Microfluidic engineering of artificial stem cell niches

Stem cells play a key role in a wide range of biological processes, in large part due to their ability to self-renew or differentiate into specialized cell types in response to various biological cues. In vivo, stem cells reside in a complex microenvironment, termed niche, that regulates cell fate through intricate combinations of biophysical and biochemical factors. To recapitulate some of these crucial interactions ex vivo and to facilitate a quicker transition to clinical and pharmaceutical applications of stem cells, numerous technologies have been developed in recent years to better control and manipulate the cellular microenvironment. In particular, combining synthetic hydrogels having controllable mechanical and biochemical signaling with microfluidic technologies that can automatically and precisely handle fluids results in unprecedented control over in vitro microenvironmental conditions. In this thesis, novel microfluidic approaches were developed to engineer stem cell niches presenting defined and modular cell-instructive physicochemical cues. In a first approach, a novel platform based on computer-controlled hydrodynamic flow focusing was developed in order to tether steady-state gradients of tagged proteins, using Fc or biotin tags, onto the surface of poly(ethylene glycol) (PEG)-based hydrogels displaying selective capturing proteins (ProteinA or NeutrAvidin). This versatile patterning strategy permitted the generation of complex biomolecule gradients, with fine control over the patterning resolution and shape. Furthermore, the chosen binding schemes enabled parallel and orthogonal gradients of multiple proteins to be formed. As a proof-of-principle, we employed this technology to assess the influence of immobilized leukemia inhibitor factor (LIF) concentration on mouse embryonic stem cell self-renewal. While this technology allowed biomolecule dose effect on stem cell behavior to be investigated, it was limited in its ability to modulate multiple microenvironmental factors simultaneously. To address this limitation, droplet-based microfluidic technology was adopted which allows perturbing microenvironmental conditions in a combinatorial fashion and with nearly unmatched precision and throughput. To this end, microfluidic chips were fabricated for the generation of PEG-based microgels with precisely controlled dimensions and physico-chemical properties. First, we developed a versatile biofunctionalization technique for tethering biomolecules of interest to the reactive PEG microgels. Then, selective peptide- and protein-modified formulations were tested for their ability to promote adhesion and proliferation of various stem cell types in a bioreactor-based suspension culture. Next, programmable modulation of the flows was adopted to generate microgels with varying elasticity, biochemical ligands or both. The different microgel properties were encoded with specific fluorescent markers such that the microenvironment properties could be readily identified via microscopy or flow cytometry. Controlling syringe pumps through computer programming allowed us to generate up to 100 populations of microgels with different elasticities and bioactive ligand concentrations in a single experiment. Thousands of compositionally distinct microgels were then analyzed by the intensity levels of two fluorescent moieties incorporated in the hydrogel network. As a proof-of-principle, we demonstrated that this technology can be adopted to [...

Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels

Droplet microfluidic technology is applied for the high-throughput synthesis via Michael-type addition of reactive, micrometer-sized poly(ethylene glycol) (PEG) hydrogels (“microgels”) with precisely controlled dimension and physicochemical properties. A versatile chemical scheme is used to modify the reactive PEG microgels with tethered biomolecules to tune their bioactive properties for the bioreactor culture and manipulation of various (stem) cell types

Stem cell niche engineering through droplet microfluidics

Stem cells reside in complex niches in which their behaviour is tightly regulated by various biochemical and biophysical signals. In order to unveil some of the crucial stem cell-niche interactions and expedite the implementation of stem cells in clinical and pharmaceutical applications, in vitro methodologies are being developed to reconstruct key features of stem cell niches. Recently, droplet-based microfluidics has emerged as a promising strategy to build stem cell niche models in a miniaturized and highly precise fashion. This review highlights current advances in using droplet microfluidics in stem cell biology. We also discuss recent efforts in which microgel technology has been interfaced with high-throughput analyses to engender screening paradigms with an unparalleled potential for basic and applied biological studies

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

Cell-Instructive Microgels with Tailor-Made Physicochemical Properties

A microfluidic in vitro cell encapsulation platform to systematically test the effects of microenvironmental parameters on cell fate in 3D is developed. Multiple cell types including fibroblasts, embryonic stem cells, and cancer cells are incorporated in enzymatically cross-linked poly(ethylene glycol)-based microgels having defined and tunable mechanical and biochemical properties. Furthermore, different approaches to prevent cell "escape" from the microcapsules are explored and shown to substantially enhance the potential of this technology. Finally, coencapsulation of microgels within nondegradable gels allows cell viability, proliferation, and morphology to be studied in different microenvironmental conditions up to two weeks in culture

Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels

Droplet microfluidic technology is applied for the high-throughput synthesis via Michael-type addition of reactive, micrometer-sized poly­(ethylene glycol) (PEG) hydrogels (“microgels”) with precisely controlled dimension and physicochemical properties. A versatile chemical scheme is used to modify the reactive PEG microgels with tethered biomolecules to tune their bioactive properties for the bioreactor culture and manipulation of various (stem) cell types

High-throughput stem cell-based phenotypic screening through microniches

As the field of tissue engineering develops, methods for screening combinations of signals for their effects on stem cell behavior are needed. We introduce a microgel-based screening platform for testing combinations of in situ-generated proteins on stem cell fate in ultrahigh-throughput. Compartmentalizing individual sets of growth factors was addressed by encapsulating aggregates of stable recombinant cell lines secreting individual glycoproteins into microgels through an on-chip polymerization. When these 'microniches' are cultured with a cell type of interest, fluorescence reporters indicate positive niches that perform the desired function, and the underlying producer cell lines of these selected microniches are analyzed by barcoded RNA sequencing. The microniche-based screening work-flow was validated via a model system based on engineered mammalian cells expressing yellow fluorescent protein (YFP) upon anti-inflammatory cytokine interleukin 4 (IL4)-based activation