a-novel-method-for-localizing-reporter-fluorescent-beads-near-cell

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

University of Illinois at Urbana-Champaign

In order to achieve a higher axial resolution for displacement measurement, a novel method is proposed based on total internal reflection filter and confocal microscope principle. A theoretical analysis of the basic measurement principles is presented. The analysis reveals that the proposed confocal detection scheme is effective in enhancing the resolution of nonlinearity of the reflectance curve greatly. In addition, a simple prototype system has been developed based on the theoretical analysis and a series of experiments have been performed under laboratory conditions to verify the system feasibility, accuracy, and stability. The experimental results demonstrate that the axial resolution in displacement measurements is better than 1 nm in a range of 200 nm which is threefold better than that can be achieved using the plane reflector.

Superresolution confocal technology for displacement measurements based on total internal reflection.

Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. Traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. Fibronectin (FN), Laminin (LN) and Collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). Pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers strong enough adhesion with ECM proteins such that a pattern can be printed, but weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5-400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces.

A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels.

A variety of cell types exhibit phenotype changes in response to the mechanical stiffness of the substrate. Many cells excluding neurons display an increase in the spread area, actin stress fiber formation and larger focal adhesion complexes as substrate stiffness increases in a sparsely populated culture. Cell proliferation is also known to directly correlate with these phenotype changes/changes in substrate stiffness. Augmented spreading and proliferation on stiffer substrates require nuclear transcriptional regulator YAP (Yes associated protein) localization in the cell nucleus and is tightly coupled to larger traction force generation. In this study, we show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrates (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented spreading and other hard substrate cytoskeleton architectures on soft collagen gels are not correlated with the cell proliferation pattern and do not require YAP localization in the cell nucleus. Finally, we examine the response of human colon carcinoma (HCT-8) cells on soft collagen gels. Recent studies show that human colon carcinoma (HCT-8) cells form multicellular clusters by 2-3 days when cultured on soft polyacrylamide (PA) gels with a wide range of stiffness (0.5-50 kPa) and coated with an extracellular matrix, ECM (collagen monomer/fibronectin). These clusters show limited spreading/wetting on PA gels, form 3D structures at the edges, and eventually display a remarkable, dissociative metastasis like phenotype (MLP), i.e., epithelial to rounded morphological transition after a week of culture on PA gels only, but not on collagen monomer coated stiff polystyrene/glass where they exhibit enhanced wetting and form confluent monolayers. Here, we show that HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels and eventually form confluent monolayers as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results suggest that cell-material interactions (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.

M. Yakut Ali

Department of Mechanical Science and Engineering

University of Illinois at Urbana-Champaign

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

M. Yakut Ali

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Mechanical Science and Engineering

University of Illinois at Urbana-Champaign

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Department of Mechanical Science and Engineering