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.

Microscale screening systems for 3D cellular microenvironments: platforms, advances, and challenges.

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.

Toward Assembly of Non-close-packed Colloidal Structures from Anisotropic Pentamer Particles.

Particles combining multiple anisotropy dimensions offer possibilities for self-assembly that have not been extensively explored to date. The scope for assembly of microparticles in which the anisotropy dimensions of internal bond angle and chemical ordering have been varied is investigated. Colloidal assemblies with interesting open (i.e., non-close-packed) structures can be assembled from these building blocks. The structure of the assemblies formed is linked to the building block anisotropy because the steric constraints introduced induce deviations from close packing. Key challenges addressed in pursuit of these structures are parallelization of microfluidic synthesis methods, simulation to efficiently search the available anisotropy space, and methods that characterize the properties of the resulting assemblies. This combined program of synthesis, simulation, assembly, and characterization may be applied to develop design rules that guide efforts to fabricate microparticle building blocks and their assemblies.

Programmable fluidic production of microparticles with configurable anisotropy.

We report a technique for continuous production of microparticles of variable size with new forms of anisotropy including alternating bond angles, configurable patchiness, and uniform roughness. The sequence and shape of the anisotropic particles are configured by exploiting a combination of confinement effects and microfluidics to pack precursor colloids with different properties into a narrow, terminal channel. The width and length of the channel relative to the particle size fully specify the configuration of the anisotropic particle that will be produced. The precursor spheres packed in the production zone are then permanently bonded into particles by thermal fusing. The flow in the production zone is reversed to release the particles for collection and use. Particles produced have linear chain structure with precisely configured, repeatable bond angles. With software programmable microfluidics, sequence and shape anisotropy are combined to yield synthesized homogeneous (type "A"), surfactantlike (type "A-B") or triblock (type "A-B-A") internal sequences in a single device. By controlling the dimensions of the microfluidic production zone, triangular prisms and particles with controlled roughness and patchiness are produced. The fabrication method is performed with precursors spheres with diameter as small as 3.0 microm.

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.

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.

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.

Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices.

Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5-8.5) and pre-incubation at low temperature (approximately 4 degrees C) before polymerization at 37 degrees C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.

Fluidic assembly and packing of microspheres in confined channels.

We study fluidic assembly and packing of spherical particles in rectilinear microchannels that are terminated by a flow constriction. First, we introduce a method for active assembly of particles in the confined microchannels by triggering a local constriction in the fluid channel using a partially closed membrane valve. This microfluidic valve allows active, on-demand particle assembly as opposed to previous passive assembly methods based on terminal channels and weirs. Second, we study the three-dimensional assembly and packing of particles against a weir in confined rectilinear microchannels. The packings result in achiral particle chains with alternating (zigzag) structure. This structure is characterized by a single, repeated bond angle whose components projected into the frame of the channel are quantified by confocal microscopy and image processing. Brownian dynamics simulation of the packing comprehensively delineates the range of bond angles possible in narrow, rectilinear microchannels as well as the complex dependence of these angles on the relative dimensions of the channel and particles. The simulations of the three-dimensional packings are accurately modeled by a compact theory based on trigonometric relationships. The experimentally measured bond angles show excellent agreement with the simulations, thereby validating the functional dependence of the achiral packing bond angles on channel dimensions. This functional relationship is immediately useful for the design of anisotropic particles by microfluidic synthesis.

Optimization of dielectrophoretic DNA stretching in microfabricated devices.

We have found that the surface and bulk solution properties in a microfabricated device affect the degree and probability of electrostretching of DNA molecules. Using lambda phage DNA, we found that significantly hydrophilic surfaces between the electrodes decrease the efficiency of stretching. Surfaces treated with higher silane (trimethylchlorosilane) concentrations performed better presumably due to the decreased nonspecific adsorption of DNA on these surfaces compared to their more hydrophilic counterparts. The shape and dimensions of the electrodes also affected the efficiency of stretching. Both liftoff and metal etching methods produced electrodes with random microscopic peaks along the electrode's edge and were poorly suited for stretching. Annealing the electrodes (450 degrees C for 10 min) removed most of these peaks and allowed for more controlled stretching to be obtained. We also found that thin electrodes (65 nm) gave close to a 90% success rate of DNA stretching but stretching with thick electrodes (350 nm) produced only a 20% success rate.

Microstencils for the patterning of nontraditional materials.

A microfabrication technique that uses a photolithographically patterned film as a microstencil has been developed. This microstencil has a bilayer structure comprised of parylene and SU-8 films with thicknesses from 4 to 100 microm. The parylene layer enables the microstencil to be mechanically peeled from hydrophilic substrates. Since no chemicals are required to release the microstencil, this technique can be used to pattern chemically and biologically sensitive materials. The amount of material deposited can be automatically controlled by the height of the SU-8 structures or externally controlled by spin coating or other thin film deposition techniques. This patterning method is very versatile and has been used to pattern features as small as 25 by 25 microm on silicon, glass, and polymer substrates. As an initial demonstration, we have patterned wax, cells, proteins, sol, and CYTOP.