Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. 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 often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

A 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.

a b s t r a c tThe rapid increase in the number of approved therapeutic proteins, including recombinant antibodies, for diseases necessitating chronic treatments raises the question of the overall costs imposed on healthcare systems. It is therefore important to investigate alternative methods for recombinant protein administration. The implantation of genetically engineered cells is an attractive strategy for the chronic long-term delivery of recombinant proteins. Here, we have developed a high-capacity cell encapsulation system for the implantation of allogeneic myoblasts, which survive at high density for at least one year. This flat sheet device is based on permeable polypropylene membranes sealed to a mechanically resistant frame which confine cells seeded in a tailored biomimetic poly(ethylene glycol) (PEG)-based hydrogel matrix. In order to quantitate the number of cells surviving in the device and optimize initial conditions leading to high-density survival, we implant devices containing C2C12 mouse myoblasts expressing a luciferase reporter in the mouse subcutaneous tissue. We show that initial cell load, hydrogel stiffness and permeable membrane porosity are critical parameters to achieve long-term implant survival and efficacy. Optimization of these parameters leads to the survival of encapsulated myogenic cells at high density for several months, with minimal inflammatory response and dense neovascularization in the adjacent host tissue. Therefore, this encapsulation system is an effective platform for the implantation of genetically engineered cells in allogeneic conditions, which could be adapted to the chronic administration of recombinant proteins.

The potential for the clinical application of stem cells in tissue regeneration is clearly significant. However, this potential has remained largely unrealized owing to the persistent challenges in reproducibly, with tight quality criteria, and expanding and controlling the fate of stem cells in vitro and in vivo. Tissue engineering approaches that rely on reformatting traditional Food and Drug Administrationapproved biomedical polymers from fixation devices to porous scaffolds have been shown to lack the complexity required for in vitro stem cell culture models or translation to in vivo applications with high efficacy. This realization has spurred the development of advanced mimetic biomaterials and scaffolds to increasingly enhance our ability to control the cellular microenvironment and, consequently, stem cell fate. New insights into the biology of stem cells are expected to eventuate from these advances in material science, in particular, from synthetic hydrogels that display physicochemical properties reminiscent of the natural cell microenvironment and that can be engineered to display or encode essential biological cues. Merging these advanced biomaterials with high-throughput methods to systematically, and in an unbiased manner, probe the role of scaffold biophysical and biochemical elements on stem cell fate will permit the identification of novel key stem cell behavioral effectors, allow improved in vitro replication of requisite in vivo niche functions, and, ultimately, have a profound impact on our understanding of stem cell biology and unlock their clinical potential in tissue engineering and regenerative medicine. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:156-164