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.

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.

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.

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.

培养模式控制对人牙周膜和骨髓间充质干细胞生物电特性的影响

目的:探索微模式培养控制和导电基底对人牙周膜干细胞和骨髓间充质干细胞生物电特性的影响。方法:通过光刻技术在盖玻片和导电玻璃两种透明材料表面构造与细胞自身宽度相当的微模式化培养基底,微模式图案分为两种:单种细胞的单格阵列结构、双细胞共培养的交叉指贯通结构。应用全细胞膜片钳技术记录两种细胞在不同培养模式下的静息电位、钾通道电流等生物电特性。实验分9组:培养皿对照组、盖玻片牙周膜干细胞组、盖玻片骨髓间充质干细胞组、盖玻片共培养牙周膜干细胞组、盖玻片共培养骨髓间充质干细胞组、导电玻璃牙周膜干细胞组、导电玻璃骨髓间充质干细胞组、导电玻璃共培养牙周膜干细胞组、导电玻璃共培养骨髓间充质干细胞组。结果:对于牙周膜干细胞,与对照组自由铺展培养模式相比,微模式化形态控制导致细胞膜电容减小,钾通道电流密度增大(P<0.01),导电基底导致细胞静息膜电位绝对值增大;两种微模式化培养模式相比,双细胞交叉指贯通培养使得细胞静息膜电位绝对值减小。对于骨髓间充质干细胞,与对照组自由铺展培养模式相比,导电基底表面细胞膜电容增大、钾通道电流密度减小和静息膜电位绝对值减小,但双细胞交叉指贯通培养则导致细胞膜电容减小、钾电流密度增大和静息膜电位绝对值增大;与盖玻片共培养模式相比,导电玻璃共培养细胞的钾电流密度显著增大(P<0.01)。结论:导电培养微模式能够调控人牙周膜和骨髓间充质干细胞的生物电特性,从利用内源电场角度,为深入探索两种干细胞促进口腔软硬组织再生修复机制提供新的思路。

材料表面微图案结构成型技术及其研究进展

表面微图案结构成型技术作为实现材料表面纹理化并促进材料先进化、功能化的一种手段已经广泛应用到实际生产制造中。根据材料应用领域的差异,表面微图案成型的主要方法包括热压花、化学蚀刻、电火花加工、光刻和3D打印。本文综述了不同表面微图案结构成型技术及制备表面纹理化功能材料的基本原理,并列举其在光伏、电子信息、智能制造、超精密制造等领域的实际应用。总结了不同表面微图案结构成型技术的特点及适用范围,提出了实现材料表面纹理化和功能化所面临的关键问题,为表面微图案结构的微加工技术提供了一定参考。

Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo

Both of the surface topographical features and distribution of biochemical cues can influence the cell-substrate interactions and thereby tissue regeneration in vivo.However,they have not been combined simultaneously onto a biodegradable scaffold to demonstrate the synergistic role so far.In this study,a proof-of-concept study is performed to prepare micropatterns and peptide gradient on the inner wall of a poly(D,L-lactide-co-caprolactone)(PLCL)guidance conduit and its advantages in regeneration of peripheral nerve in vivo.After linear ridges/grooves of 20/40μm in width are created on the PLCL film,its surface is aminolyzed in a kinetically controlled manner to obtain the continuous gradient of amino groups,which are then transferred to CQAASIKVAV peptide density gradient via covalent coupling of glutaraldehyde.The Schwann cells are better aligned along with the stripes,and show a faster migration rate toward the region of higher peptide density.Implantation of the nerve guidance conduit made of the PLCL film having both the micropatterns and peptide gradient can significantly accelerate the regeneration of sciatic nerve in terms of rate,function recovery and microstructures,and reduction of fibrosis in muscle tissues.Moreover,this nerve conduit can also benefit the M2 polarization of macrophages and promote vascularization in vivo.

PEGMA刷微图案诱导聚苯乙烯纳米颗粒阵列化

基于光引发原子转移自由基聚合(ATRP)反应,在疏水性引发剂表面制备亲水性聚乙二醇甲基丙烯酸酯(PEGMA)聚合物刷微图案,以PEGMA聚合物刷图案化表面为模板,借助亲疏水区域间表面能的差异使聚苯乙烯(PS)纳米颗粒在亲水区域精准组装制备纳米颗粒微阵列。采用X射线光电子能谱仪(XPS)与拉曼光谱仪(Raman)对各阶段基体表面化学组分进行表征,结果表明,聚多巴胺/3-(三甲氧基硅烷基)丙基2-溴-2-甲基丙酸酯(PDA/SiBr)复合涂层、PEGMA聚合物刷以及PS纳米颗粒微阵列被成功制备。采用扫描电子显微镜(SEM)和光学显微镜对PEGMA聚合物刷图案化表面及PS纳米颗粒阵列化表面进行观察,结果表明PS纳米颗粒在PEGMA聚合物刷微图案区域选择性组装。研究了PS纳米颗粒微阵列表面BSA蛋白吸附行为,荧光图像结果表明PS纳米颗粒在PEGMA聚合物刷微图案表面组装后可以有效促进BSA蛋白在该区域的黏附。相关研究工作拓展了图案化聚合物刷在纳米颗粒微阵列制备中的应用,为纳米颗粒在高质量生物微阵列、生物探针等领域的应用提供了技术支持。

