Microfluidics-Generated Immobilized Biomolecule Gradients on Hydrogels

Extracellular matrix (ECM)-tethered morphogen gradients play an important role in tissue development and regeneration. There is currently a limited set of model systems that can mimic these gradients in an artificial ECM-like environment. Microfluidics offers several possibilities to create soluble or substrate-adsorbed protein gradients, which does not exactly represent the in vivo conditions where many proteins are immobilized in a three-dimensional environment. For this reason, the aim of this project has been to develop a model system that immobilizes a microfluidics-generated protein gradient on a poly (ethylene) glycol (PEG) hydrogel. To achieve this, thin films of a PEG hydrogel with incorporated neutravidin were cast on a glass slide and pressure sealed with a polydimethylsyloxane piece containing the microfluidic gradient generator. A stable gradient of biotinylated proteins was then applied and monitored under the microscope. The biotinylated proteins were captured by the neutravidin within the hydrogel thereby immobilizing the gradient. After disassembling the microfluidic device, the hydrogels could directly be used for cell-based assays. As a proof of principle, hydro gels were patterned with biotinylated fibronectin gradients and seeded with primary human dermal fibroblasts. Preliminary time-lapse microscopy experiments demonstrated a fibronectin concentration-dependent behavior of the fibroblasts. This novel model system enhances microfluidically generated protein gradients in several ways, because the patterned hydrogel mimics much better the in vivo situation than adsorped protein gradients on hard substrates. This platform can serve for cell migration and differentiation studies that more closely reproduce the in-vivo microenvironment

Hydrogel Microfluidics to Control Stem Cell Fate

Biomolecular signaling is of utmost importance in governing many biological processes such as morphogenesis during tissue development where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion and in the context of a soft and hydrated microenvironment. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro- scale is far from ideal due to issues related to medium evaporation, limited space for cell growth, shear stress and a lack of cell-instructive microenvironments that might adversely impact cell-fate. As a result, microfluidic cell culture systems are not yet suitable to unravel complex multicellular phenomena. This may explain why they are not yet widely used in biology laboratories. Consequently, the overall goal of this thesis was to overcome this technology gap by developing next generation microfluidics through a combination of microfabrication and smart biomaterials. A special emphasis was placed on decoupling biomolecule presentation at micro-scale from macro-scale cell culture in order to enable long-term stem cell culture within user-friendly multiwell plate formats. First, a novel microfluidic concept was invented to rapidly immobilize linear protein gradients on the surface of poly(ethylene glycol) (PEG)-based hydrogels whose biophysical properties are reminiscent of natural extracellular matrices. This method allows efficient capture of steady-state gradients of tagged proteins (e.g. using Fc or biotin tags) in just a few minutes on engineered hydrogels that display the corresponding auxiliary proteins (e.g. ProteinA or NeutrAvidin). The selectivity and orthogonality of the chosen binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is impossible using existing platforms. After patterning, the microfluidic chip can be readily removed from the gel for cell culture applications. Using quantitative single-cell time-lapse microscopy, this platform was validated here by probing the effect of fibronectin concentration on the directionality and speed of cell migration. Next, the resolution, flexibility and throughput of the protein patterning on hydrogels was substantially enhanced by the introduction of hydrodynamic flow focusing, that is, the generation of user-defined patterns by spatially controlled step- wise deposition of biomolecules. To this end, a microfluidic device was conceived that enabled the generation of arrays of parallel and crossed overlapping gradients. Application of the platform to generate gradients of immobilized leukemia inhibitory factor (LIF) showed an influence of the LIF concentration on ESC self-renewal. This platform should be useful to systematically probe the effect of biomolecule dose, singly or in combinations, on stem cell behavior in vitro; however, it lacks the ability to dynamically vary the presentation of biomolecules that might be crucial to control stem cell fate towards establishing functional in vitro tissue models. To address this limitation, in the last part of the thesis work a hydrogel-based microfluidic chip was developed that decouples macro-scale cell culture on the gel surface from the precise spatiotemporal biomolecule delivery at the micro-scale, i.e. through the gel layer. The hydrogel chip, used here as a simple insert that is compatible with conventional multiwell plate formats, was optimized to support long- term ESC maintenance in both adherent format and as uniformly sized ESC aggregates termed embryoid bodies. The platform was successfully used for the spatially controlled neuronal commitment of ESC via delivery of gradients of the morphogen retinoic acid. Taken together, in this thesis several innovative microengineered cell culture platforms are presented which enable, perhaps for the first time, the long-term culture and manipulation of stem cell fate at the micro-scale. These systems should be useful to systematically probe in vitro the effect of biomolecule dose and delivery dynamics on stem cell behavior, ultimately facilitating the development of complex in vitro tissue models

