the course of millions of years,nature has evolved to ensure survival and presents us with a myriad of functional surfaces and structures that can boast high efficiency,multifunctionality,and sustainability.What makes these surfaces particularly practical and effective is the intricate micropatterning that enables selective interactions with microstructures.Most of these structures have been realized in the laboratory environment using numerous fabrication techniques by tailoring specific surface properties.Of the available manufacturing methods,additive manufacturing(AM)has created opportunities for fabricating these structures as the complex architectures of the naturally occurring microstructures far exceed the traditional ways.This paper presents a concise overview of the fundamentals of such patterned microstructured surfaces,their fabrication techniques,and diverse applications.A comprehensive evaluation of micro fabrication methods is conducted,delving into their respective strengths and limitations.Greater emphasis is placed on AM processes like inkjet printing and micro digital light projection printing due to the intrinsic advantages of these processes to additively fabricate high resolution structures with high fidelity and precision.The paper explores the various advancements in these processes in relation to their use in microfabrication and also presents the recent trends in applications like the fabrication of microlens arrays,microneedles,and tissue scaffolds.

Surface-tension-confined droplet microfluidics

This article is a concise overview about the developing microfluidic systems named surface-tension-confined droplet microfluidics （STORMs）. Different from traditional complexed droplet microfluidics which generated and confined the droplets by three-dimensional （3D） poly（dimethylsiloxane）-based microchannels, STORM systems provide two- dimensional （2D） platforms for control of droplets. STORM devices utilize surface energy, with methods such as surface chemical modification and mechanical processing, to control the movement of fluid droplets. Various STORM devices have been readily prepared, with distinct advantages over conventional droplet microfluidics, which generated and confined the droplets by 3D poly（dimethylsiloxane）-based microchannels, such as significant reduction of energy consumption neces- sary for device operation, facile or even direct introduction of droplets onto patterned surface without external driving force such as a micropump, thus increased frequency or efficiency of droplets generation of specific STORM device, among others. Thus, STORM devices can be excellent alternatives for majority areas in droplet microfluidics and irreplaceable choices in certain fields by contrast. In this review, fabrication methods or strategies, manipulation methods or mechanisms, and main applications of STORM devices are introduced.

Optimization of micropatterned poly(lactic-coglycolic acid) films for enhancing dorsal root ganglion cell orientation and extension

Nerve conduits have been a viable alternative to the ‘gold standard’ autograft for treating small peripheral nerve gap injuries. However, they often produce inadequate functional recovery outcomes and are ineffective in large gap injuries. Ridge/groove surface micropatterning has been shown to promote neural cell orientation and guide growth. However, optimization of the ratio of ridge/groove parameters to promote orientation and extension for dorsal root ganglion （DRG） cells on poly（lactic-co-glycolic acid） （PLGA） films has not been previously conducted. Photolithography and micro-molding were used to define various combinations of ridge/groove dimensions on PLGA films. The DRG cells obtained from chicken embryos were cultured on micropatterned PLGA films for cell orientation and migration evaluation.Biodegradation of the films occurred during the test period, however, this did not cause deformation or distortion of the micropatterns. Results from the DRG cell orientation test suggest that when the ridge/groove ratio equals 1 （ridge/groove width parameters are equal, i.e., 10 μm/10 μm （even））, the degree of alignment depends on the size of the ridges and grooves, when the ratio is smaller than 1 （groove controlled） the alignment increases as the ridge size decreases, and when the ratio is larger than 1 （ridge controlled）, the alignment is reduced as the width of the grooves decreases. The migration rate and neurite extension of DRG neurons were greatest on 10 μm/10 μm and 30 μm/30 μm micropatterned PLGA films. Based on the data, the 10 μm/10 μm and 30 μm/30 μm micropatterned PLGA films are the optimized ridge/groove surface patterns for the construction of nerve repair devices.

Controlled release of FK506 from micropatterned PLGA films:potential for application in peripheral nerve repair

