Microchamber Cultures of Bladder Cancer: A Platform for Characterizing Drug Responsiveness and Resistance in PDX and Primary Cancer Cells.

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.

Harnessing endogenous signals from hepatocytes using a low volume multi-well plate.

Hepatocytes are highly differentiated epithelial cells that lose their phenotype and function when removed from the in vivo environment. Given the importance of hepatic cultures for drug toxicity, bioartificial liver assist devices and basic biology studies, considerable efforts have been focused on the maintenance of hepatic function in vitro. The methods used to date include co-cultivation of hepatocytes with stromal cells, organizing these cells into spheroids and imbedding them into bioactive gels. Our team has recently demonstrated that primary rat hepatocytes confined to microfluidic channels in the absence of convection maintained the epithelial phenotype through upregulation of endogenous signals including hepatocyte growth factor (HGF). The objective of the present study was to transition from microfluidic devices, which are somewhat specialized and challenging to use, towards low volume multiwell plates ubiquitous in biology laboratories. Using a combination of 3D printing and micromolding we have constructed inserts that can be placed into standard 12-well plates and can be used to create low volume culture conditions under which primary hepatocytes maintained a differentiated phenotype. This phenotype enhancement was confirmed by hepatic function assays including albumin synthesis and expression. Importantly we confirmed upregulation of HGF inside the low volume culture plates and demonstrated that inhibition of HGF signaling degraded the hepatic phenotype in our cell culture platform. Overall, this study outlines a new cell culture system that leverages the low volume effects of microfluidic channels in a multiwell plate format. Beyond hepatocytes, such a system may be of use in the maintenance of other difficult-to-culture cells including stem cells and primary cancer cells.

Detecting cell-secreted growth factors in microfluidic devices using bead-based biosensors.

Microfluidic systems provide an interesting alternative to standard macroscale cell cultures due to the decrease in the number of cells and reagents as well as the improved physiology of cells confined to small volumes. However, the tools available for cell-secreted molecules inside microfluidic devices remain limited. In this paper, we describe an integrated microsystem composed of a microfluidic device and a fluorescent microbead-based assay for the detection of the hepatocyte growth factor (HGF) and the transforming growth factor (TGF)-ß1 secreted by primary hepatocytes. This microfluidic system is designed to separate a cell culture chamber from sensing chambers using a permeable hydrogel barrier. Cell-secreted HGF and TGF-ß1 diffuse through the hydrogel barrier into adjacent sensing channels and are detected using fluorescent microbead-based sensors. The specificity of sensing microbeads is defined by the choice of antibodies; therefore, our microfluidic culture system and sensing microbeads may be applied to a variety of cells and cell-secreted factors.

Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels.

The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-ß1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.

Embryonic Stem Cells Cultured in Microfluidic Chambers Take Control of Their Fate by Producing Endogenous Signals Including LIF.

It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced ∼140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. Stem Cells 2016;34:1501-1512.

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.

Impact of Nanotopography, Heparin Hydrogel Microstructures, and Encapsulated Fibroblasts on Phenotype of Primary Hepatocytes.

Hepatocytes, the main epithelial cell type in the liver, perform most of the biochemical functions of the liver. Thus, maintenance of a primary hepatocyte phenotype is crucial for investigations of in vitro drug metabolism, toxicity, and development of bioartificial liver constructs. Here, we report the impact of topographic cues alone and in combination with soluble signals provided by encapsulated feeder cells on maintenance of the primary hepatocyte phenotype. Topographic features were 300 nm deep with pitches of either 400, 1400, or 4000 nm. Hepatocyte cell attachment, morphology and function were markedly better on 400 nm pitch patterns compared with larger scale topographies or planar substrates. Interestingly, topographic features having biomimetic size scale dramatically increased cell adhesion whether or not substrates had been precoated with collagen I. Albumin production in primary hepatocytes cultured on 400 nm pitch substrates without collagen I was maintained over 10 days and was considerably higher compared to albumin synthesis on collagen-coated flat substrates. In order to investigate the potential interaction of soluble cytoactive factors supplied by feeder cells with topographic cues in determining cell phenotype, bioactive heparin-containing hydrogel microstructures were molded (100 µm spacing, 100 µm width) over the surface of the topographically patterned substrates. These hydrogel microstructures either carried encapsulated fibroblasts or were free of cells. Hepatocytes cultured on nanopatterned substrates next to fibroblast carrying hydrogel microstructures were significantly more functional than hepatocytes cultured on nanopatterned surfaces without hydrogels or stromal cells significantly elevated albumin expression and cell junction formation compared to cells provided with topographic cues only. The simultaneous presentation of topographic biomechanical cues along with soluble signaling molecules provided by encapsulated fibroblasts cells resulted in optimal functionality of cultured hepatocytes. The provision of both topographic and soluble signaling cues could enhance our ability to create liver surrogates and inform the development of engineered liver constructs.

