3D collagen cultures under well-defined dynamic strain: A novel strain device with a porous elastomeric support

The field of mechanobiology has grown tremendously in the past few decades, and it is now well accepted that dynamic stresses and strains can impact cell and tissue organization, cell-cell and cell-matrix communication, matrix remodeling, cell proliferation and apoptosis, cell migration, and many other cell behaviors in both physiological and pathophysiological situations. Natural reconstituted matrices like collagen and fibrin are often used for three-dimensional (3D) mechanobiology studies because they naturally form fibrous architectures and are rich in cell adhesion sites; however, they are physically weak and typically contain >99% water, making it difficult to apply dynamic stresses to them in a truly 3D context. Here we present a composite matrix and strain device that can support natural matrices within a macroporous elastic structure of polyurethane. We characterize this system both in terms of its mechanical behavior and its ability to support the growth and in vivo-like behaviors of primary human lung fibroblasts cultured in collagen. The porous polyurethane was created with highly interconnected pores in the hundreds of mm size scale, so that while it did not affect cell behavior in the collagen gel within the pores, it could control the overall elastic behavior of the entire tissue culture system. In this way, a well-defined dynamic strain could be imposed on the 3D collagen and cells within the collagen for several days (with elastic recoil driven by the polyurethane) without the typical matrix contraction by fibroblasts when cultured in 3D collagen gels. We show lung fibroblast-to-myofibroblast differentiation under 30%, 0.1 Hz dynamic strain to validate the model and demonstrate its usefulness for a wide range of tissue engineering applications. © 2008 Wiley Periodicals, Inc.

New Tools for Hydrogel-Based 3D Bioprinting

Andrea Negro

The in vitro recapitulation of tissue and organ function represents one of the main objectives of tissue engineering. Developments in this field have numerous applications, from alleviating the shortage of donor organs to providingmore representative platforms for drug testing. As such, there is an increasingly significant body of literature devoted to the development of better strategies to reconstruct living matter outside of the body. The three-dimensional (3D) architecture of a tissue, its histoarchitecture, is a key aspect to consider when engineering a tissue. Indeed, tissue function not only arises from individual elements that compose the tissue, its different cell types and their extracellular matrices (ECM), but also fromtheir precise spatial arrangement and interactions. Bioprinting is considered as one of the most promising techniques to recapitulate the complexity of in vivo histoarchitectures. Early bioprinting examples have shown successful application of inkjet printing to geometrically arrange cells and biomolecules in 2D and, to a much lesser extent, in 3D. However, no group has shown yet a technological platformable to print a living tissue construct whose make-up could even closelymimic the histoarchitecture of a native tissue. In this thesis the most significant challenges in using inkjet printers for tissue engineering were tackled. Perhaps the most critical bottleneck of state-of-the-art bioprinting is the lack of suitable bioinks; precursor liquids with distinct physico-chemical properties that very rapidly transforminto solid hydrogels and also possess the necessary bioactive characteristics to guide cell development into a functional tissue. To this end, a novel double network hydrogel systemwas conceived which combines ultra-rapid cross-linking of alginate, a biologically inert polysaccharide network, with the slower enzymatic crosslinking of a synthetic but highly bioactive poly(ethylene glycol) (PEG) gel. The latter could be tailored virtually on demand tomimic a desired ECM in a tissue. Based on this bioink concept it was demonstrated that complex multicomponent 3D shapes could be printed. Importantly, using primary human fibroblasts as model system, the bioinks showed outstanding characteristics as cellular microenvironments, facilitating rapid 3D cell migration and self-organization into multicellular networks. To mimic the spatial complexity of living tissues, it is crucial to master the deposition of multiple, cell-containing bioinks into user-defined 3D structures. Each bioink could thus mimic a specific cell/ECMcomponent present in a native tissue. To be able to reproduce an appropriate histoarchitecture, bioinks have to be deposited precisely enough to approximate their natural 3D spatial arrangement down to a cellular scale. Technically, this implies that the dispensing units must be extremely well aligned and synchronized to one another in order to pattern according to a specified design. In order to address these challenges, several novel bioprinting concepts were developed, which allowed the patterning of multiple bioinks containing living cells into 3D tissue-like geometries. In the last part of this thesis, an important first step towards the engineering of macroscopic tissue models was taken. Accordingly, a fundamental requirement for the viability and maturation of a printed tissue construct is the supply of nutrients and growth factors through a microfluidic network, similar to the vascularization in a native tissue. To this end, a novel approach for the printing of 3D constructs with an interconnected channel network enabling rapid nutrient transport was devised. This was achieved by exploiting the multicomponent printing developed earlier. A channel network inside the construct was patterned using a degradable hydrogel that could be selectively removed to leave behind the perfusable microfluidic system. Moreover, it was possible to combine this sacrificial layer approach with cell-containing bioinks to generate a cellularized perfusable tissue model. Encouraging preliminary long-termculture experiments revealed that dynamic cell culture conditions could be maintained, leading to viable cell growth over more than one week under perfusion conditions. Taken together, this thesis presents several important steps toward an inkjet-based platform for in vitro tissue engineering. It is expected that the strategies developed here will provide a solid basis for future advances in the emerging bioprinting field.

