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      The revolution will be open-source: how 3D bioprinting can change 3D cell culture

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          Abstract

          The development of three-dimensional culture scaffolds represents a revolutionary step forward for in vitro culture systems. Various synthetic and naturally occurring substrates have been developed that support 3D growth of cells. In most fields, including mammary gland biology and tumorigenesis, the two most common substrates used are the basement membrane rich extracellur matrix (ECM) isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcomas (e.g. Matrigel) and collagen extracted from rat-tails. The processes of 3D culture in these two substrates has remained unchanged for nearly half a century: cells are either mixed with unpolymerized matrix to disperse them randomly throughout the substrate upon polymerization or overlaid randomly on top of a preformed hydrogel. While effective in generating organoid/tumoroid structures, the random nature of these processes has many drawbacks that limit the reproducibility and tunability of the experimental design. Furthermore, random cellular distributions limit the utility of these substrates for studying interactions within the cellular microenvironment, which have been shown to be critical for the control of stem and cancer cell function [1]. To overcome these issues, computer numerically controlled (CNC) devices can be adapted to precisely control cellular deposition within hydrogels. An example of these devices can be found in the three-axis control of modern 3D fusion deposition method (FDM) printers. Despite a rapid drop in cost of 3D printing technology, printers specifically engineered for bioprinting purposes generally remain unattainably expensive for general biological research laboratories. Thus the technology has been limited to specific biofabrication applications in specialty biomedical engineering laboratories. Furthermore, commercially available bioprinters are exclusively designed for printing “bioinks” (unpolymerized scaffoldings with or without cells) into shapes. While potentially useful for medical reconstructive procedures, the shape of the hydrogel is meaningless to cell biologists seeking to understand basic questions of cell biology, or for engineering applications seeking to direct specific differentiation of cells. To this end, we recently developed a low-cost open access 3D bioprinting system that can be used for scientists applications (Figure 1) [2]. The printer is an open source project that allows other laboratories to build their own system. Initially, all necessary parts can be printed using a standard off-the-shelf 3D FDM printer. The same printer can then be modified with these parts into a 3D bioprinter. In essence, you can 3D print your own 3D bioprinter. This system is designed to be adaptable to any application the end user requires, and we have described its use for both printing cells as well as guiding electrodes for directed electrical pulsing of cells [3, 4]. To increase precision and maintain integrity of printed cells, we use pulled glass micropipette syringes as our cell injection “print-head” [2]. Compared to standard steel needles attached to luer lock syringes, these glass micropipettes have a finer point and reduce sheer force on the cells. Combined, this minimizes disruptions to the cells and allows the hydrogel to seal behind the print. Thus, our system allows for the precise placement of cells, that then self-organize into organoids/tumoroids, making functional structures [4]. This differs from many bioprinting approaches that print cell free or cell-laden “bioinks” into 3D shapes. Again, while these approachees are potentially useful for specific medical reconstruction procedures, they have little utility for most biological applications. For example, cells printed in the shape of a mammary gland are not a mammary gland; it is the coordinated function and differentiation of cells through development that make tissues and organs functional. Figure 1 Custom benchtop 3D bioprinter and its application for printing large mammary organoids Top: Image of an example 3D bioprinter constructed off of the Felix 3.0 (FELIXrobotics, NL) platform. Middle: Example of coordinated print of clusters of red fluorescent protein (RFP) labeled MCF12a cells at distances of 200μm in linear array. Image taken 24 hours post-print. Scale bar = 200μm. Bottom: Mature organoid (21 days post-print) formed from coordinated print of clusters of RFP MCF12a cells into a circular array. Resulting organoids have been shown to have contiguous lumens stretching > 3mm in length. We have recently described the use of this system for printing mammary organoids in standard 3D hydrogels [4]. By depositing specific numbers of cells at controllable distances we could guide the growth of organoids into predictable sizes and shapes (Figure 1). The key to the guided growth was the fact that mammary epithelial cells (MCF12a and MCF10a) would preferentially grow towards neighboring prints, forming single contiguous organoids. Using this strategy, we generated large contiguous luminal mammary organoids (> 5mm in length). This is in clear contrast to random culture where the dispersion of cells results in random organoid shape and size, with organoids never forming more than a couple of hundred microns in size. Controlling for organoid distancing, shaping, and sizing is thus not feasible in standard culture models and therefore interpretation of studies where these factors may play a role becomes difficult. This is particularly true for experiments on microenvironmental forces and cancer/epithelial cell growth. Because surrounding organoids can influence the rigidity of the microenvironment, control of the placement, spacing, and size is critical. In an era of poor data reproducibility in science [5], instruments and methods designed to limit lab-to-lab variability are in need. The open source nature of our bioprinter helps facilitate standardization of experimental parameters across laboratories. This is because the machine instructions or GCODE generated for printing experiments can be shared once data is published. We have shared our files through our website http://www.odustemcell.org. Researchers can go to this website, download the files needed to print their own bioprinter, and then use the GCODE files necessary to repeat our experiments exactly using their own bioprinter. While this certainly doesn’t eliminate all inter-laboratory variability, it helps simplify the process of reproducing an experiment. Our current focused application of this printing technology is to understand the role of the cellular microenvironment in controlling differentiation of stem and cancer cells (Reid et al., Submitted). We have explored this topic in in vivo models [6–10], but our bioprinting platform allows for mechanistic insights into the process. These studies are ongoing, but one can imagine our printing platform can be used to improve any application where the random nature of traditional 3D culture is a confounding variable. And, one could argue, nearly every study is potentially confounded by this factor. A common issue with modern science/scientist is the tendency to assign radical ideas to the “science fiction” classification; however, bioprinting needs not be the stuff of science fiction. Our studies highlight the ease of access and the utility of the technology for basic cell and cancer biology studies. Thus, we hope to lower the bar of entry further by developing easier-to-access solutions, such as ready built kits, and a graphic user interface (GUI) to simplify the experimental programming. The system offers a potentially revolutionary step forward for 3D culture models of development and cancer.

