Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Biomacromolecules ; 18(3): 709-718, 2017 03 13.
Article in English | MEDLINE | ID: mdl-28157290

ABSTRACT

As a result of improved relevance to in vivo physiology, in vitro studies are increasingly performed in diverse, three-dimensional (3D) biomaterials. However, material-cell type pairing effects on cytokine availability remain unclear. We cultured five cell types in agarose, alginate, collagen, Matrigel, or RGD-functionalized polyethylene glycol (PEG) hydrogels. We measured 21 cytokines in the conditioned media, and we identified differences in measured cytokine levels that were cell-type- or material-dependent. We further evaluated our data using principal component analysis. Interestingly, component one identified two classes of biomaterials with characteristic cytokine expression levels. Component two identified cell-type-dependent differences in cytokines related to the wound response. Although elements of soluble cytokine availability are shared despite parameter differences, material and cellular properties variably influenced cytokine levels, underlining the influence of biomaterial-cell type pairings on in vitro assay outcomes. Relationships between material properties, cellular responses, and cytokine availability in 3D in vitro models warrant further investigation.


Subject(s)
Biocompatible Materials/chemistry , Cytokines/biosynthesis , Cell Culture Techniques , Cell Line, Tumor , Culture Media/chemistry , Epithelial Cells/metabolism , Humans , Hydrogels/chemistry , Polyethylene Glycols/chemistry , Principal Component Analysis
2.
Cell Mol Life Sci ; 72(2): 237-49, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25274061

ABSTRACT

The increasing interest in studying cells using more in vivo-like three-dimensional (3D) microenvironments has created a need for advanced 3D screening platforms with enhanced functionalities and increased throughput. 3D screening platforms that better mimic in vivo microenvironments with enhanced throughput would provide more in-depth understanding of the complexity and heterogeneity of microenvironments. The platforms would also better predict the toxicity and efficacy of potential drugs in physiologically relevant conditions. Traditional 3D culture models (e.g., spinner flasks, gyratory rotation devices, non-adhesive surfaces, polymers) were developed to create 3D multicellular structures. However, these traditional systems require large volumes of reagents and cells, and are not compatible with high-throughput screening (HTS) systems. Microscale technology offers the miniaturization of 3D cultures and allows efficient screening of various conditions. This review will discuss the development, most influential works, and current advantages and challenges of microscale culture systems for screening cells in 3D microenvironments.


Subject(s)
Cell Culture Techniques/methods , Cellular Microenvironment/physiology , High-Throughput Screening Assays/methods , Microfluidic Analytical Techniques/methods , Microtechnology/methods , Tissue Array Analysis/methods
3.
PLoS One ; 8(12): e81689, 2013.
Article in English | MEDLINE | ID: mdl-24349113

ABSTRACT

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced ß1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with ß1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.


Subject(s)
Cell Movement/genetics , Cell Transformation, Neoplastic , Fibroblasts/pathology , Phenotype , Actins/genetics , Actins/metabolism , CD146 Antigen/genetics , CD146 Antigen/metabolism , Cell Adhesion , Cell Culture Techniques , Cell Line, Tumor , Extracellular Matrix/chemistry , Fibroblasts/metabolism , Gene Expression , Humans , Hydrogels , Integrin beta1/genetics , Integrin beta1/metabolism , Matrix Metalloproteinases/chemistry , Molecular Mimicry , Primary Cell Culture , Vinculin/genetics , Vinculin/metabolism
4.
Proc Natl Acad Sci U S A ; 110(25): 10111-6, 2013 Jun 18.
Article in English | MEDLINE | ID: mdl-23729815

ABSTRACT

Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (µDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that µDots can also be used as a simple multiplexed 3D cellular growth platform. Using the µDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.


Subject(s)
Microfluidic Analytical Techniques , Microfluidics/instrumentation , Microfluidics/methods , Models, Biological , Adrenal Cortex/cytology , Breast Neoplasms/pathology , Capillaries/metabolism , Cell Biology/instrumentation , Cell Line, Tumor , Cell Membrane/physiology , Cell Movement , Collagen Type I/metabolism , Computer Simulation , Female , Humans , Hydrocortisone/analysis , Hydrocortisone/metabolism , Male , Matrix Metalloproteinase Inhibitors/pharmacology , Metabolomics/instrumentation , Metabolomics/methods , Prostatic Neoplasms/pathology , Steroids/analysis , Steroids/metabolism , Toxicology/instrumentation , Toxicology/methods
5.
Integr Biol (Camb) ; 5(3): 631-40, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23340769

ABSTRACT

During breast carcinoma progression, the three-dimensional (3D) microenvironment is continuously remodeled, and changes in the composition of the extracellular matrix (ECM) occur. High throughput screening platforms have been used to decipher the complexity of the microenvironment and to identify ECM components responsible for cancer progression. However, traditional screening platforms are typically limited to two-dimensional (2D) cultures, and often exclude the influence of ECM and stromal components. In this work, a system that integrates 3-dimensional cell culture techniques with an automated microfluidic platform was used to create a new ECM screening platform that cultures cells in more physiologically relevant 3D in vitro microenvironments containing stromal cells and different ECM molecules. This new ECM screening platform was used to culture T47D breast carcinoma cells in mono- and co-culture with human mammary fibroblasts (HMF) with seven combinations of three different ECM proteins (collagen, fibronectin, laminin). Differences in the morphology of T47D clusters, and the proliferation of T47D cells were found in ECM compositions rich in fibronectin or laminin. In addition, an MMP enzyme activity inhibition screening showed the capabilities of the platform for small molecule screening. The platform presented in this work enables screening for the effects of matrix and stromal compositions and show promises for providing new insights in the identification of key ECM components involved in breast cancer.


Subject(s)
Breast Neoplasms/pathology , Cell Culture Techniques , Extracellular Matrix/metabolism , Automation , Breast/cytology , Cell Line, Tumor , Cell Proliferation , Coculture Techniques , Disease Progression , Female , Fibronectins/metabolism , Humans , Image Processing, Computer-Assisted , Laminin/metabolism , Polymers/chemistry
6.
J Lab Autom ; 16(3): 171-85, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21609700

ABSTRACT

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions.


Subject(s)
Automation, Laboratory/methods , Microfluidic Analytical Techniques , Cell Culture Techniques/methods , Cell Line, Tumor , Coculture Techniques/methods , Collagen Type I/metabolism , Culture Media/chemistry , Fibroblasts/physiology , Humans , Temperature
SELECTION OF CITATIONS
SEARCH DETAIL
...