After decades of research,peripheral nerve injury and repair still frequently results in paralysis,chronic pain and neuropathies leading to severe disability in patients.Current clinically available nerve conduits only provide crude guidance of regenerating axons across nerve gap without additional functionality.FK506（Tacrolimus）,an FDA approved immunosuppressant,has been shown to enhance peripheral nerve regeneration but carries harsh side-effects when delivered systemically.The objective of this study was to develop and evaluate a bioresorbable drug delivery system capable of local extended delivery of FK506 that also provides topological guidance cues to guide axon growth via microgrooves.Photolithography was used to create micropatterned poly（lactide-co-glycolic acid）（PLGA） films embedded with FK506.Non-patterned,10/10 μm（ridge/groove width）,and 30/30 μm patterned films loaded with 0,1,and 3 μg/cm2 FK506 were manufactured and characterized.In vitro FK506 rate of release testing indicated that the films are capable of an extended（at least 56 days）,controlled,and scalable release of FK506.Neurite extension bioactivity assay indicated that FK506 released from the films（concentration of samples tested ranged between 8.46–19.7 ng/m L） maintained its neural bioactivity and promoted neurite extension similar to control FK506 dosages（10 ng/m L FK506）.The multi-functional FK506 embedded,micropatterned poly（lactide-co-glycolic acid） films developed in this study have potential to be used in the construction of peripheral nerve repair devices.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment

BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.

Standing surface acoustic wave-assisted fabrication of patterned microstructures for enhancing cell migration

Microfluidic device with patterned microstructures on the substrate surface was used to regulate cell adhesion,morphology,and functions in tissue engineering.We developed a microfluidic device which employing microscale patterned microstructures to achieve enhanced cell adhesion and migration.Biocompatible hydrogel substrates with micro-wavy and lattice-patterned microstructures were fabricated using standing surface acoustic waves and ultraviolet solidification.After seeding the L929 mouse fibroblast cells onto the patterned substrate of the microfluidic device,we determined that the viability and proliferation rate of cell migration can be greatly enhanced.Furthermore,L929 cells showed two types of gathering modes after 48 h of culturing.Cell growth was guided by the patterned substrate used in the microfluidic device and showed differences in the location distribution.Therefore,the developed microfluidic device with patterned microstructures can extend the application of in vitro cell culturing for future drug development and disease diagnosis.

Anisotropic cell growth-regulated surface micropatterns in flower petals

Flower petals have not only diverse macroscopic morphologies but are rich in microscopic surface patterns, which are crucial to their biological functions. Both experimental measurements and theoretical analysis are conducted to reveal the physical mechanisms underlying the formation of minute wrinkles on flower petals. Three representative flowers, daisy, kalanchoe blossfeldiana, and Eustoma grandiflorurn, are investigated as examples. A surface wrinkling model, incorporating the measured mechanical properties and growth ratio, is used to elucidate the difference in their surface morphologies. The mismatch between the anisotropic epidermal cell growth and the isotropic secretion of surficial wax is found to dictate the surface patterns.

Biofabrication of aligned structures that guide cell orientation and applications in tissue engineering

The organized alignment of cells in various tissues plays a significant role in the maintenance of specific functions.To induce such an alignment,ideal scaffolds should simulate the characteristics and morphologies of natural tissues.Aligned structures that guide cell orientation are used to facilitate tissue regeneration and repair.We here review how various aligned structures are fabricated,including aligned electrospun nanofibers,aligned porous or channeled structures,micropatterns and combinations thereof,and their application in nerve,skeletal muscle,tendon,and tubular dentin regeneration.The future use of aligned structures in tissue engineering is also discussed.

Status of the Streamer Mode in Gas Detectors

This work shows the results of a streamer discharge mode studies in various gas detectors developed up-to-date. The results are based on a new experimental data from high-speed thin-gap gas detector application developments as well as on basic knowledge of multi-wire devices operations.

Transparent micropatterned conductive films based on highlyordered nanowire network

Transparent conductive films that are based on nanowire networks are essential to construct flexible,wearable,and even stretchable electronics.However,large-scale precise micropatterning,especially with regard to the controllability of the organizing orientation of nanowires,is a critical challenge.Herein,we proposed a liquid film rupture self-assembly approach for manufacturing transparent conductive films with microstructure arrays based on a highly ordered nanowire network.The large-scale microstructure conductive films were fabricated through air-liquid interface self-assembly and liquid film rupture self-assembly.Six typical micropattern morphologies,including square,hexagon,circle,serpentine,etc.,were prepared to reveal the universal applicability of the proposed approach.The homogeneity and controllability of this approach were verified for multiple assemblies.With the assembly cycles increasing,the optical transmittance decreases slightly.In addition,theoretical model analysis is carried out,and the analytical formula of the speed of the film moving with the surface tension and the density of the liquid film is presented.Finally,the feasibility of this approach for piezoresistive strain sensors is verified.This fabrication approach demonstrated a cost-effective and efficient method for precisely arranging nanowires,which is useful in transparent and wearable applications.

