Dual-chambered membrane bioreactor for coculture of stratified cell populations.

We have developed a dual-chambered bioreactor (DCB) that incorporates a membrane to study stratified 3D cell populations for skin tissue engineering. The DCB provides adjacent flow lines within a common chamber; the inclusion of the membrane regulates flow layering or mixing, which can be exploited to produce layers or gradients of cell populations in the scaffolds. Computational modeling and experimental assays were used to study the transport phenomena within the bioreactor. Molecular transport across the membrane was defined by a balance of convection and diffusion; the symmetry of the system was proven by its bulk convection stability, while the movement of molecules from one flow line to the other is governed by coupled convection-diffusion. This balance allowed the perfusion of two different fluids, with the membrane defining the mixing degree between the two. The bioreactor sustained two adjacent cell populations for 28 days, and was used to induce indirect adipogenic differentiation of mesenchymal stem cells due to molecular cross-talk between the populations. We successfully developed a platform that can study the dermis-hypodermis complex to address limitations in skin tissue engineering. Furthermore, the DCB can be used for other multilayered tissues or the study of communication pathways between cell populations.

Implementation of a Dynamic Co-Culture Model Abated Silver Nanoparticle Interactions and Nanotoxicological Outcomes In Vitro.

The incorporation of engineered nanoparticles (NPs) into everyday consumer goods, products, and applications has given rise to the field of nanotoxicology, which evaluates the safety of NPs within biological environments. The unique physicochemical properties of NPs have made this an insurmountable challenge, as their reactivity and variable behavior have given rise to discrepancies between standard cell-based in vitro and animal in vivo models. In this study, enhanced in vitro models were generated that retained the advantages of traditional cell cultures, but incorporated the modifications of (1) inclusion of an activated immune element and (2) the presence of physiologically-relevant dynamic flow. Following verification that the human alveolar epithelial and macrophage (A549/U937) co-culture could be successfully sustained under both static and dynamic conditions, these cultures, in addition to a standard A549 static model, were challenged with 10 nm citrate coated silver NPs (AgNPs). This work identified a reshaping of the AgNP-cellular interface and differential biological responses following exposure. The presence of dynamic flow modified cellular morphology and reduced AgNP deposition by approximately 20% over the static exposure environments. Cellular toxicity and stress endpoints, including reactive oxygen species, heat shock protein 70, and secretion of pro-inflammatory cytokines, were found to vary as a function of both cellular composition and flow conditions; with activated macrophages and fluid flow both mitigating the severity of AgNP-dependent bioeffects. This work highlights the possibility of enhanced in vitro systems to assess the safety of engineered NPs and demonstrates their effectiveness in elucidating novel NP-cellular interactions and toxicological profiles.

3D Cell Culture Models as Recapitulators of the Tumor Microenvironment for the Screening of Anti-Cancer Drugs.

Today, innovative three-dimensional (3D) cell culture models have been proposed as viable and biomimetic alternatives for initial drug screening, allowing the improvement of the efficiency of drug development. These models are gaining popularity, given their ability to reproduce key aspects of the tumor microenvironment, concerning the 3D tumor architecture as well as the interactions of tumor cells with the extracellular matrix and surrounding non-tumor cells. The development of accurate 3D models may become beneficial to decrease the use of laboratory animals in scientific research, in accordance with the European Union's regulation on the 3R rule (Replacement, Reduction, Refinement). This review focuses on the impact of 3D cell culture models on cancer research, discussing their advantages, limitations, and compatibility with high-throughput screenings and automated systems. An insight is also given on the adequacy of the available readouts for the interpretation of the data obtained from the 3D cell culture models. Importantly, we also emphasize the need for the incorporation of additional and complementary microenvironment elements on the design of 3D cell culture models, towards improved predictive value of drug efficacy.

Tissue-engineered arterial intima model exposed to steady wall shear stresses.

The arterial intima is continuously under pulsatile wall shear stresses (WSS) imposed by the circulating blood. The knowledge of the contribution of smooth muscle cells (SMC) to the response of endothelial cell (EC) to WSS is still incomplete. We developed a co-culture model of EC on top of SMC that mimics the inner in vivo structure of the arterial intima of large arteries. The co-cultured model, as well as a monolayer model of EC, were developed in custom-designed wells that allowed for mechanobiology experiments. Both the monolayer and co-culture models were exposed to steady flow induced WSS of up to 24 dyne/cm2 and for lengths of 60 min. Quantification of WSS induced alterations in the cytoskeletal actin filaments (F-actin) and vascular endothelial cadherin (VE-cadherin) junctions were utilized from confocal images and flow cytometry. High confluency of both models was observed even after exposure to the high WSS. The quantitive analysis revealed larger post WSS amounts of EC F-actin polymerization in the monolayer, which may be explained by the relative help of the SMC to resist the external load of WSS. The VE-cadherin demonstrated morphological alterations in the monolayer model, but without significant changes in their content. The SMC in the co-culture maintained their contractile phenotype post high WSS which is more physiological, but not post low WSS. Generally, the results of this work demonstrate the active role of SMC in the intima performance to resist flow induced WSS.

Tissue-engineered multi-cellular models of the uterine wall.

The human uterus is composed of three layers: endometrium, myometrium and perimetrium. It remodels during the monthly menstrual cycle and more significantly during the complex stages of reproduction. In vivo studies of the human uterine wall are yet incomplete due to ethical and technical limitations. The objective of this study was to develop in vitro uterine wall models that mimic the in vivo structure in humans. We co-cultured multiple cellular models of endometrial epithelial cells, endometrial stromal cells and smooth muscle cells on a synthetic membrane mounted in multi-purpose custom-designed wells. Immunofluorescence staining and confocal imaging confirmed that the new model represents the in vivo anatomical architecture of the inner uterine wall. Hormonal treatment with progesterone and ß-estradiol demonstrated increased expression of progestogen-associated endometrial protein, which is associated with the in vivo receptive uterus. The new tissue-engineered in vitro models of the uterine wall will enable deeper investigation of molecular and biomechanical aspects of the blastocyst-uterus interaction during the window of implantation.