Towards single-cell bioprinting:micropatterning tools for organ-on-chip development

Organs-on-chips (OoCs) hold promise to engineer progressively more human-relevant in vitro models for pharmaceutical purposes. Recent developments have delivered increasingly sophisticated designs, yet OoCs still lack in reproducing the inner tissue physiology required to fully resemble the native human body. This review emphasizes the need to include microarchitectural and microstructural features, and discusses promising avenues to incorporate well-defined microarchitectures down to the single-cell level. We highlight how their integration will significantly contribute to the advancement of the field towards highly organized structural and hierarchical tissues-on-chip. We discuss the combination of state-of-the-art micropatterning technologies to achieve OoCs resembling human-intrinsic complexity. It is anticipated that these innovations will yield significant advances in realization of the next generation of OoC models.</p

Tumor cell migration in complex microenvironments

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable.National Science Foundation (U.S.) (Graduate Research Fellowship)Charles Stark Draper Laboratory (Research and Development Program (N.DL-H-550151))National Cancer Institute (U.S.) (R21CA140096

Parsing the Effects of Physical Microenvironmental Cues on Stem Cell Adhesion and Lineage Specification

Tissue engineering is a broad field geared toward improving or replacing biological material and comprises an immense collection of biological nuances to consider before strategies for clinical applications can be fully realized. Physical and biochemical signals are responsible for making up a cell\u27s microenvironment to guide morphology and function through cell-extracellular matrix signaling, cell-cell signaling, and soluble signaling. In particular, a deeper understanding of these cell-extra cellular matrix factors guiding stem cell adhesion, spreading, and differentiation is crucial to harnessing the potential to develop tissue for regenerative purposes. Mounting evidence suggests that physical cues are a key to understanding the potential of stem cells and significant efforts have been made to begin to parse the effects of cell-matrix interactions, yet little is known about the interplay in guiding cell signaling. The work presented here focuses on utilizing novel methods and materials to deconstruct individual cell-matrix interactions and gain a deeper understanding of the cooperative signaling behaviors for mesenchymal and embryonic stem cells. Micropatterning studies utilizing dip pen nanolithography showed that physical signals in the microenvironment are vital to regulating mesenchymal stem cell adhesion. Matrix elasticity, ligand density, and adhesion topography were individually altered to observe single cell adhesion and spreading with matrix elasticity proving to regulate the adhesion and spreading of the cells. Photolithography based studies detailing cell spreading and matrix elasticity showed that when confining single cells into different geometric shapes and sizes on a matrix of tunable elasticity, cell shape and size ultimately became responsible for stem cell lineage commitment over matrix elasticity. Signaling pathway inhibition experiments utilizing nocodazole and Y-27632 suggested that RhoA is a key regulator of cell response to the cooperative effect of these tunable substrate variables. Embryonic stem cells were then micropatterned on novel UV/ozone modified polystyrene to detail and observe the physical effects on single embryonic stem cells. Micropatterned cells were able to be cultured for up to 48 hours on patterns while forming stress fibers and focal adhesions similar to somatic cells, thereby demonstrating their responsiveness to extracellular matrix cues while maintaining expression of pluripotency transcription factor Oct4. The results from this work validate the immense importance of physical signaling and the effects on mesenchymal and embryonic stem cells. By understanding the effects of physical signaling in conjunction with biochemical signaling in controlling cell spreading and lineage commitment, tissue engineering is able to draw one step closer to potential applications for repairing and replacing biological function

Bio-Inspired Materials For Parsing Matrix Physicochemical Control Of Cell Migration: A Review

Cell motility is ubiquitous in both normal and pathophysiological processes. It is a complex biophysical response elicited via the integration of diverse extracellular physicochemical cues. The extracellular matrix directs cell motilityvia gradients in morphogens (a.k.a. chemotaxis), adhesive proteins (haptotaxis), and stiffness (durotaxis). Three-dimensional geometrical and proteolytic cues also constitute key regulators of motility. Therefore, cells process a variety of physicochemical signals simultaneously, while making informed decisions about migration viaintracellular processing. Over the last few decades, bioengineers have created and refined natural and synthetic in vitro platforms in an attempt to isolate these extracellular cues and tease out how cells are able to translate this complex array of dynamic biochemical and biophysical features into functional motility. Here, we review how biomaterials have played a key role in the development of these types of model systems, and how recent advances in engineered materials have significantly contributed to our current understanding of the mechanisms of cell migration

ナノ及びマイクロパターン化表面による間葉系幹細胞の形態と機能の制御

筑波大学 (University of Tsukuba)201

Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

BACKGROUND: The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells. PRINCIPAL FINDINGS: Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication. CONCLUSION/SIGNIFICANCE: We demonstrated for the first time the modulation of HepG2 cells' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics

Electrical Coupling Between Micropatterned Cardiomyocytes and Stem Cells

To understand how stem cells functionally couple with native cardiomyocytes is crucial for cell-based therapies to restore the loss of cardiomyocytes that occurs during heart infarction and other cardiac diseases. Due to the complexity of the in vivo environment, our knowledge of cell coupling is heavily dependent on cell-culture models. However, conventional in vitro studies involve undefined cell shapes and random length of cell-cell contacts in addition to the presence of multiple homotypic and heterotypic contacts between interacting cells. Thus, it has not been feasible to study electrical coupling corresponding to isolated specific types of cell contact modes. To address this issue, we used microfabrication techniques to develop different geometrically-defined stem cell-cardiomyocyte contact assays to comparatively and quantitatively study functional stem cell-cardiomyocyte electrical coupling. Through geometric confinements, we will construct a matrix of identical microwells, and each was constructed as a specific microenvironment. Using laser-guided cell micropatterning technique, individual stem cells or cardiomyocytes can be deposited into the microwells to form certain contact mode. In this research, we firstly constructed an in-vivo like cardiac muscle fiber microenvironment, and the electrical conductivity of stem cells was investigated by inserting stem cells as cellular bridges. Then, the electrical coupling between cardiomyocytes and stem cells was studied at single-cell level by constructing contact-promotive/-preventive microenvironments

Engenharia de estruturas semelhantes a capilares incorporadas em hidrogéis para cultura de células 3D

Nowadays, the biggest challenge in tissue engineering consists in developing structures and in the application of strategies to emulate the anatomical and cellular complexity and vascularization of native tissues to maintain cell viability and functionality. The presence of functional blood vessel networks is essential to ensure adequate nutrient flow and oxygen diffusion throughout the support structure, two key requirements for maintaining cell viability. This work aimed to develop a complex in vitro model that mimics the native vascular network. To this end, a multilayered membrane made of six bilayers of chitosan (CHI)/alginate (ALG) or CHI/ALG-RGD (tripeptide of Arginine (R)-Glycine (G)- Aspartic acid (D) responsible for the cellular adhesion to the extracellular matrix (ECM)) were produced via Layer-by-Layer (LbL) assembly technology on the ALG printed structures. The ALG structures coated with the multilayered membranes were embedded in xanthan gum, chemically modified with methacrylated groups in order to obtain a mechanically robust hydrogel structure after photocrosslinking by UV light exposure. The liquification of the ALG printed structures, coated with the CHI/ALG, CHI/ALG-RGD or without the multilayers membranes, with ethylenediaminetetraacetic acid (EDTA), led to the formation of microchannels in which human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours. The obtained results demonstrate that the microchannels encompassing CHI/ALG-RGD multilayered membranes contributed to a larger cellular adhesion, demonstrating their potential to be applied in tissue engineering and regenerative medicine strategies.Atualmente, o maior desafio em engenharia de tecidos consiste no desenvolvimento de estruturas e aplicação de estratégias que visem mimetizar a complexidade anatómica e celular, assim como a vascularização de tecidos nativos, de forma a manter a viabilidade e funcionalidade das células. A presença de estruturas funcionais à base de vasos sanguíneos é essencial para garantir o fluxo adequado de nutrientes, assim como a difusão de oxigénio em toda a estrutura de suporte, dois requisitos essenciais para manter a viabilidade celular. Este trabalho teve como objetivo desenvolver um modelo complexo in vitro que mimetize a rede vascular nativa. Com esse intuito, membranas multicamadas compreendendo seis bicamadas de quitosana (CHI)/alginato (ALG) e CHI/ALG-RGD (tripéptido de Arginina (R)-Glicina (G)-Ácido aspártico (D) responsável pela adesão de células à matriz extracelular) foram produzidas, via tecnologia de deposição camada-a-camada (do inglês Layer-by-Layer assembly technology), em estruturas impressas de ALG. As fibras de ALG revestidas com os filmes multicamadas foram embebidas em goma xantana, quimicamente modificada com grupos metacrilatos, de modo a obter uma estrutura de hidrogel mecanicamente robusta após foto-reticulação por ação da luz UV. A liquefação das estruturas impressas de ALG, contendo as multicamadas de CHI/ALG ou CHi/ALG-RGD, com ácido etilenodiamino tetra-acético (EDTA), levou à formação de microcanais nos quais se cultivaram células endoteliais humanas, extraídas da veia umbilical durante 24 horas. Os resultados obtidos demonstraram que os microcanais compreendendo as membranas multicamadas à base de CHI/ALG-RGD contribuíram para uma maior adesão celular, demonstrando o seu potencial para estratégias de engenharia de tecidos e medicina regenerativa.Mestrado em Biotecnologi

Ultrashort-pulsed laser ablation of poly-L-lactide (PLLA) for cell and tissue engineering applications

El contenido de los capítulos 3 y 4 están sujetos a confidencialidad 167 p.La tecnología de ablación láser es una herramienta bien establecida para la modificación superficial de materiales de distinta naturaleza (metales, polímeros, cerámicas, vidrio¿). La ablación de material mediante láseres de pulso ultracorto (menor que 10 picosegundos) es capaz de generar motivos topográficos micrométricos con una alta precisión debido a un proceso de ablación ¿frío¿ minimizando los efectos térmicos en el material sin producir cambios químicos en el mismo. Es por tanto una tecnología versátil para la fabricación de superficies microestructuradas en un proceso directo y sin contacto y aplicable a una gran variedad de materiales para generar motivos con distintas geometrías sobre superficies no planas. En este trabajo de tesis se aplica la tecnología de ablación mediante láser pulsado de picosegundos para la creación de micro-patrones topográficos en planchas de ácido poli-L-láctico (PLLA), para investigar el mecanismo de ablación del mismo y el efecto de los micro-patrones en el comportamiento de varios tipos de células mediante ensayos in vitro, con el objetivo final de elucidar el alcance de la influencia de estos micro-patrones en el comportamiento celular y evaluar la tecnología como método de fabricación de soportes en la ingeniería de tejidos