Polyetheretherketone and titanium surface treatments to modify roughness and wettability–Improvement of bioactivity and antibacterial properties

Among the materials available for implant production,titanium is the most used while polyetheretherketone(PEEK)is emerging thanks to its stability and to the mechanical properties similar to the ones of the bone tissue.Material surface properties like roughness and wettability play a paramount role in cell adhesion,cell proliferation,osteointegration and implant stability.Moreover,the bacterial adhesion to the biomaterial and the biofilm formation depend on surface smoothness and hydrophobicity.In this work,two different treatments,sandblasting and air plasma,were used to increase respectively roughness and wettability of two materials:titanium and PEEK.Their effects were analyzed with profilometry and contact angle measurements.The biological properties of the material surfaces were also investigated in terms of cell adhesion and proliferation of NIH-3T3 cells,MG63 cells and human Dental Pulp Stem Cells.Moreover,the ability of Staphylococcus aureus to adhere and form a viable biofilm on the samples was evaluated.The biological properties of both treatments and both materials were compared with samples of Synthegra;titanium,which underwent laser ablation to obtain a porous micropatterning,characterized by a smooth surface to discourage bacterial adhesion.All cell types used were able to adhere and proliferate on samples of the tested materials.Cell adhesion was higher on sandblasted PEEK samples for both MG63 and NIH-3T3 cell lines,on the contrary,the highest proliferation rate was observed on sandblasted titanium and was only slightly dependent on wettability;h DPSCs were able to proliferate similarly on sandblasted samples of both tested materials.The highest osteoblast differentiation was observed on laser micropatterned titanium samples,but similar effects,even if limited,were also observed on both sandblasted materials and air plasma treated titanium.The lowest bacterial adhesion and biofilm formation was observed on micropatterned titanium samples whereas,the highest biofilm formation was detected on sandblasted PEEK samples,and in particular on samples not treated with air-plasma,which displayed the highest hydrophobicity.The results of this work showed that all the tested materials were able to sustain osteoblast adhesion and promote cell proliferation;moreover,this work highlights the feasible PEEK treatments which allow to obtain surface properties similar to those of titanium.The results here reported,clearly show that cell behavior depends on a complex combination of surface properties like wettability and roughness and material nature,and while a rough surface is optimal for cell adhesion,a smooth and less hydrophilic surface is the best choice to limit bacterial adhesion and biofilm formation.

An inkjet printing soft photomask and its application on organic polymer substrates

This article presents a simple,fast and low-cost method to fabricate a flexible UV light photomask.The designed micropatterns were directly printed onto transparent hybrid composite film of biaxially oriented polypropylene coated with silica oxide (BOPP-SiOx) by an inkjet printer.Compared to the conventional chrome-mask,it is of advantages such as suitable for non-planar substrates,scalable for large area production,and extreme low cost.Combined with the confined photo-catalytic oxidation (CPO) reaction,the printed flexible BOPP-SiOx photomask was successfully used to pattern the shape of wettability of organic polymer surfaces,and then polyaniline patterns were deposited on the modified substrates with strong adhesion.With the above photomasks,the polyacrylic acid graft chains were duplicated on the poly (ethylene terephthalate) (PET) and BOPP substrates by photografting polymerization.We grafted polyacrylic acid (PAA) on a non-planar plastic substrate with this soft and thin plastic photomask.Scanning electron microscopy (SEM) and optical microscopy were used to characterize the surface morphology and thickness of ink layers of the printed photomask.Optical microscopy was used to characterize the deposition polyaniline micropatterns.It was found that the desired patterns were precisely printed on the modified polymer films and were applied in modifying organic polymer substrates.The printed photomask could be exploited in the fields such as prototype microfluidics,micro-sensors,optical structures and any other kind of microstructures which does not require high durability and dimensional stability.

Functional imaging of neuron-astrocyte interactions in a compartmentalized microfluidic device

Traditional approaches in cultivating neural cells in a dish without orienting their interactions have had only limited success in revealing neural network properties.To enhance the experimental capabilities of studying neural circuitry in vitro,we designed an experimental system combining concepts of micropatterned surfaces,microfluidic devices and genetically encoded biosensors.Micropatterning was used to position neurons and astrocytes in defined locations and guide interactions between the two cell types.Microfluidic chambers were placed atop micropatterned surfaces to allow delivery of different pharmacological agents or viral vectors to the desired cell types.In this device,astrocytes and neurons communicated through grooves molded into the floor of the microfluidic device.By combining microfluidics with genetically encoded calcium indicators as functional readouts,we further demonstrated the utility of this device for analyzing neuron–neuron and neuron–astrocyte interactions in vitro under both healthy and pathophysiological conditions.We found that both spontaneous and evoked calcium dynamics in astrocytes can be modulated by interactions with neurons.In the future,we foresee employing the microdevices described here for studying mechanisms of neurological disorders.

