scholarly journals Development of Gelatin-Based Flexible Three-Dimensional Capillary Pattern Microfabrication Technology for Analysis of Collective Cell Migration

2021 ◽  
Vol 4 (1) ◽  
pp. 11
Author(s):  
Hiromichi Hashimoto ◽  
Mitsuru Sentoku ◽  
Kento Iida ◽  
Kenji Yasuda
Keyword(s):  
Cell Migration ◽  
Structural Changes ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Migration Velocity ◽  
In Vitro Assays ◽  
Collective Cell Migration ◽  
Interactive Behavior ◽  
Microfabrication Technology

Collective cell migration is thought to be a dynamic and interactive behavior of cell cohorts that is essential for diverse physiological developments in living organisms. Recent studies revealed that the topographical properties of the environment regulate the migration modes of cell cohorts, such as diffusion versus contraction relaxation transport and the appearance of vortices in larger available space. However, conventional in vitro assays fail to observe changes in cell behavior in response to the structural changes. In this study, we developed a method to fabricate the flexible three-dimensional structures of capillary microtunnels to examine the behavior of vascular endothelial cells (ECs). Microtunnels with altering diameters were formed inside gelatin gel through spot heating a portion of gelatin by irradiating the µm-sized absorption at the tip of the microneedle with a focused permeable 1064 nm infrared laser. The ECs moved and spread two-dimensionally on the inner surface of the capillary microtunnels as a monolayer instead of filling the capillary. In contrast to the 3D straight topographical constraint, which exhibited width-dependent migration velocity, the leading ECs altered its migration velocity according to the change in the supply of cells behind the leading ECs, caused by their progression through the diameter-altering structure. Our findings provide insights into the collective migration modes inside 3D confinement structures, including their fluid-like behavior and the conservation of cell numbers.

scholarly journals Modeling Liver Organogenesis by Recreating Three-Dimensional Collective Cell Migration: A Role for TGFβ Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Ogechi Ogoke ◽  
Osama Yousef ◽  
Cortney Ott ◽  
Allison Kalinousky ◽  
Wayne Lin ◽  
...  
Keyword(s):  
Cell Migration ◽  
Liver Cell ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Three Dimensional ◽  
3D Models ◽  
Branching Morphogenesis ◽  
Lung Fibroblasts ◽  
Collective Cell Migration

Three-dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development and organogenesis. Mechanisms of CCM differ in 2D vs. 3D systems, and existing models are limited to 2D or transwell-based systems, suggesting there is a need for improved 3D models of CCM. To recreate liver 3D CCM, we engineered in vitro 3D models based upon a morphogenetic transition that occurs during liver organogenesis, which occurs rapidly between E8.5 and E9.5 (mouse). During this morphogenetic transition, 3D CCM exhibits co-migration (multiple cell types), thick-strand interactions with surrounding septum transversum mesenchyme (STM), branching morphogenesis, and 3D interstitial migration. Here, we engineer several 3D in vitro culture systems, each of which mimics one of these processes in vitro. In mixed spheroids bearing both liver cells and uniquely MRC-5 (fetal lung) fibroblasts, we observed evidence of co-migration, and a significant increase in length and number of liver spheroid protrusions, which was highly sensitive to transforming growth factor beta 1 (TGFβ1) stimulation. In MRC-5-conditioned medium (M-CM) experiments, we observed dose-dependent branching morphogenesis associated with an upregulation of Twist1, which was inhibited by a broad TGFβ inhibitor. In models in which liver spheroids and MRC-5 spheroids were co-cultured, we observed complex strand morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor and thicken to form permanent and thick anchoring contacts between the two spheroids. In these spheroid co-culture models, we also observed spheroid fusion and strong evidence for interstitial migration. In conclusion, we present several novel cultivation systems that recreate distinct features of liver 3D CCM. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases like cancer, and liver cell therapy, and will also serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.

scholarly journals Mesenchyme modulates three-dimensional, collective cell migration of liver cells in vitro - a role for TGFß pathway

2020 ◽  
Author(s):  
Ogechi Ogoke ◽  
Osama Yousef ◽  
Cortney Ott ◽  
Allison Kalinousky ◽  
Lin Wayne ◽  
...  
Keyword(s):  
Cell Migration ◽  
Liver Cell ◽  
Three Dimensional ◽  
Branching Morphogenesis ◽  
Lung Fibroblasts ◽  
Collective Cell Migration ◽  
Paracrine Effects ◽  
Cell Therapies

ABSTRACTThree dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development/ organogenesis. Here, we modeled liver organogenesis to induce 3D CCM and improve existing models. The liver diverticulum, normally surrounded by septum transversum mesenchyme (STM) at E8.5, was modeled with a miniature liver spheroid surrounded by mesenchymal cells and matrix. In mixed spheroid models with both liver and uniquely MRC5 (fetal lung) fibroblasts, we observed co-migration of cells, and a significant increase in length and number of liver spheroid protrusions, and this was highly sensitive to TGFB1 stimulation. To understand paracrine effects between MRC-5 cells and liver, we performed conditioned medium (M-CM) experiments. Interestingly, the addition of M-CM increased liver 3D CCM, with thin, 3D, dose-dependent branching morphogenesis, an upregulation of Twist1, and a sensitivity to a broad TGFB inhibitor. To test the effects of cell-cell interactions of 3D CCM, the STM was modeled with a spheroid of MRC-5 cells, and we performed co-spheroid culture of liver with MRC-5. We observed a complex morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor, and thicken to form permanent and thick anchoring contacts between the two spheroids. We also observed spheroid fusion, a form of interstitial migration. In conclusion, we present several novel cultivation systems that induce distinct features of 3D CCM, as judged by the presence of branching, linearity, thickness, and interstitial migration. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases, and liver cell therapy, and will serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.

