2D Stem Cell Culture on Microfluidic Generated Hydrogel Beads

Stem cells capacity to self-renew and differentiate into specialized cell types, endow them with great promises in regenerative medicine. It is believed that the stem cells microenvironment plays a significant role in regulating their fate. However, with exception to embryonic stem cells, there is currently no tractable methods to control stem cells expansion in vitro. Microcarriers were shown to yield enhanced cell expansion compared to conventional cell culture. Nevertheless, the materials used for microcarriers fabrication do not offer the possibility to tailor their physicochemical properties to cell-specific requirements. To fill this technological gap, we propose to develop versatile hydrogel microcarriers with tunable mechanical properties and tailored surface modification. The combination of microfluidic droplet generation and poly(ethylene glycol)-based hydrogels allow the generation of monodisperse hydrogel microcarriers. Chemical moieties on the microcarriers surface enable versatile plug-n-play surface modifications. Microcarriers functionalized with thiolated adhesion ligand were successfully used for cell culture. Furthermore, magnetic particle incorporation within hydrogel microcarriers facilitated their handling during cell culture. To validate our hydrogel microcarriers, we characterized the culture of fibroblasts and HEK cells over 5 days. An increased number of cells were recovered with the hydrogel microcarriers compared to conventional culture. Finally, we were able to culture human mesenchymal stem cells on our hydrogel microcarriers. Altogether, we demonstrated the proof-of-principle experiments for the generation of customized hydrogel microcarriers for stem cell expansion in vitro. The presented technology will offer great promises in the future.

Hydrogel Microfluidics to Control Stem Cell Fate

Steffen Cosson

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.

EPFL2012

Bioengineering stem cell niches in 2D and 3D

Nora Leonardi

Muscle degenerative disorders, such as Duchenne muscular dystrophy, have a profound impact on the quality of life and survival of patients. Transplantation of myoblasts to restore muscle function has been discouraging since these cells die and don't migrate but it has more recently been reported that muscle stem cells, known as satellite cells, have an extraordinary potential to contribute to muscle fiber regeneration, home to their niche and replenish the stem cell pool when injected freshly into damaged muscle. These cells however quickly lose their regenerative potential when cultured in vitro. Adult stem cells are known to be regulated by their microenvironment through a complex mixture of signals including soluble factors, matrix bound proteins, neural input and mechanical properties and the identification of extrinsic factors that can regulate muscle stem cell quiescence and self-renewal has been a focus of research in the Blau lab. Strikingly, recent studies in the Blau lab have shown that muscle stem cells cultured on compliant hydrogels coated with laminin were more viable than those cultured on tissue culture plastic and robustly contributed to muscle regeneration when transplanted into irradiated mouse leg muscles. The aim of this project was to follow up on these findings and to further elucidate the role biophysical and biochemical microenvironmental cues play in the regulation of muscle stem cell fate. Hydrogels have emerged as a popular material to recreate the stem cell niche in vitro due to their biocompatibility, high water content, tissue-like elasticity and diffusive properties and were employed here to fabricate substrates of varying rigidities. The physicochemical properties and tethered protein densities of a range of poyl(ethylene glycol) hydrogels were first characterized to define a suitable set of cell culture substrates. Gel stiffness could be linearly modulated from 2.5 to 45 kPa and the ligand density was found to be in the low femtomolar range. ELISA showed similar protein density on gels of different stiffnesses but immunofluorescence could not fully confirm this. Committed myoblasts were then seeded on these gels and shown to respond to substrate stiffness and ligand density in terms of morphology. Lastly, freshly isolated muscle stem cells were cultured on these gels and on rigid tissue culture plastic and shown to spread more, be more viable and grow more rapidly on stiffer gels as compared to soft ones. Myogenic progression was retrospectively analysed using immunohistochemistry but no differences between culture conditions were observed. Based on the influences of gel rigidity and ligand density observed in vitro, an in vivo assay, the ultimate test of stem cell function, is currently underway.

2009

Stem cell niche engineering through photopatterning and microfluidics

Laura Kolb

The application of stem cells in drug screening and regenerative therapy has led to important advances in basic biology and biomedicine. Such strategies require high cell numbers and the efficient maturation into faithful functional organ or tissue units. An important aspect to gain control over stem cell fate is the recapitulation of their natural instructive extracellular matrix (ECM). In vivo, stem cells reside in microenvironments, termed niches, providing distinct, tissue-specific signals. In a complex interplay, the fate decisions are tightly regulated by various parameters including biophysical and biochemical signals. Biomaterial science interfaced with sophisticated bioengineering techniques has provided powerful tools to recreate stem cell niches in vitro. However, major challenges for the efficient stem cell expansion and differentiation still have not been sufficiently addressed. In this thesis, platforms have been developed to help to reconstruct this dynamic and niche-specific signaling panoply. In one approach, a hydrogel crosslinking scheme based on a Michael-type addition reaction was combined with photosensitive caging molecules. By masking thiol moieties of a poly(ethylene glycol) (PEG) macromer with photolabile groups, the covalent crosslinking with vinylsulfone groups of complementary PEG macromers was spatiotemporally controlled by light illumination. By the generation of precisely controlled stiffness gradients, we were able to spatially direct human mesenchymal stem cell (hMSC) migration. Along the same lines, light-controlled uncaging was applied to tether biomolecules into a hydrogel matrix. PEG macromers were covalently functionalized with a peptide substrate of activated transglutaminase factor XIII (FXIIIa). This allowed for any desired ECM-derived peptide or protein fused to the counter-reactive peptide substrate to be enzymatically tethered into the hydrogel network. Photoprotection of the hydrogel-bound peptide substrate enabled exquisite dynamic control of biomolecule distribution. The patterning platform was exemplified by the guided three-dimensional (3D) invasion of encapsulated hMSCs into the hydrogel matrix. The second part of this work addressed the challenge of dissecting the vast molecular signaling networks that influence stem cell behavior. A droplet microfluidic-based screening platform was developed to study the effect of pivotal niche proteins in unparalleled combinatorial throughput via micron-sized hydrogel beads termed microniches. Randomized compartmentalization of stable inducible mammalian cell lines into microniches translated into myriads of combinations of secreted proteins tethered to the hydrogel matrix. Selected microniches with a desired phenotypic effect were analyzed for the protein combinations by targeted genotypic analysis of the underlying encapsulated stable mammalian lines. Positive instructive effects of microniches could be demonstrated on a model system of engineered HEK cells and are currently under evaluation for mesendodermal differentiation of a human embryonic stem cell (hESC) reporter cell line. The novel light-responsive materials as well as microfluidic-based screening approach presented here strive for a near-physiological niche signaling architecture. These tools can help to fully exploit the stem cell potential and accelerate the translation of stem cell-based applications from the laboratory towards the clinic.

EPFL2016