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          Most cited references 3

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          Accessible bioprinting: adaptation of a low-cost 3D-printer for precise cell placement and stem cell differentiation.

          The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive 'off-the-shelf' commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, 'luer-lock' syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering.
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            Reprogramming non-mammary and cancer cells in the developing mouse mammary gland.

            The capacity of any portion of the murine mammary gland to produce a complete functional mammary outgrowth upon transplantation to an epithelium-divested fat pad is unaffected by the age or reproductive history of the donor. Likewise, through serial transplantations, no loss of potency is detected when compared to similar transplantations of the youngest mammary tissue tested. This demonstrates that stem cell activity is maintained intact throughout the lifetime of the animal despite aging and the repeated expansion and depletion of the mammary epithelium through multiple rounds of pregnancy, lactation and involution. These facts support the contention that mammary stem cells reside in protected tissue locales (niches), where their reproductive potency remains essentially unchanged through life. Disruption of the tissue, to produce dispersed cells results in the desecration of the protection afforded by the "niche" and leads to a reduced capacity of dispersed epithelial cells (in terms of the number transplanted) to recapitulate complete functional mammary structures. Our studies demonstrate that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary cells, including mouse and human cancer cells, may be sequestered and reprogrammed to perform mammary epithelial cell functions including those ascribed to mammary stem/progenitor cells. Published by Elsevier Ltd.
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              • Article: not found

              3D bioprinter applied picosecond pulsed electric fields for targeted manipulation of proliferation and lineage specific gene expression in neural stem cells

              Objective Picosecond pulse electric fields (psPEF) have the potential to elicit functional changes in mammalian cells in a non-contact manner. Such electro-manipulation of pluripotent and multipotent cells could be a tool in both neural interface and tissue engineering. Here, we describe the potential of psPEF in directing neural stem cells (NSCs) gene expression, metabolism, and proliferation. As a comparison mesenchymal stem cells (MSCs) were also tested. Approach A psPEF electrode was anchored on a customized commercially available 3-D printer, which allowed us to deliver pulses with high spatial precision and systematically control the electrode position in three-axes. When the electrodes are continuously energized and their position is shifted by the 3-D printer, large numbers of cells on a surface can be exposed to a uniform psPEF. With two electric field strengths (20 and 40 kV/cm), cell responses, including cell viability, proliferation, and gene expression assays, were quantified and analyzed. Main Results Analysis revealed both NSCs and MSCs showed no significant cell death after treatments. Both cell types exhibited an increased metabolic reduction; however, the response rate for MSCs was sensitive to the change of electric field strength, but for NSCs, it appeared independent of electric field strength. The change in proliferation rate was cell-type specific. MSCs underwent no significant change in proliferation whereas NSCs exhibited an electric field dependent response with the higher electric field producing less proliferation. Further, NSCs showed an upregulation of glial fibrillary acidic protein (GFAP) after 24 hours to 40 kV/cm, which is characteristic of astrocyte specific differentiation.
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                Author and article information

                Journal
                Oncotarget
                Oncotarget
                Oncotarget
                ImpactJ
                Oncotarget
                Impact Journals LLC
                1949-2553
                30 July 2019
                30 July 2019
                : 10
                : 46
                : 4724-4726
                Affiliations
                1 School of Medical Diagnostic & Translational Sciences, Old Dominion University, Norfolk, VA, USA
                Author notes
                Correspondence to: Robert D. Bruno, rbruno@ 123456odu.edu
                Patrick C. Sachs, psachs@ 123456odu.edu
                Article
                27099
                10.18632/oncotarget.27099
                6730589
                321e21a4-4327-4851-bfad-90c7b0941e1f
                Copyright: © 2019 Bruno et al.

                This article is distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.

                Categories
                Editorial

                Oncology & Radiotherapy

                3d bioprinting, 3d culture, mammary gland, breast cancer, open-source

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