EPFL2014

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

Most tissue engineering approaches include the delivery of reparative cells to a damaged host tissue. Generally these cells are sought to be of a stem cell character since they retain a high potential for proliferation and differentiation into diverse phenotypes. To restrict cell function to the desired phenotype and to avoid anti-immune responses, autologous stem cells are frequently isolated from intact regions of the same (damaged) organ, such as keratinocytes from the basal layer of the skin and hair follicles or from the same germ layer origin such as mesenchymal stem cells (MSC) that are obtained from the bone marrow. This approach faces two major problems: 1) The number of source cells obtained from biopsies is generally too low for direct implantation, which requires cell multiplication in culture. 2) The standard culture conditions, including growth in plastic dishes, passaging, and the composition of the growth medium, never correspond to the natural microenvironment and influence cell differentiation, often resulting in non-desired phenotypes. The objective of my thesis was to provide a solution for both problems that is applicable for the average cell culture laboratory. Our technique reduces traumatic enzymatic passaging, provides constant cell densities to keep cells in the exponential growth phase and at the same time avoids contact inhibition of cell growth. This novel approach comprises the development of a new elastic culture surface, produced from a high extension silicone rubber (HESR) that we here for the first time applied for cell culture. The HESR is enlarged by a novel mechanical approach permitting to visualize and control cells under expansion. Untreated silicone, the polymer component of the HESR, does not promote cell attachment and must therefore be functionalized. In the first part of my thesis (Chapter 2) we establish a surface treatment for silicone membranes that will resist large surface expansions. We compare established coating techniques that have originally been developed to provide static silicone surfaces with cell adhesive molecules by 1) hydrophobic, 2) electrostatic and 3) covalent interactions. Cell attachment, spreading and proliferation was evaluated between these three treatments as a function of the coating in static and stretched conditions. We show that covalent immobilization of collagen type I is most suitable to stretch cells cultured on elastic silicone substrates. We applied this technique to functionalize the HESR culture surface in all later applications. The second central part of my thesis (Chapter 3) develops a new culture method consisting of a motorized device that isotropically expands the coated HESR surface to yet unmatched 1000% of its initial surface. Proof-of-principle of this method is given by culturing human MSC, which are sensitive to passaging during expansion in vitro and that are therapeutically relevant cells to repair bone, cartilage, and cardiovascular tissue. MSC were cultured three consecutive passages on the culture surface that has been expanded in steps of 5% every 6 h for 20 days, respectively. After nine weeks of expansion culture, MSC exhibited consistently high proliferation rates, fully retained their pluripotent stem cell character and were inducible to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. The cell surface expansion culture surface produced ∼10-times higher cell numbers compared with nine weeks of standard culture, which required nine events of passaging to attain the same culture surface. We propose our method to produce MSC for clinical cell therapy. In the third part of my thesis (Chapter 4) we implement our novel surface expansion method to culture fibroblasts with the aim to: 1) rapidly expand primary fibroblasts, 2) achieve higher cell yields than obtained with conventional culture, and 3) obtain homogeneous cell populations with non-fibrogenic character. The expansion of primary autologous dermal fibroblasts in culture is a pivotal step to obtain sufficiently high cell numbers for application in skin esthetics and for the treatment of large burn wounds. Dynamically enlarging the culture surface by 800% over 20 d, yielded ∼1.5 higher fibroblasts numbers than the equivalent three standard passages. By further optimizing the surface expansion speed according to the cell growth rates, the same high cell numbers were produced after only 9 d of culture. The highly compliant nature of the HESR surface had the beneficial effect of completely abolishing the formation of 'fibrotic' myofibroblasts. Hence, our novel method for fibroblast culture is faster and yields higher numbers of more homogeneous cells than standard culture passaging. It delivers a fibroblast quality and quantity that is appealing for cell therapeutic purposes. The aim of the last part of my thesis (Chapter 5) was to provide highly compliant silicone elastomers, such as the HESR, with a micropattern of cell adhesive proteins. In particular we aim to produce arrays of extracellular matrix proteins with a size and morphology to promote formation of single focal adhesions on highly elastic and expandable surfaces. This will allow future studies on cell mechanosensing through focal adhesions by discriminating between effects of focal adhesion size changes and stretch. An existing method to pattern rigid surfaces with proteins, so-called microcontact printing, is not suitable to transfer proteins onto surfaces that are softer than the stamp. We here establish a new transfer technique that is based on a stencil with micron-sized openings. The new stencil is used to expose the surface of compliant silicone substrates with Young's modulus of 10-100 kPa to a protein solution of fibronectin, collagen I and protein-repellent polymers, leaving behind the desired protein pattern. Fibroblasts were successfully attached onto the resulting focal islet arrays on compliant substrates.