Durable metal-enhanced fluorescence flexible platform by in-situ growth of micropatterned Ag nanospheres

Here, we demonstrate an in-situ growth of micropatterned silver nanospheres(Ag NS) array on transparent polyimide(PI), namely PI-Ag substrate, applied as a flexible platform for metal-enhanced fluorescence(MEF). The scheme proposed here can easily control the grid formation of Ag NSs and the particle size inside, thus achieving patterned fluorescence imaging and MEF efficiency optimization. Meanwhile, the magnitude of enhanced intensity, relying on the distance between Ag NSs and emissive molecules, was systematically investigated by exploring diverse polyethyleneimine(PEI) spacer thickness. Consequently,an optimal enhancement factor of 7.9 and a pattern of grid fluorescence imaging was obtained with an insertion of 10 nm PEI on the PI-Ag(25 nm) platform. Moreover, owing to robust adhesion between Ag NSs and PI film by in-situ growth, this flexible PI-Ag MEF platform maintained a stable MEF efficiency even after taking mechanical bending for 1000 cycles. This new surface-confined micropatterned Ag NSs PI film provides a promising candidate in design flexible MEF platforms for future analytical and clinical sensing applications.

Simulation study of the performance of new micropattern gaseous detectors

Background The micropattern gaseous detectors(MPGDs)are widely used in high-energy physics experiment,such as detector upgrade projects in LHC,due to its excellent performance on rate capability,spatial and time resolutions.Method In this paper,we studied the performances of GEM,FTM andμ-RWELL detectors on time and spatial resolutions using Monte Carlo simulation methods and compared their performances and characteristics at various working conditions.Result Result shows that time resolution of MPGDs improves with the increase of electric field intensity in drift region,while spatial resolution shows the reverse tendency.In addition,detectors operating with an electronegative gas mixture show better performances on both time and spatial resolution.Conclusion We studied the performance of triple-GEM,FTM andμ-RWELL detectors with Monte Carlo simulation.In this paper,ANSYS and GARFIELD are used to build full electric field model of the detector.The time resolution and spatial resolution are derived,which are very important for triggering performance and track reconstruction ability.These results will provide references on detector design and the technology chosen in LHC detector upgrade projects.

Cell orientation on a stripe-micropatterned surface

Stripe-micropatterned surfaces have recently been a unique tool to study cell orientation. In this paper, we prepared, by the photolithography transfer technique, stable gold (Au) micropatterns on PEG hydrogel surfaces with defined cell-resistant (PEG hydrogel) and cell-adhesive (gold microstripes) properties. 3T3 fibroblasts were cultured on Au-microstripe surfaces to observe cell adhesion and orientation. Five statistical parameters were defined and used to describe cell orientation on micropatterns. With the increase of inter-stripe distance, the orientational order parameter, the ratio of long and short axes of a cell, and the occupation fraction of cells on stripes increased gradually, whereas the spreading area of a single cell decreased. The abrupt changes of these four parameters did not happen at the same inter-distance. The adhesion ratio of a cell on Au stripes over cell spreading area did not change monotonically as a function of inter-stripe distance. The combination of the 5 statistical parameters represented well the cell orientation behaviors semi-quantitatively.

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 characterized by GPC and 1H 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.

Investigating design principles of micropatterned encapsulation systems containing high-density microtissue arrays

Immunoisolation is an important strategy to protect transplanted cells from rejection by the host immune system.Recently,microfabrication techniques have been used to create hydrogel membranes to encapsulate microtissue in an arrayed organization.The method illustrates a new macroencapsulation paradigm that may allow transplantation of a large number of cells with microscale spatial control,while maintaining an encapsulation device that is easily maneuverable and remaining integrated following transplantation.This study aims to investigate the design principles that relate to the translational application of micropatterned encapsulation membranes,namely,the control over the transplantation density/quantity of arrayed microtissues and the fidelity of pre-formed microtissues to micropatterns.Agarose hydrogel membranes with microwell patterns were used as a model encapsulation system to exemplify these principles.Our results show that high-density micropatterns can be generated in hydrogel membranes,which can potentially maximize the percentage volume of cellular content and thereby the transplantation efficiency of the encapsulation device.Direct seeding of microtissues demonstrates that microwell structures can efficiently position and organize pre-formed microtissues,suggesting the capability of micropatterned devices for manipulation of cellular transplants at multicellular or tissue levels.Detailed theoretical analysis was performed to provide insights into the relationship between micropatterns and the transplantation capacity of membrane-based encapsulation.Our study lays the ground for developing new macroencapsulation systems with microscale cellular/tissue patterns for regenerative transplantation.

