The Influence of Biomaterials on Cytokine Production in 3D Cultures.

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.

Microfluidic 3D models of cancer.

Despite advances in medicine and biomedical sciences, cancer still remains a major health issue. Complex interactions between tumors and their microenvironment contribute to tumor initiation and progression and also contribute to the development of drug resistant tumor cell populations. The complexity and heterogeneity of tumors and their microenvironment make it challenging to both study and treat cancer. Traditional animal cancer models and in vitro cancer models are limited in their ability to recapitulate human structures and functions, thus hindering the identification of appropriate drug targets and therapeutic strategies. The development and application of microfluidic 3D cancer models have the potential to overcome some of the limitations inherent to traditional models. This review summarizes the progress in microfluidic 3D cancer models, their benefits, and their broad application to basic cancer biology, drug screening, and drug discovery.

Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

Enabling screening in 3D microenvironments: probing matrix and stromal effects on the morphology and proliferation of T47D breast carcinoma cells.

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.

Automation of three-dimensional cell culture in arrayed microfluidic devices.

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.

Transition to invasion in breast cancer: a microfluidic in vitro model enables examination of spatial and temporal effects.

The transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer progression. We introduce a simple microfluidic 3D compartmentalized system in which mammary epithelial cells (MCF-DCIS) are co-cultured with human mammary fibroblasts (HMFs), which promotes a transition from DCIS to IDC in vitro. The model enables control of both spatial (distance-dependence) and temporal (transition from larger clusters) aspects within the microenvironment, allowing recapitulation of the in vivo environment in ways not practical with existing experimental models. When HMFs were cultured some distance (0.5-1.5 mm) from the MCF-DCIS cells, we observed an initial morphological change, suggesting soluble factors can begin the transition. However, cell-cell contact with HMFs allowed the MCF-DCIS cells to complete the transition to invasion. Uniquely, the compartmentalized platform enables the analysis of the intrinsic second harmonic generation signal of collagen, providing a label-free quantitative analysis of DCIS-associated collagen remodeling. The arrayed microchannel-based model is compatible with existing infrastructure and, for the first time, provides a cost effective approach to test for inhibitors of pathways involved in DCIS progression to IDC allowing a screening approach to the identification of potential therapeutic targets. Importantly, the model can be easily adapted and generalized to a variety of cell-cell signaling studies.