Open-source design data files are provided, as well as any files required for fabrication, including .STL files for printed pieces and .NC files for CNC machining. (dashed black line) can be used to estimate the depth at which there is 10% of the initial pyruvate concentration at any given time point. Supporting Information Figure S3: The 3D nature of optical sectioning. Cutaway view of the sample area for the FLIM experiments (left). Medium was removed and reserved to ensure that the gel contacted the cover glass for imaging. The region of the collagen gel that can be probed by optical imaging is shown in red. An orthogonal view of a z-stack of images taken through a collagen gel (inlay, right). Each image was a taken at a different depth into the sample. The signal is from NADH intensity to show the cells inside the collagen gel. Supporting Information Figure S4: Assessment of MDA-231 cell growth on various materials. A) Brightfield images of cells grown for 3 days in wells either with no material or in the presence of Amyloid b-Peptide (1-40) (human) materials potentially utilized for the bioreactor, including polystyrene (PS) (cell culture plastic control), polypropylene (PP), silicone rubber (SR), Delrin (del) or RC31 (RC31). (B) Graph showing the change, over 3 days, in the density of cells grown in the presence of Amyloid b-Peptide (1-40) (human) various materials, normalized to the cell density of that treatment on day 1. (P=0.0113 for materials comparison, two-way ANOVA; * P<0.05, **<0.01, Dunnetts multiple comparison test vs. no material control, day 3 only). C) Graph showing the cell density on day 3 relative to PS control, which takes into account mechanical disruption of cell contacts resulting from physical presence of the material wafer in the well. (P=0.008, one-way ANOVA; Dunnetts multiple comparisons test indicate no significant differences when compared to control PS). Scale bar is 100 microns. NIHMS1000763-supplement-Supp_info.pdf (1.1M) GUID:?9DDAF1FA-890D-44CB-84C7-A0535F56BB0D Abstract Purpose: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism and have emerged. Specifically, magnetic resonance spectroscopy (MRS) of hyperpolarized 13C-labeled pyruvate allows for the real-time monitoring of LDH activity [11]C[13], while optical fluorescence lifetime ERCC6 imaging (FLIM) of Amyloid b-Peptide (1-40) (human) the intrinsically fluorescent NADH [14], [15] allows for the measurement of its chemical state, whether protein-bound or free in the cytosol [16]. These two metabolic measurement techniques yield complementary information, by probing organ and cellular scales, respectively. Therefore, combined studies that utilize both methods may add value for quantitatively investigating enzyme activity and cofactor status for various metabolic pathways. Hyperpolarized MRS imaging Amyloid b-Peptide (1-40) (human) studies with 13C-pyruvate are moving rapidly to clinical translation [12], principally because of their ability to measure LDH activity and upregulation of glycolysis of cancer [17], [18]. These recent advances are supported by pre-clinical studies as well as studies of cell cultures [19] and tumor biopsy tissues [20] using MRS of three dimensional (3D) sample volumes. In contrast, optical imaging experiments are often performed in adherent 2D cell cultures on glass bottom dishes at sub-cellular resolution [21]. Although the cellular resolution is desirable, cells cultured directly on conventional glass bottom dishes lack the 3D microenvironment encountered [22], [23]. Collagen gels that more closely resemble the native (breast) tumor microenvironment [24] can improve the biological relevance of optical imaging experiments (Supporting Information Figure S1). While optical experiments using imaging windows implanted above tumors in small animal models enable direct imaging within the tumor microenvironment [25], they have intrinsic limitations including poor depth of field and increased cost and.