Photoinduced micropattern in an azobenzene polymer film

The micropattern observed in the amorphous azobenzene polymer film by degenerated four-wave mixing has been reported. Patterns with well-defined structures are examined with the scanning electron microscopy and the polarizing optical microscopy. It is demonstrated that the control of photoinduced micropattern in the azobenzene polymer film is possible by using appropriate polarized writing beams with total incident power exceeding a certain threshold.

具有表面微图形化胰肠吻合口支架的构建及研究

胰十二指肠切除术后胰瘘极大地影响了患者的术后生存率及生活质量,目前临床上选择聚氨酯(TPU)管作为胰肠吻合口支架以应对这一并发症,但存在规格不匹配、易脱落等问题。受到树蛙趾垫表面微图形增强湿摩擦力的启发,设计了三种不同的微图形,并利用具有形状记忆功能的生物可降解材料制备了具有不同微图形的胰肠吻合口支架,然后对三种不同微图形的支架进行对比研究。使用6臂聚乙二醇作为引发剂,与己内酯发生开环聚合反应,加入丙烯酰氯接上双键,通过光交联,利用材料的形状记忆性能制备了印有表面微图形的6臂聚乙二醇-聚己内酯胰肠吻合口支架。考察了6臂聚乙二醇-聚己内酯支架的形状记忆性能、力学性能、亲疏水性等,证明该支架具有良好的形状记忆性能,有足够的刚性为胰管提供有效支撑,且能够引流胰液,维持胰管畅通。研究了模拟支架与胰管组织之间的摩擦行为,结果表明,表面微图形改性的6臂聚乙二醇-聚己内酯交联支架比TPU支架具有更大的湿摩擦力,同时对胰管组织造成的磨损较小,且正六边形柱体微图形为表面改性的最佳选择。最后评估了材料的生物相容性,证实材料具备植入体内的安全性,具有潜在的临床应用价值。

喷印系统喷印头结构参数对聚焦性能影响研究

喷印头的设计极大程度上影响着气溶胶喷印系统喷印线条精度的高低。为了优化喷印头的结构参数,应用仿真模拟软件Fluent对某型喷印头喷印过程进行仿真模拟。通过内部流场和颗粒轨迹分析,研究了出口直径一定情况下出口倾斜角、喷管长度、过渡收缩角、入口直径对喷印系统聚焦性能的影响。结果表明,在外界参数不变的条件下,出口倾斜角α=1.5°、喷管长度L=12.3mm、过渡段收缩角β=22°与入口直径D=2.3mm时,该系统喷印头具有最佳聚焦性能。本研究为气溶胶喷印系统喷印头结构参数的选取提供参考依据。

气道平滑肌细胞长距离促进胶原基质的重构作用

本研究利用微加工图形技术,设计了气道平滑肌细胞团的点阵排布,通过荧光标记胶原蛋白,以可控和可视化的方式研究细胞长距离促进胶原基质重构的力学机制。实验结果显示,24 h内,相距300μm的细胞之间能有效生成胶原纤维条带,在相距800μm的细胞团组之间也有此现象,表明细胞能长距离地精确调控胶原基质的重构作用。进一步探索该现象的细胞力学机制,发现加入肌动蛋白骨架和收缩力的抑制剂均阻止了胶原纤维的发生,而添加钙离子抑制剂的实验结果证明细胞钙信号参与了胶原重构。本研究初步验证了细胞收缩力和钙信号的传导作用,可为进一步深入研究细胞群体力学交流水平上的力学信号转导通路提供思路。

高分辨率熔融金属电流体喷印直写工艺

固态金属材料以其高导电性和低成本等特点在柔性电子器件的制造中具有巨大的应用潜力,但现有熔融金属微制造工艺的制造精度有限。为了提高熔融金属在导电结构制备中的分辨率,研发了一种熔融金属电流体喷印直写打印方法。对该方法的基本原理进行阐述。利用自主研发的熔融金属电流体喷印喷头装置,以金属锡为原料进行实验,对打印工艺参数进行了研究。探究了脉冲峰值电压、喷嘴高度、打印速度对锡线成型质量的影响规律,研究结果表明:当脉冲峰值电压4.0 kV,喷嘴高度0.20 mm,打印速度2.0 mm/s时,锡线成型质量最好,能实现线宽约为50μm的锡线制备。最后,结合优化的工艺参数在聚酰亚胺(PI)薄膜上制作了高分辨率导电图案,验证了该方法的可行性,为熔融金属材料在柔性电子器件制造中的应用提供了新途径。