Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture

© 2018 Elsevier B.V. A cell spheroid is a three-dimensional (3D) aggregation of cells. Synthetic, in-vitro spheroids provide similar metabolism, proliferation, and species concentration gradients to those found in-vivo. For instance, cancer cell spheroids have been demonstrated to mimic in-vivo tumor microenvironments, and are thus suitable for in-vitro drug screening. The first part of this paper discusses the latest microfluidic designs for spheroid formation and culture, comparing their strategies and efficacy. The most recent microfluidic techniques for spheroid formation utilize emulsion, microwells, U-shaped microstructures, or digital microfluidics. The engineering aspects underpinning spheroid formation in these microfluidic devices are therefore considered. In the second part of this paper, design considerations for microfluidic spheroid formation chips and microfluidic spheroid culture chips (μSFCs and μSCCs) are evaluated with regard to key parameters affecting spheroid formation, including shear stress, spheroid diameter, culture medium delivery and flow rate. This review is intended to benefit the microfluidics community by contributing to improved design and engineering of microfluidic chips capable of forming and/or culturing three-dimensional cell spheroids

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines

International audienceThe application of nanotechnology to medicine, usually termed nanomedicine, has given a crucial impulse to the design of various drug-loaded nanocarriers driven by the aim to overcome the limits associated with traditional drug delivery modalities, in particular, in the field of cancer treatment. However, an appropriate preclinical evaluation of the real therapeutic potential of nanomedicines suffers from the lack of relevant models that are well representative of the human disease and good predictors of the therapeutic response in patients. In this context, great emphasis has been directed toward 3D tumor models aiming to surmount the insufficient predictive power of traditional 2D monolayer cultures of cancer cells. This review focuses on multicellular tumor spheroids (MCTS), which are currently the most widely employed 3D tumor model in preclinical studies. After a brief discussion on spheroid construction strategies and analytical/imaging techniques employed in experimental settings, the application of 3D MCTS to the evaluation of nanomedicines displaying various physico-chemical properties is reviewed. Finally, relevant examples of scaffold and microfluidic systems in which MCTS have been included are described

Identifcation and stablization of a novel 3D hepatocyte monolayer for hepatocyte-based applications