A microsystem integrating photodegradable hydrogel microstructures and reconfigurable microfluidics for single-cell analysis and retrieval.

We developed a micropatterned photodegradable hydrogel array integrated with reconfigurable microfluidics to enable cell secretion analysis and cell retrieval at the single-cell level. The activity of protease molecules secreted from single cells was monitored using FRET peptides entrapped inside microfabricated compartments. Antibody-modified gel islands tethering cells to the surface could be degraded by UV exposure to release specific single cells of interest.

Micropatterned sensing hydrogels integrated with reconfigurable microfluidics for detecting protease release from cells.

Matrix metalloproteinases (MMPs) play a central role in the breakdown of the extracellular matrix and are typically upregulated in cancer cells. The goal of the present study is to develop microwells suitable for capture of cells and detection of cell-secreted proteases. Hydrogel microwells comprised of poly(ethylene glycol) (PEG) were photopatterned on glass and modified with ligands to promote cell adhesion. To sense protease release, peptides cleavable by MMP9 were designed to contain a donor/acceptor FRET pair (FITC and DABCYL). These sensing molecules were incorporated into the walls of the hydrogel wells to enable a detection scheme where cells captured within the wells secreted protease molecules which diffused into the gel, cleaved the peptide, and caused a fluorescence signal to come on. By challenging sensing hydrogel microstructures to known concentrations of recombinant MMP9, the limit of detection was determined to be 0.625 nM with a linear range extending to 40 nM. To enhance sensitivity and to limit cross-talk between adjacent sensing sites, microwell arrays containing small groups (∼20 cells/well) of lymphoma cells were integrated into reconfigurable PDMS microfluidic devices. Using this combination of sensing hydrogel microwells and reconfigurable microfluidics, detection of MMP9 release from as few as 11 cells was demonstrated. Smart hydrogel microstructures capable of sequestering small groups of cells and sensing cell function have multiple applications ranging from diagnostics to cell/tissue engineering. Further development of this technology will include single-cell analysis and function-based cell sorting capabilities.

Induced transition of CdSe nanoparticle superstructures by controlling the internal flow of colloidal solution.

The self-assembly behaviors of flow-enhanced CdSe nanoparticle (NP) colloidal systems were investigated, which were systemically prepared by adding ethylene glycol (EG) or acetic acid (AA) to NP suspensions with deionized water (DI water) base. The additive solvents, which had higher boiling points and lower surface tensions than those of the DI water, modified the internal flow of the NP colloidal system, consequently affecting the morphologies of the generated NP superstructures after the full evaporation of their droplets. In flow-enhanced systems, NPs were formed into highly elongated dendrites that stretched from the center region to the edges along the direction of convective flow inside the droplet, while NPs in random drift system were easily aggregated to form cluster-shaped thick dendritic structures. When the volume fraction of EG was increased, the dominant superstructures were changed from dendrites to clusters, which can be mainly attributed to the changes in the dielectric properties of the NP droplets as evaporation proceeded because of the large discrepancy in the vapor pressures of EG and DI water. The balance between the interparticle potentials of electrostatic repulsion and van der Waals attraction was continuously altered, resulting in the formation of clusters with increasing EG ratio. Contrastively, the transition of superstructures could not be observed in the case of colloidal system prepared by mixing DI water and AA, which can be ascribed to the similar vapor pressures of the two solvents; the dielectric properties of the solution mixture was barely changed throughout the steady evaporation process, which resulted in the formation of uniformly distributed highly elongated dendrites. Polarization-dependent imaging experiments and photoluminescence measurements revealed that the stretched dendrites formed under the flow-enhanced conditions showed higher crystallinity than that of the clusters.

