Effects of physicochemical properties of polyacrylamide (PAA) and (polydimethylsiloxane) PDMS on cardiac cell behavior.

In vitro cell culture is commonly applied in laboratories around the world. Cultured cells are either of primary origin or established cell lines. Such transformed cell lines are increasingly replaced by pluripotent stem cell derived organotypic cells with more physiological properties. The quality of the culture conditions and matrix environment is of considerable importance in this regard. In fact, mechanical cues of the extracellular matrix have substantial effects on the cellular physiology. This is especially true if contractile cells such as cardiomyocytes are cultured. Therefore, elastic biomaterials have been introduced as scaffolds in 2D and 3D culture models for different cell types, cardiac cells among them. In this review, key aspects of cell-matrix interaction are highlighted with focus on cardiomyocytes and chemical properties as well as strengths and potential pitfalls in using two commonly applied polymers for soft matrix engineering, polyacrylamide (PAA) and polydimethylsiloxane (PDMS) are discussed.

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels.

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell-cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testingin vitro(organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapiesin vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells inpoly-N-isopropylacrylamid(PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Impact of Poly(dimethylsiloxane) Surface Modification with Conventional and Amino Acid-Conjugated Self-Assembled Monolayers on the Differentiation of Induced Pluripotent Stem Cells into Cardiomyocytes.

Cardiomyocytes, differentiated from induced pluripotent stem cells (iPSCs), have the potential to produce patient- and disease-specific pharmacological and toxicological platforms, in addition to their cardiac cell therapy applications. However, the lack of both a robust and a simple procedure for scalable cell substrate production is one of the major limitations in this area. Mimicking the natural healthy myocardium extracellular matrix (ECM) properties by altering the cell substrate properties, such as stiffness and chemical/biochemical composition, can significantly affect cell substrate interfacial characteristics and potentially influence cellular behavior and differentiation of iPSCs to cardiomyocytes. Here, we propose a systematic and biomimetic approach, based on the preparation of poly(dimethylsiloxane) (PDMS) substrates having the similar stiffness as healthy heart tissue and a well-defined surface chemistry obtained by conventional [(3-aminopropyl)triethoxysilane (APTES) and octadecyltrimethoxysilane (OTS)] and amino acid (histidine and leucine)-conjugated self-assembled monolayers (SAMs). Among a wide range of different concentrations, the 50:1 prepolymer cross-linker ratio of PDMS allowed adaptation of the myocardium stiffness with a Young's modulus of 23.79 ± 0.61 kPa. Compared with conventional SAM modification, amino acid-conjugated SAMs greatly improved iPSC adhesion, viability, and cardiac marker expression by increasing surface biomimetic properties, whereas all SAMs enhanced cell behavior, with respect to native PDMS. Furthermore, leucine-conjugated SAM modification provided the best environment for cardiac differentiation of iPSCs. This optimized approach can be easily adapted for cardiac differentiation of iPSCs in vitro, rendering a very promising tool for microfluidics, drug screening, and organ-on-chip platforms.

The Potential Application of Biomaterials in Cardiac Stem Cell Therapy.

Biomaterials play a vital role in the field of regenerative medicine and tissue engineering. To date, a large number of biomaterials have been used in cardiovascular research and application. Recently, biomaterials have held a lot of promise in cardiac stem cell therapy. They are used in cardiac tissue engineering to form scaffolds for cellular transplantation, promote angiogenesis, enhance transplanted cell engraftment or influence cell migration. The science of biomaterial designing has evolved to an extent where they can be designed to mimic the microenvironment of a cardiac tissue in vivo and contribute in deciding the fate of transplanted stem cells and induce cardiac lineage oriented stem cell differentiation. In this review, we focus on biomaterials used in cardiovascular stem cell research, tissue engineering and regenerative medicine and conclude with an outlook on future impacts of biomaterial in medical sciences.

The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.