The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo-relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested, and evaluated 1) a novel removable microfluidics-based cell-delivery biochip and 2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both 2D and 3D arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm/s in the radial plane and 50 μm/s in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 seconds, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system.

Traditional cell-culture techniques lack the spatial control of single cells that is necessary to recreate the cell-cell contact arrangement found in tissue. Since Ashkins developed optical force-based cellmanipulation techniques [1], the laser microbeam has been used in a microscopic system to explore various biological interactions at molecular and cellular levels. These explorations of a single cell or subcellular organelle involve trapping (using a laser-tweezers microscope [2]) and guidance (using a laser-guided direct writing microscope [3,4]). When laser tweezers are used, a high numerical aperture (NA) microscope objective generates a strongly focused laser beam, which 3D traps a particle, such as a biological cell, in the beam's focal point. In laser-guided direct writing, a low NA microscope objective generates a weakly focused laser beam, which traps a particle in the beam axis and guides it to move along the direction of beam propagation.Based on these techniques, we have developed a laser cell-micropatterning system in which the laser beam is focused in a transition state between generating an optical trap and optical guidance [5]. Using this system, a single biological cell can be trapped in a typical (e.g., 30mm) cell culture dish and patterned onto a designated cell culture niche with very high spatial and temporal resolution [6][7][8].However, this and the other currently available laser-based single-cell-manipulation techniques can be used to pattern only spherical cells, not irregularly shaped cells such as rod-shape cardiomyocytes. The work stems from the use of a spatial light modulator (SLM) loaded with a computer generated phase map to shape a single laser beam into multiple microbeams analogous to techniques used in holographic optical tweezers (HOT) [9]. In contrast to the optical configuration in HOT, our system uses a low NA objective to produce multiple weakly focused laser microbeams in our multiple beam laser guidance system. Based on the cell image acquired during laser cell patterning, the multiple beams can be distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, here we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes (ACMs) in an end-to-end connected alignment to replicate the in vivo heart-muscle structure without use of substrate surface modifications that may interfere with in vivo-like cell-cell and cell-extracellular matrix interactions.The basic structure of the multiple-beam laser cell-patterning system is schematically shown in Figure 1. The cell suspension was loaded into the microfluidics-based cell feeding system built in the celldeposition chamber, with a microfluidic channel width of 200 µm. The guidance region (also the imaging region of the objective) was initially focused onto the outlet of the cell-feeding microfluidic channel by controlling the movement of the ce...

Breast cancer survival has drastically improved over the past decades; however, drug resistance and subsequent disease progression is responsible for the incurability of advanced disease. While the focus of many drug response studies is the transformed tumor cells, there is increasing evidence suggesting a role for stromal cells in tumorigenesis and drug resistance. Microenvironmental components, including extracellular matrix, fibroblasts, leukocytes, and adipocytes, all contribute to physiological mammary gland biogenesis. Accordingly, these stromal elements contribute to disease progression and resistance. However, many in vitro drug response studies still utilize 2D monolayer cultures with purified breast tumor cells. In vivo studies remain the gold standard for drug development, even though they are performed with immune-compromised mice that may not reflect the physiological tumor microenvironment and have been repeatedly shown to be a poor representation of clinical outcomes. Thus, there is a need for more complex in vitro models to test drug response effectively and accurately. We have previously demonstrated the benefits of using a patient-derived, tri-culture (3x), 3D perfusion microtumor (3DpMT) system. To further replicate the complex tumor microenvironment, we have expanded to a penta-culture (5x) model by incorporating macrophages and lymphocytes alongside the tumor cells, fibroblasts, and adipocytes of the 3x model. We have accrued over 207 primary tumor samples, including both resected tumor and core biopsies, from which we have generated 12 stable PDX models (~50% ER+) and >20 3x, 4x, and 5x 3DpMT with a focus on triple negative (TNBC). The 5x patient-derived 3DpMT tissues represent our most complex breast cancer in vitro model and have been cultured successfully for up to 5 weeks allowing for high-throughput, long term drug response testing with different dosing strategies. They have been characterized by flow cytometry, IHC, RNA expression, NGS, DNA methylation patterns, and cytokine/chemokine secretion. When possible, marker expression has been compared to the primary tumor. Furthermore, the accuracy of our models to replicate clinical tissue is evident in the similar toxicities of chemotherapies observed in clinical use. With these models we can replicate physiological processes including cell migration, polarization of macrophages, activation of lymphocytes, and changes in molecular profiles throughout the duration of our 5x culture assays. Our model has the potential to test a myriad of drugs, from conventional chemotherapies to novel immunotherapies over extended time periods with different dosing strategies in order to provide a more accurate prediction of patient-specific clinical response. Citation Format: Qi Guo, Melissa Millard, Christine Wilhelm, Ashley Elrod, Nick Erdman, Lacey E. Dobrolecki, Brian McKinley, Mary Rippon, Wendy Cornett, John Rinkliff, Amanda Scopteuolo, Linda Gray, James Epling, Barbara Garner, Jeff Hanna, Eric McGill, C. David Williams, David Schammel, David L. Kaplan, Christopher Corless, Jeff Edenfield, Michael T. Lewis, Howland E. Crosswell, Teresa M. DesRochers. Complex, patient-derived, multi-cell type, 3D models of breast cancer for personalized prediction of therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5673.

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