Graft copolymer-templated mesoporous TiO(2) films micropatterned with poly(ethylene glycol) hydrogel: novel platform for highly sensitive protein microarrays.

In this study, we describe the use of organized mesoporous titanium oxide (TiO(2)) films as three-dimensional templates for protein microarrays with enhanced protein loading capacity and detection sensitivity. Multilayered mesoporous TiO(2) films with high porosity and good connectivity were synthesized using a graft copolymer consisting of a poly(vinyl chloride) (PVC) backbone and poly(oxyethylene methacrylate) (POEM) side chains as a structure-directing template. The average pore size and thickness of the TiO(2) films were 50-70 nm and 1.5 µm, respectively. Proteins were covalently immobilized onto mesoporous TiO(2) film via 3-aminopropyltriethoxysilane (APTES), and protein loading onto TiO(2) films was about four times greater than on planar glass substrates, which consequently improved the protein activity. Micropatterned mesoporous TiO(2) substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on TiO(2) films using photolithography. Because of non-adhesiveness of PEG hydrogel towards proteins, proteins were selectively immobilized onto surface-modified mesoporous TiO(2) region, creating protein microarray. Specific binding assay between streptavidin/biotin and between PSA/anti-PSA demonstrated that the mesoporous TiO(2)-based protein microarrays yielded higher fluorescence signals and were more sensitive with lower detection limits than microarrays based on planar glass slides.

Non-positional cell microarray prepared by shape-coded polymeric microboards: A new microarray format for multiplex and high throughput cell-based assays.

A non-positional (or suspension) cell microarray was developed using shape-coded SU-8 photoresist microboards for potential application in multiplex and high-throughput cell-based assays. A conventional photolithography process on glass slides produced various shapes of SU-8 micropatterns that had a lateral dimension of 200 µm and a thickness of 40 µm. The resultant micropatterns were detached from the slides by sonication and named "microboards" due to the fact that had a much larger lateral dimension than thickness. The surfaces of the SU-8 microboards were modified with collagen to promote cell adhesion, and it was confirmed that collagen-coated SU-8 microboards supported cell adhesion and proliferation. Seeding of cells into poly(ethylene glycol)(PEG) hydrogel-coated well plates containing collagen-modified microboards resulted in selective cell adhesion onto the microboards due to the non-adhesiveness of PEG hydrogel toward cells, thereby creating non-positional arrays of microboards carrying cells. Finally, two different cell types (fibroblasts and HeLa cells) were separately cultured on different shapes of microboards and subsequently mixed together to create a non-positional cell microarray consisting of multiple cell types where each cell could be easily identified by the shape of the microboard to which they had adhered. Because numerous unique shapes of microboards can be fabricated using this method by simply changing the photomask designs, high throughput and multiplex cell-based assays would be easily achieved with this system in the future.

Phenol biosensor based on hydrogel microarrays entrapping tyrosinase and quantum dots.

This paper describes the use of microarray-based biosensor system for the determination of phenol. Microarrays based on poly(ethylene glycol)(PEG) hydrogel were prepared by photopatterning of a solution containing PEG diacrylate (PEG-DA), photoinitiator, tyrosinase, and CdSe/ZnS quantum dots (QDs). During photo-induced crosslinking, tyrosinase and QDs were entrapped within the hydrogel microarrays, making the hydrogel microarray fluorescent and responsive to phenol. The entrapped tyrosinase could carry out enzyme-catalyzed oxidation of phenol to produce quinones, which subsequently quenched the fluorescence of QDs within hydrogel microarray. The fluorescence intensity of the hydrogel microarrays decreased linearly according to phenol concentration and the detection limit of this system was found to be 1.0 µM. The microarray system presented in this study could be combined with a microfluidic device as an initial step to create a phenol-detecting "micro-total-analysis-system (µ-TAS)".