Giant enhancement of photoluminescence emission in monolayer WS_(2)by femtosecond laser irradiation

Monolayer transition metal dichalcogenides have emerged as promising mat erials for opt oelectTonic and nanophotonic devices.However,the low photoluminescence(PL)quantum yield(QY)hinders their various potential applications.Here we engineer and enhance the PL intensity of monolayer WS_(2)by femtosecond laser irradiation.More than two orders of magnitude enhancement of PL intensity as compared to the as-prepared sample is determined.Furthermore,the engineering time is shortened by three orders of magnitude as compared to the improvement of PL intensity by continuous-wave laser irradiation.Based on the evolution of PL spectra,we attribute the giant PL enhancement to the conversion from trion emission to exciton,as well as the improvement of the QY when exciton and trion are localized to the new-formed defects.We have created microstructures on the monolayer WS_(2)based on the enhancement of PL intensity,where the engineered structures can be stably stored for more than three years.This flexible approach with the feature of excellent long-term storage stability is promising for applications in information storage,display technology,and opto electronic devices.

Micro and Nanofabrication methods to control cell-substrate interactions and cell behavior: A review from the tissue engineering perspective

Cell-substrate interactions play a crucial role in the design of better biomaterials and integration ofimplants with the tissues. Adhesion is the binding process of the cells to the substrate through interactionsbetween the surface molecules of the cell membrane and the substrate. There are severalfactors that affect cell adhesion including substrate surface chemistry, topography, and stiffness. Thesefactors physically and chemically guide and influence the adhesion strength, spreading, shape and fate ofthe cell. Recently, technological advances enabled us to precisely engineer the geometry and chemistry ofsubstrate surfaces enabling the control of the interaction cells with the substrate. Some of the mostcommonly used surface engineering methods for eliciting the desired cellular responses on biomaterialsare photolithography, electron beam lithography, microcontact printing, and microfluidics. Thesemethods allow production of nano- and micron level substrate features that can control cell adhesion,migration, differentiation, shape of the cells and the nuclei as well as measurement of the forces involvedin such activities. This review aims to summarize the current techniques and associate these techniqueswith cellular responses in order to emphasize the effect of chemistry, dimensions, density and design ofsurface patterns on cell-substrate interactions. We conclude with future projections in the field of cellsubstrateinteractions in the hope of providing an outlook for the future studies.

Preparation of stable micropatterns of gold on cell-adhesion-resistant hydrogels assisted by a hetero-bifunctional macromonomer linker

Surface patterning is very useful in biomaterial studies, yet it is not easy to prepare a micropattern with cell-adhesion contrast that is stable in a wet environment. Recently, a platform technique of transfer photolithography was invented to fabricate stable metal microarrays on the surface of a cell-adhesion resistant and mechanically biomimetic poly（ethylene glycol） hydrogel; the linker is the key chemical in such a transfer strategy. This article reports the design and synthesis of a hetero-bifunctional macromonomer linker with a thiol group at one end and an acryloyl group at the other end. The bifunctional linker was char- acterized by GPC and ~H NMR, and the average number of thiol groups in the bifunctional linker was detected by Ellman＇s reagent. The regent stability under wet conditions was also confirmed by the model reactants. The resultant micropatterned surfaces are meaningful for future studies of cell behaviors on mechanically biomimetic matrixes.

Nature-inspired topographies on hydroxyapatite surfaces regulate stem cells behaviour

Surface topography is one of the key factors in regulating interactions between materials and cells.While topographies presented to cells in vivo are non-symmetrical and in complex shapes,current fabrication techniques are limited to replicate these complex geometries.In this study,we developed a microcasting technique and successfully produced imprinted hydroxyapatite(HAp)surfaces with nature-inspired(honeycomb,pillars,and isolated islands)topographies.The in vitro biological performance of the developed non-symmetrical topographies was evaluated using adipose-derived stem cells(ADSCs).We demonstrated that ADSCs cultured on all HAp surfaces,except honeycomb patterns,presented well-defined stress fibers and expressed focal adhesion protein(paxillin)molecules.Isolated islands topographies significantly promoted osteogenic differentiation of ADSCs with increased alkaline phosphatase activity and upregulation of key osteogenic markers,compared to the other topographies and the control unmodified(flat)HAp surface.In contrast,honeycomb topographies hampered the ability of the ADSCs to proliferate and differentiate to the osteogenic lineage.This work presents a facile technique to imprint nature-derived topographies on the surface of bioceramics which opens up opportunities for the development of bioresponsive interfaces in tissue engineering and regenerative medicine.