scholarly journals Theory of mechano-chemical patterning and optimal migration in cell monolayers

2020 ◽  
Author(s):  
Daniel Boocock ◽  
Naoya Hino ◽  
Natalia Ruzickova ◽  
Tsuyoshi Hirashima ◽  
Edouard Hannezo
Keyword(s):  
Cell Migration ◽  
Mapk Pathway ◽  
Three Dimensional ◽  
Collective Cell Migration ◽  
Chemical Patterning ◽  
Collective Behaviors ◽  
Erk Mapk ◽  
Spatio Temporal

AbstractCollective cell migration offers a rich field of study for non-equilibrium physics and cellular biology, revealing phenomena such as glassy dynamics [1], pattern formation [2] and active turbulence [3]. However, how mechanical and chemical signaling are integrated at the cellular level to give rise to such collective behaviors remains unclear. We address this by focusing on the highly conserved phenomenon of spatio-temporal waves of density [2, 4–8] and ERK/MAPK activation [9–11], which appear both in vitro and in vivo during collective cell migration and wound healing. First, we propose a biophysical theory, backed by mechanical and optogenetic perturbation experiments, showing that patterns can be quantitatively explained by a mechano-chemical coupling between three-dimensional active cellular tensions and the mechano-sensitive ERK/MAPK pathway. Next, we demonstrate how this biophysical mechanism can robustly induce migration in a desired orientation, and we determine a theoretically optimal pattern for inducing efficient collective migration fitting well with experimentally observed dynamics. We thereby provide a bridge between the biophysical origin of spatio-temporal instabilities and the design principles of robust and efficient long-ranged migration.

scholarly journals Weakening of resistance force by cell–ECM interactions regulate cell migration directionality and pattern formation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masaya Hagiwara ◽  
Hisataka Maruyama ◽  
Masakazu Akiyama ◽  
Isabel Koh ◽  
Fumihito Arai
Keyword(s):  
Cell Migration ◽  
Pattern Formation ◽  
Epithelial Cells ◽  
Optical Tweezers ◽  
Three Dimensional ◽  
Lung Epithelial Cells ◽  
Resistance Force ◽  
Collective Cell Migration ◽  
Physical Forces ◽  
Cellular Movement

AbstractCollective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell–cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.

scholarly journals Self-organized cell migration across scales – from single cell movement to tissue formation

Development ◽  
2021 ◽  
Vol 148 (7) ◽  
pp. dev191767
Author(s):  
Jessica Stock ◽  
Andrea Pauli
Keyword(s):  
Cell Migration ◽  
Multiple Scales ◽  
Single Cells ◽  
Cell Movement ◽  
Biological Response ◽  
Collective Cell Migration ◽  
Theoretical Concepts ◽  
Self Organizing

ABSTRACTSelf-organization is a key feature of many biological and developmental processes, including cell migration. Although cell migration has traditionally been viewed as a biological response to extrinsic signals, advances within the past two decades have highlighted the importance of intrinsic self-organizing properties to direct cell migration on multiple scales. In this Review, we will explore self-organizing mechanisms that lay the foundation for both single and collective cell migration. Based on in vitro and in vivo examples, we will discuss theoretical concepts that underlie the persistent migration of single cells in the absence of directional guidance cues, and the formation of an autonomous cell collective that drives coordinated migration. Finally, we highlight the general implications of self-organizing principles guiding cell migration for biological and medical research.

Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

1983 ◽  
Vol 60 (1) ◽  
pp. 89-102
Author(s):  
D de Bono ◽  
C. Green
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Pure Cultures ◽  
Species Specific ◽  
Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

scholarly journals Cell migration and organization in three-dimensional in vitro culture driven by stiffness gradient

2016 ◽  
Vol 113 (11) ◽  
pp. 2496-2506 ◽  
Author(s):  
Danielle Joaquin ◽  
Michael Grigola ◽  
Gubeum Kwon ◽  
Christopher Blasius ◽  
Yutao Han ◽  
...  
Keyword(s):  
Cell Migration ◽  
In Vitro Culture ◽  
Three Dimensional

scholarly journals Validation of in vitro assays in three-dimensional human dermal constructs

2018 ◽  
Vol 41 (11) ◽  
pp. 779-788 ◽  
Author(s):  
Ayesha Idrees ◽  
Valeria Chiono ◽  
Gianluca Ciardelli ◽  
Siegfried Shah ◽  
Richard Viebahn ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
In Vitro Assays ◽  
Dimensional System ◽  
Type I ◽  
Human Dermal Fibroblasts ◽  
Cell Viability Assay ◽  
Normal Human ◽  
Normal Human Dermal Fibroblasts

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially “murine in vitro dermal construct” based on L929 cells was generated, leading to the development of “human in vitro dermal construct” consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue®, RealTime-Glo™ MT, and CellTiter-Glo® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the “shaking time” to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

2019 ◽  
Vol 44 (6) ◽  
pp. 810-821
Author(s):  
Edibe Avci ◽  
Yeliz Z. Akkaya-Ulum ◽  
Digdem Yoyen-Ermis ◽  
Gunes Esendagli ◽  
Banu Balci-Peynircioglu
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Neutrophil Migration ◽  
Cost Effective ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

Sign in / Sign up

Export Citation Format

Share Document