Triple-negative breast cancer cells invade adipocyte/preadipocyte-encapsulating geometrically inverted mammary organoids.

This paper describes the manufacture of geometrically inverted mammary organoids encapsulating primary mammary preadipocytes and adipocytes. Material manipulation in an array of 192 hanging drops induces cells to self-assemble into inside-out organoids where an adipose tissue core is enveloped by a cell-produced basement membrane, indicated by laminin V staining and then a continuous layer of mammary epithelial cells. This inverted tissue structure enables investigation of multiple mammary cancer subtypes, with a significantly higher extent of invasion by triple-negative MDA-MB-231 breast cancer cells compared to MCF7 cells. By seeding cancer cells into co-culture around pre-formed organoids with encapsulated preadipocytes/adipocytes, invasion through the epithelium, then into the adipose core is observable through acquisition of confocal image stacks of whole mount specimens. Furthermore, in regions of the connective tissue core where invasion occurs, there is an accumulation of collagen in the microenvironment. Suggesting that this collagen may be conducive to increased invasiveness, the anti-fibrotic drug pirfenidone shows efficacy in this model by slowing invasion. Comparison of adipose tissue derived from three different donors shows method consistency as well as the potential to evaluate donor cell-based biological variability. Insight box Geometrically inverted mammary organoids encapsulating primary preadipocytes/adipocytes (P/As) are bioengineered using a minimal amount of Matrigel scaffolding. Use of this eversion-free method is key to production of adipose mammary organoids (AMOs) where not only the epithelial polarity but also the entire self-organizing arrangement, including adipose position, is inside-out. While an epithelial-only structure can analyze cancer cell invasion, P/As are required for invasion-associated collagen deposition and efficacy of pirfenidone to counteract collagen deposition and associated invasion. The methods described strike a balance between repeatability and preservation of biological variability: AMOs form consistently across multiple adipose cell donors while revealing cancer cell invasion differences.

Engineering cell heterogeneity into organs-on-a-chip.

Organ-on-a-chip development is an application that will benefit from advances in cell heterogeneity characterization because these culture models are intended to mimic in vivo microenvironments, which are complex and dynamic. Due in no small part to advances in microfluidic single cell analysis methods, cell-to-cell variability is an increasingly understood feature of physiological tissues, with cell types from as common as 1 out of every 2 cells to as rare as 1 out of every 100 000 cells having important roles in the biochemical and biological makeup of tissues and organs. Variability between neighboring cells can be transient or maintained, and ordered or stochastic. This review covers three areas of well-studied cell heterogeneity that are informative for organ-on-a-chip development efforts: tumors, the lung, and the intestine. Then we look at how recent single cell analysis strategies have enabled better understanding of heterogeneity within in vitro and in vivo tissues. Finally, we provide a few work-arounds for adapting current on-chip culture methods to better mimic physiological cell heterogeneity including accounting for crucial rare cell types and events.

Building an experimental model of the human body with non-physiological parameters.

New advances in engineering and biomedical technology have enabled recent efforts to capture essential aspects of human physiology in microscale, in-vitro systems. The application of these advances to experimentally model complex processes in an integrated platform - commonly called a 'human-on-a-chip (HOC)' - requires that relevant compartments and parameters be sized correctly relative to each other and to the system as a whole. Empirical observation, theoretical treatments of resource distribution systems and natural experiments can all be used to inform rational design of such a system, but technical and fundamental challenges (e.g. small system blood volumes and context-dependent cell metabolism, respectively) pose substantial, unaddressed obstacles. Here, we put forth two fundamental principles for HOC design: inducing in-vivo-like cellular metabolic rates is necessary and may be accomplished in-vitro by limiting O2 availability and that the effects of increased blood volumes on drug concentration can be mitigated through pharmacokinetics-based treatments of solute distribution. Combining these principles with natural observation and engineering workarounds, we derive a complete set of design criteria for a practically realizable, physiologically faithful, five-organ millionth-scale (× 10-6) microfluidic model of the human body.

Collagen Type II enhances chondrogenic differentiation in agarose-based modular microtissues.

BACKGROUND AIMS: Cell-based therapies have made an impact on the treatment of osteoarthritis; however, the repair and regeneration of thick cartilage defects is an important and growing clinical problem. Next-generation therapies that combine cells with biomaterials may provide improved outcomes. We have developed modular microenvironments that mimic the composition of articular cartilage as a delivery system for consistently differentiated cells. METHODS: Human bone marrow-derived mesenchymal stem cells (MSC) were embedded in modular microbeads consisting of agarose (AG) supplemented with 0%, 10% and 20% collagen Type II (COL-II) using a water-in-oil emulsion technique. AG and AG/COL-II microbeads were characterized in terms of their structural integrity, size distribution and protein content. The viability of embedded MSC and their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages over 3 weeks in culture were also assessed. RESULTS: Microbeads made with <20% COL-II were robust, generally spheroidal in shape and 80 ± 10 µm in diameter. MSC viability in microbeads was consistently high over a week in culture, whereas viability in corresponding bulk hydrogels decreased with increasing COL-II content. Osteogenic differentiation of MSC was modestly supported in both AG and AG/COL-II microbeads, whereas adipogenic differentiation was strongly inhibited in COL-II containing microbeads. Chondrogenic differentiation of MSC was clearly promoted in microbeads containing COL-II, compared with pure AG matrices. CONCLUSIONS: Inclusion of collagen Type II in agarose matrices in microbead format can potentiate chondrogenic differentiation of human MSC. Such compositionally tailored microtissues may find utility for cell delivery in next-generation cartilage repair therapies.