Ph.DDOCTOR OF PHILOSOPH

Thermoresponsive Microgels for Multicellular Spheroids Formation

Multicellular spheroids (MCS) are considered as the most promising three dimensional (3D) in-vitro model which will narrow down the gap between in-vitro two-dimensional culture and in-vivo animal models. They exhibit physiologically relevant cell-cell and cell-matrix interactions, and present similar gene expression, heterogeneity and structural complexity as in-vivo tissues. Multicellular spheroids have been attempted for drug screening and evaluation, mechanical studies on cancer cell invasion and migration, and regeneration medicine. However, fabrication of uniform-sized MCSs at a high throughput platform, and evaluation of MCSs for clinical relevance are two main challenges. In this thesis, thermally responsive microgels were employed as physical supports to culture multicellular spheroids from both tumor cells and stem cells, which are potentially applied in anti-cancer drug evaluation, tissue engineering and regeneration medicine. The thermally reversible poly (N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-AA)) microgels were first employed to fabricate HeLa MCSs. This microgel approach restricted cell mobility at a lower initial cell density due to a large volume in the microgel networks, which resulted in uniform-sized spheroids formation compared to non-adhesive culture. Moreover, because of thermal reversibility of this microgel, spheroids were released from the physical supports via cooling down the system to room temperature. After demonstrating the formation of tumor spheroids in the microgel, HeLa cells were further encapsulated inside microgel-droplets generated from flow focusing microfluidics to obtain controllable uniform-sized spheroids. This approach combined the benefit of using thermal sensitive microgels as physical supports for MCS formation and droplet generation at a high throughput platform. Highly uniform-sized MCSs were obtained through this method. Importantly, the MCSs were easily released from the droplets by reducing the culture temperature to room temperature without using strong chemical or enzyme reagents. This approach may be used for generation of uniform-sized MCSs for drug screening and evaluation. The microenvironment generated from the microgel plays an important role in MCS formation. The key characteristics of the microenvironment, such as surface charge density, hydrophobicity, mechanical strength, and the microstructure of the microgels, were investigated by synthesizing a range of poly(N-isopropylacrylamide) (P(NIPAM)) based microgels, including P(NIPAM), P(NIPAM-co-methacrylic acid) (P(NIPAM-MAA)), P(NIPMAM-co-acrylic acid) (P(NIPAM-AA)), P(NIPAM-co-malic acid) (P(NIPAM-MA)) and P(NIPAM-co-itaconic acid)(P(NIPAM-IA)). It was found that the moderate negatively charged surface with high hydrophilicity P(NIPAM-IA) microgels was beneficial for cellular growth. The high or low charge density resulted in slow cell proliferation. The hydrophobicity of microgels had a negative impact on cell growth. The large pore size of the P(NIPAM-IA) networks also allowed cell migration which promoted MCSs formation. Different cell types (HEK 293, U87, HeLa and mesenchymal stem cells) have been demonstrated to successfully form MCSs within the P(NIPAM-IA) microgel. The thermal sensitive microgels were further applied to form stem cell MCSs. Human cardiac stem cells (hCSCs) were cultured in the P(NIPAM)) based microgel networks including P(NIPAM-co-dimethyl amino ethyl methacrylate) (P(NIPAM-DMAEMA)), P(NIPAM-IA), (P(NIPAM-co-2-hydroxyethyl methacrylate) (P(NIPAM-HEMA)), P(NIPAM-co-poly(ethylene glycol) methyl ether acrylate) (P(NIPAM-PEGA)). These microgels displayed different charges (cationic, anionic, and neutral) and different degrees of hydrophobicity. Through evaluation of hCSCs viability, proliferation and release of regenerative factors, P(NIPAM-IA) was identified as one of the best candidates for forming hCSCs spheroids because of its negatively charged surface with high hydrophilicity. The thermal reversibility of P(NIPAM-IA) renders it as injectable hydrogels. Initial results showed that injection of this microgel into mice did not elicit immune system responses, reduced myocardial apoptosis and promoted angiogenesis in the mice. In summary, we have fabricated MCSs in different types of thermal responsive microgels through either physical control of the uniform size by confining cells in the microgel-droplets generated from microfluidics or fine tune of the microenvironment for MCS formation. The P(NIPAM-IA) microgel with moderated anionic charge and high hydrophilicity was found to promote MCSs formation. This microgel did not elicit any immune response, which indicates the potential of using this microgel for future clinical studies.Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 201

Control of flow and oxygen in a 3-D perfused micro-environment fosters balanced survival of hepatocyte-non-parenchymal cell co-cultures

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references (p. 141-153).Creating a physiologically relevant in vitro liver model requires reproducing the cellular heterogeneity of in vivo liver in a functional state. However differentiated sinusoidal endothelial cells (SECs), marked by SE-1 expression are difficult to maintain in culture while stellate cells easily activate and over-proliferate. We hypothesized that recreating a liver tissue system that captured in vivo like paracrine influences would foster survival of these cells, and predicted that stimuli resulting from flow and oxygen gradients close to physiological conditions would preserve the delicate balance between the cell types. Spheroids containing hepatoctyes with incorporated non-parenchymal cells (NPCs) were seeded into capillary bed sized channels in polycarbonate scaffolds, housed in a three-dimensional perfused system, and maintained for two weeks. Micro-flow rates of different media through the formed tissue units in scaffolds were controlled using pneumatic pumps and microfluidics. Staining and confocal imaging of endpoint tissue showed that lower flow rates closer to physiological regimes allowed the survival of SE-1+ SECs, regardless of exogenously added growth factors in the medium. Higher flow rates, exogenous growth factors, and scaffold contact were associated with activation of stellate cells (alpha-smooth muscle actin staining).Since oxygen measurements in the system coupled low flow rates with hypoxic tissue outlet concentrations, we parsed out these variables by repeating flow experiments in low oxygen environments. Retention of SE-i staining cells even in higher flow rates demonstrated that hypoxic conditions in the tissue could play a role in aiding their survival by overcoming negative effects brought about by high flow. The relationship of stellate cells with flow rate was unaffected by oxygen concentrations. To explore if the negative effects of high flow on SE-i expression were mediated by transforming growth factor-beta (TGF-[beta]), we added a TGF-[beta] inhibitor SB-431542 in our cultures, and found that it greatly enhanced the presence of SE-1 staining SECs at high flow rates. In conclusion we successfully created a three-dimensional flow controlled hepatic culture system that allows balanced survival of hepatocytes and non-parenchymal cells, making it useful as a potential model for studies such as cancer metastasis that require interactions between tumor cells and heterotypic host tissue. Key Words: Liver, In vitro, co-culture, sinusoidal, endothelial, stellate, oxygen, flow, shear.by Ajit Dash.Ph.D

Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models

Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases

INVESTIGATING BIOLOGICAL EFFECTS OF NANOPARTICLES WITH 3-DIMENSIONAL CELL MODELS

Ph.DDOCTOR OF PHILOSOPH

Cellular biomechanics in 2D and 3D epithelial model tissues : from keratin intermediate filaments to breast gland in vitro reconstructed basement membranes

The mechanical organization of biological tissue is crucial to the load-transmitting capacity of our bodies, and follows a hierarchical architecture that macroscopically results in organ’s formation and function. At the base of such a tightly regulated structure we find single cells, whose mechanical properties are decisive in shaping their interaction with the surrounding environment. Developing a fundamental understanding of this interplay over a wide range of length scales is essential to reach the deep working knowledge of the biomechanics of multicellular systems required for tissue engineering, surface design and nanomedicine. In this work, the mechanical properties of epithelial model systems were analyzed at different organizational scales (namely, from single cells to microtissue) by means of atomic force microscopy (AFM) micro- and nano-indentation experiments. Despite the intrinsic difficulty in characterizing soft and heterogeneous biological samples in terms of mechanical response, this technique still offers an exciting possibility to quantitatively probe their viscoelastic behavior. At first, a murine epidermal cell line completely devoid of keratin intermediate filaments (knock-out keratinocytes) was compared to its wild type counterpart in order to assess the role played by this cytoskeletal component in conferring mechanical stability to single cells. Then, cellular monolayers were analyzed, to validate the relevance of our findings also in a more physiological context. Despite its presence in organs such as skin and nails, which obviously serve a barrier function, the mechanical role of keratin in deeper tissue remained controversial for a long time. Reconstructed keratin polymer gels in fact display properties resemblant of viscoelastic solids; in vivo, the networks are formed of bundles that are relatively sparse and show lower connectivity than other cytoskeletal components. This fact, together with the low values of bending stiffness and extremely high extensibility reported for these filaments, would suggest that keratin networks confer resilience and elasticity to cells, rather than a scaffolding function against compressive stress. Our results though clearly pointed at a substantial softening of keratin-lacking cells, with elasticity moduli differences of 25% to 35% between wild type and knock-out according to the cellular region probed. The presented data represent the first proof of this effect on the single cell level. Validation of this result further came from the observation that the difference could be partially suppressed by reintroducing a single keratin protein in the mutant cells. In the second part of this work, a three-dimensional cell culture system mimicking the elementary unit of a human breast gland was analyzed in terms of its biomechanical and permeation properties. The cell line used for this purpose (MCF10A), when grown in an extracellular matrix-resembling environment, can develop into growth-arrested acinar structures which follow the same substantial maturation steps of a human breast gland; cells organize according to an apico-basal polarization scheme, secrete a dense matrix of cross-linked extracellular matrix proteins to surround them (the so called basement membrane) and finally develop a hollow lumen necessary, in vivo, for milk production and secretion. The centrality of breast gland tissue in a context of cancer research cannot be overstated: alveolar units are the hotspot for tumor formation, and as such have been the focus of much attention in the past years. Relatively little effort, though, has been dedicated to understanding the mechanical interplay of healthy breast gland microtissues with their surrounding environment, despite the fact that one of the hallmarks of cancer progression is a set of strong alterations in the mechanical phenotype of aberrant cells. Here, we offer an experimental analysis of the mechanical properties of healthy 3D acinar structures at different developmental stages, and briefly compare them with those of invasive microtissues. The application of different hyperelastic models to the interpretation of nanoindentation experiments is discussed, along with a tentative clarification of some of controversies arising during AFM data analysis. Additionally, a characterization of isolated basement membranes performed by means of atomic force microscopy imaging, scanning electron microscopy and superresolution light microscopy is reported; experimental evidence suggests that basement membranes act as fundamentally elastic materials whose thickness and structural stability change throughout the different developmental stages. To complement this biomechanical analysis, we investigated the acinar permeation properties; in short, data elucidate that the basement membrane acts as a passive diffusion barrier with a size-selectivity threshold for the retardation of macromolecular permeation of about 40 kDa and a pore size of at least 9 nm. At the same time, it offers a fundamental mechanical shielding function, reaching elastic modulus values of up to about 400 kPa in the fully matured state. Taken together, the presented data underline how intra- and extra-cellular polymer networks serve a crucial function in defining the mechanical properties of epithelial tissue.</p