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.
Highlights
Standard three-dimensional (3D) in vitro culture techniques, such as those used for mammary epithelial cells, rely on random distribution of cells within hydrogels
We demonstrate the superiority of our printing process over manual matrix embedding techniques in efficiency and consistency in organoid morphology
MCF12A and MCF10A cells were initially cultured in 2D on tissue culture plastic in a 75-cm2 flask supplemented with a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12), 5% Horse Serum, 20 ng/mL human epidermal growth factor, 0.01 mg/mL bovine insulin, 500 ng/mL hydrocortisone, and 1% ABAM
Summary
Standard three-dimensional (3D) in vitro culture techniques, such as those used for mammary epithelial cells, rely on random distribution of cells within hydrogels These systems offer advantages over traditional 2D models, limitations persist owing to the lack of control over cellular placement within the hydrogel. While some variability inevitably results from disparities in local environmental conditions, such as collagen fiber anisotropy within specific regions of a gel, a major source of potentially controllable variability results from the random distribution of cells within the gel [18,19,20,21,22,23] This variability leads to difficulty in interpreting and reproducing results, especially from laboratory to laboratory. As inter-laboratory reproducibility is a major concern in modern biomedical research, platforms that will allow better control and reproducibility are highly desired [24]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
