Consistent and reproducible cultures of large-scale 3D mammary epithelial structures using an accessible bioprinting platform

Abstract

BackgroundStandard three-dimensional (3D) in vitro culture techniques, such as those used for mammary epithelial cells, rely on random distribution of cells within hydrogels. Although these systems offer advantages over traditional 2D models, limitations persist owing to the lack of control over cellular placement within the hydrogel. This results in experimental inconsistencies and random organoid morphology. Robust, high-throughput experimentation requires greater standardization of 3D epithelial culture techniques.MethodsHere, we detail the use of a 3D bioprinting platform as an investigative tool to control the 3D formation of organoids through the “self-assembly” of human mammary epithelial cells. Experimental bioprinting procedures were optimized to enable the formation of controlled arrays of individual mammary organoids. We define the distance and cell number parameters necessary to print individual organoids that do not interact between print locations as well as those required to generate large contiguous organoids connected through multiple print locations.ResultsWe demonstrate that as few as 10 cells can be used to form 3D mammary structures in a single print and that prints up to 500 μm apart can fuse to form single large structures. Using these fusion parameters, we demonstrate that both linear and non-linear (contiguous circles) can be generated with sizes of 3 mm in length/diameter. We confirm that cells from individual prints interact to form structures with a contiguous lumen. Finally, we demonstrate that organoids can be printed into human collagen hydrogels, allowing for all-human 3D culture systems.ConclusionsOur platform is adaptable to different culturing protocols and is superior to traditional random 3D culture techniques in efficiency, reproducibility, and scalability. Importantly, owing to the low-cost accessibility and computer numerical control–driven platform of our 3D bioprinter, we have the ability to disseminate our experiments with absolute precision to interested laboratories.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1045-4) contains supplementary material, which is available to authorized users.

A 3D bioprinter platform for mechanistic analysis of tumoroids and chimeric mammary organoids

“…Movies 1 and 2). This is consistent with the activity of non-tumorigenic MCF-12A cells in previous findings which move between print locations and continue to display movement within organoids 23 . Importantly, as these structures began to generate branched extensions, tumor cells remained equally dispersed within the networked structures (Fig.…”

“…To improve efficiency of chimeric structure formation, we next sought to guide the formation of chimeric organoids using our custom bioprinting system. We previously described our ability to standardize the frequency of organoid formation through control of the initial cell quantities within bioprinted cell-deposits; cell-deposits containing at least 40 cells formed organoids within 7 days post-printing 23 . Therefore, we mixed MCF12A cells and tumorigenic MCF-7 or MDA-MB-468 cells in a 5:1 ratio as above, and then printed 66 nl of cell mixtures equivalent to 40 cells in equally-spaced linear (300 µm) and circular (500 µm) arrays inside collagen I gels.…”

“…We have recently described the adaptation of a low-cost accessible 3D bioprinter for the purpose of precise cell printing within 3D hydrogels 22,23 . This bioprinting platform was designed for use in basic cell biology laboratories and can be used to generate large 3D mammary organoids in hydrogels 23 . Unlike traditional culture, the 3D bioprinted system precisely places cells allowing for greater control of organoid formation and experimental consistency.…”

Engineering of tissue constructs using coaxial bioprinting

“…Organoids have been used in a staggering number of applications, including intestinal, cerebral, pancreatic, kidney, hepatic, retinal, lung, colonic, gastric, thyroid, prostate, salivary, mammary, lingual, placental, and spinal tissues, which are reviewed elsewhere [ 88 ]. Bioprinting has only recently seen application in organoid literature [ [89] , [90] , [91] , [92] , [93] , [94] , [95] , [96] ]. In coaxial systems, developing organoid tissues might be printed within a sacrificial sheath hydrogel, allowing for mechanical strength and hierarchal arrangement, without compromising cell viability ( Fig.…”

Correction to: Consistent and reproducible cultures of large-scale 3D mammary epithelial structures using an accessible bioprinting platform

“…Following publication of the original article [ 1 ], the authors reported a typesetting error in the spelling of the second author’s name.…”