EPFL2008

Tissue engineering complex and mechanically active microenvironments for studies in asthma and cancer

Alice Anna Tomei

The goals of tissue engineering include recapitulating specific tissue functions for regenerative medicine and developing in vitro models of human tissues to study human physiology and pathophysiology and for testing and screening drugs before expensive clinical trials. Success of these goals relies on the capability to recapitulate the complex interactions between cells and their microenvironments in vitro, which requires interdisciplinary approaches. Tissue engineering has evolved greatly in the past two decades, with notable progress in replacing function in tissues such as cartilage, bone, arteries, skin, and pancreatic islets. What is lacking, however, are examples of, and design principles for, engineering complex microenvironments to understand disease progression. The two particular examples addressed in this thesis are (1) airway remodeling in asthma and (2) tumor invasion and metastasis in cancer. These diseases involve complex interactions between different cell types as well as extracellular matrix remodeling, which, together with the mechanical environment that affects cell-cell and cell-matrix interactions and that can change in a diseased state (or in some cases induce the disease state), collectively cause the pathology. Such disease states are difficult to study in vivo because (a) one cannot easily tease out mechanisms or have much control over these complex interactions in an animal model, and (b) there are many differences between animal and human cells. The aim of this thesis was to engineer mechanically dynamic microenvironments for the study of two disease processes: viral infection in asthmatic remodeled airways and cancer metastasis to the lymph node. These involved multidisciplinary approaches and drew tools and methods from mechanobiology, materials science, cell biology, and molecular biology. First, we developed and characterized a relevant 3D in vitro model of the bronchial mucosa with a functional epithelial barrier, including improved epithelial differentiation, ciliogenesis, and mucociliary function and showed for the first time that dynamic compression, such as that seen in asthmatic bronchoconstriction, enhances viral infection of epithelial and subepithelial cells using lentiviral technology and may help explaining why asthmatic patients are more susceptible to viral infection. However, the strain device had deficiencies in reproducibility, ease of use, time to assemble, and most importantly, homogeneous distribution of strain (because collagen and other natural extracellular matrices are mechanically weak since they have >99% water and cannot retain residual strain). To address these problems, we engineered a completely different dynamic strain device, which includes an elastic porous superstructure to contain the collagen and which can modulate stress/strain to the entire culture system. We characterized this device and showed that we could impose dynamic strains to 3D soft, hydrated cultures over relatively long periods of time and with variable amplitude and frequency in a uniform way. The device has the potential to be used in a wide spectrum of applications, including the possibility to study the correlation between mechanostimulation and metastatic potential of tumor cells in the context of lung metastasis of many cancer types. Finally, we used this material as a supporting structure for tissue-generated stress such as that generated by myofibroblasts, and in particular the ones that build up the lymph node stroma in the paracortex. The importance of these cells relies on their ability to form conduits for chemokine transport to high endothelial venules, where lymphocytes enter the node, the ability to form a platform for directing immune cell migration and interactions, and the ability to secrete two chemokines that are important to regulate lymphocyte adhesion and migration: CCL19 and CCL21. Specifically, we found that lymph node stromal cells use this supporting composite to remodel and reorganize their surrounding matrix into physiologically relevant bundles. By giving these cells the relevant stimuli (matrix microenvironment and flow), we could recapitulate certain features of the lymph node microenvironment, including a physiological stromal structure and chemokine profile, that may be critical for understanding immune and cancer cell interactions there. Using this model we demonstrated for the first time that slow flow through the lymph node stroma can itself drive cell and stromal organization and upregulates CCL21 production. Finally, we incorporated this engineered lymph node model into a larger model of the tumor-lymphatic-lymph node pathway to model the relative modulation of tumor growth, migration within a lymphatic "channel" towards the lymph node, and behavior within this lymph node environment. We used human lymphatic endothelial cells and a mass of transduced tumor cells with engineered chemokine profiles. We showed how CCR7 signaling correlates with the ability of tumor cells to metastasize to the lymph node and to interact with lymph node stromal cells once they reach the node. Our findings demonstrate how a multidisciplinary tissue engineering approach allows creating complex microenvironments in vitro that include both physical and chemical cues and allows recapitulating tissue function in vitro and investigating dynamic pathophysiological diseases like asthma and cancer metastasis. It combines both the development of these model systems and some examples of their usefulness to gain insight into asthma pathophysiology and cancer biology.

EPFL2008

New Tools for Hydrogel-Based 3D Bioprinting

Andrea Negro

EPFL2014

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

EPFL2008

Tissue engineering complex and mechanically active microenvironments for studies in asthma and cancer

Alice Anna Tomei

EPFL2008