Microfluidic patterning of protein gradients on biomimetic hydrogel substrates

This protocol describes a versatile microfluidic method to generate tethered protein gradients of virtually any user-defined shape on biomimetic hydrogel substrates. It can be applied to test, in a microenvironment of physiologically relevant stiffness, how cells respond to graded biomolecular signals, for example to elucidate how morphogen proteins affect stem cell fate. The method is based on the use of microfluidic flow focusing to rapidly capture in a step-wise manner tagged biomolecules via affinity binding on the gel surface. The entire patterning process can be performed in <1 h. We illustrate one application of this method, namely, the spatial control of mouse embryonic stem cell self-renewal in response to gradients of the self-renewal-promoting signal leukemia inhibitory factor

Microfluidics-generated immobilized biomolecule gradients on hydrogels

Extra-cellular matrix (ECM) tethered morphogen gradients play an important role in tissue development and regeneration. There is currently only a limited set of model systems which can mimic these gradients in an artificial ECM-like environment. We have developed such a system that is able to stably immobilize a microfluidics-generated protein gradient on a poly(ethylene) glycol (PEG) hydrogel. After patterning, the hydrogel can be released from the chip and be directly used for cell culture

Marcuse

Computer-controlled hydrodynamic flow focusing was utilized to generate tethered protein gradients of any user-defined shape on the surface of soft synthetic hydrogels

Patterning of cell-instructive hydrogels by hydrodynamic flow focusing

Microfluidic gradient systems offer a very precise means to probe the response of cells to graded biomolecular signals in vitro, for example to model how morphogen proteins affect cell fate during developmental processes. However, existing gradient makers are designed for non-physiological plastic or glass cell culture substrates that are often limited in maintaining the phenotype and function of difficult-to-culture mammalian cell types, such as stem cells. To address this bottleneck, we combine hydrogel engineering and microfluidics to generate tethered protein gradients on the surface of biomimetic poly(ethylene glycol) (PEG) hydrogels. Here we used software-assisted hydrodynamic flow focusing for exposing and rapidly capturing tagged proteins to gels in a step-wise fashion, resulting in immobilized gradients of virtually any desired shape and composition. To render our strategy amenable for high-throughput screening of multifactorial artificial cellular microenvironments, a dedicated microfluidic chip was devised for parallelization and multiplexing, yielding arrays of orthogonally overlapping gradients of up to 4 × 4 proteins. To illustrate the power of the platform for stem cell biology, we assessed how gradients of tethered leukemia inhibitory factor (LIF) influence embryonic stem cell (ESC) behavior. ESC responded to LIF gradients in a binary manner, maintaining the pluripotency marker Rex1/Zfp42 and forming self-renewing colonies above a threshold concentration of 85 ng cm-2. Our concept should be broadly applicable to probe how complex signaling microenvironments influence stem cell fate in culture

Caratteri, stili cognitivi e insegnamento della musica

versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5 min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types

Dialect fieldworkers in nineteenth-century Ireland

The fabrication of microfluidic devices is often still a time-consuming and costly process. Here we introduce a very simple and cheap microfabrication process based on "razor writing", also termed xurography, for the ultra-rapid prototyping of microfluidic devices. Thin poly(dimethylsiloxane) (PDMS) membranes are spin-coated on flexible plastic foil and cut into user-defined shapes with a bench-top cutter plotter. The PDMS membranes can then be assembled into desirable microdevices via plasma bonding. The plastic foil allows manipulation of exceptionally thin (30-300 mu m) PDMS layers and can be readily peeled after fabrication. This versatile technique can be used to produce a wide variety of microfluidic device prototypes within just a few hours