Encoding Stem-Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides.

Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capability-a capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly(ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This "encoded" ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine.

Accelerated Combinatorial High Throughput Star Polymer Synthesis via a Rapid One-Pot Sequential Aqueous RAFT (rosa-RAFT) Polymerization Scheme.

Advanced polymerization methodologies, such as reversible addition-fragmentation transfer (RAFT), allow unprecedented control over star polymer composition, topology, and functionality. However, using RAFT to produce high throughput (HTP) combinatorial star polymer libraries remains, to date, impracticable due to several technical limitations. Herein, the methodology "rapid one-pot sequential aqueous RAFT" or "rosa-RAFT," in which well-defined homo-, copolymer, and mikto-arm star polymers can be prepared in very low to medium reaction volumes (50 µL to 2 mL) via an "arm-first" approach in air within minutes, is reported. Due to the high conversion of a variety of acrylamide/acrylate monomers achieved during each successive short reaction step (each taking 3 min), the requirement for intermediary purification is avoided, drastically facilitating and accelerating the star synthesis process. The presented methodology enables RAFT to be applied to HTP polymeric bio/nanomaterials discovery pipelines, in which hundreds of complex polymeric formulations can be rapidly produced, screened, and scaled up for assessment in a wide range of applications.

Triple Activity of Lamivudine Releasing Sulfonated Polymers against HIV-1.

In this article a library of polymeric therapeutic agents against the human immunodeficiency virus (HIV) is presented. The library of statistical copolymers of varied molar mass was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The synthesized polymers comprise pendent hydroxyl and sulfonated side chains as well as the reverse transcriptase prodrug lamivudine (3TC) attached via a disulfide self-immolative linker. The glutathione mediated release of 3TC is demonstrated as well as the antiviral efficacy against HIV entry and polymerase activity. Although a high degree of polymer sulfonation is required for effective HIV entry inhibition, polymers with approximately ∼50% sulfonated monomer demonstrated potent kinase independent reverse transcriptase inhibition. In addition, the sulfonated polymers demonstrate activity against DNA-DNA polymerase, which suggests that these polymers may exhibit activity against a broad spectrum of viruses. In summary, the polymers described provide a triple-active arsenal against HIV with extracellular activity via entry inhibition and intracellular activity by kinase-dependent lamivudine-based and kinase-independent sulfonated polymer based inhibition. Since these sulfonated copolymers are easily formulated into gels, we envision them to be particularly suited for topical application to prevent the mucosal transmission of viruses, particularly HIV.

Concise review: tailoring bioengineered scaffolds for stem cell applications in tissue engineering and regenerative medicine.

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 Administration-approved 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.

Ultra-rapid prototyping of flexible, multi-layered microfluidic devices via razor writing.

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 µm) 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.

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.

A high-capacity cell macroencapsulation system supporting the long-term survival of genetically engineered allogeneic cells.

The 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.

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.

Programmable microfluidic patterning of protein gradients on hydrogels.

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