Hmscs

Human dual-color variant PC3 prostate cancer cells expressing H2B-eGFP and DsRed2 were from Anticancer; luciferase-expressing PC3 cells were provided by Dr. Gary Gallick, UT MD Anderson Cancer Center. Cells were maintained in DMEM (Corning), 10% fetal calf serum (Sigma), penicillin and streptomycin (both 100 μg/ml, Sigma). Human C4–2B cells (provided by Dr. Timothy Thompson, UT MD Anderson Cancer Center) expressing H2B/mCherry and LifeAct-GFP were cultured in RPMI (Corning), 10% fetal calf serum (Sigma), penicillin and streptomycin (both 100 μg/ml, Sigma) and 1% HEPES. The identity of tumor cell lines was verified by Short Tandem Repeat DNA profiling (Characterized Cell Line Core Facility, M.D. Anderson Cancer Center). ASC52telo telomerase reverse transcriptase immortalized adipose tissue derived mesenchymal stem cells (hMSCs, ATCC) were maintained in Minimum Essential Medium (MEM1X, Corning), supplemented with 17% fetal calf serum, vitamins (Sigma), non-essential amino acids (Sigma), sodium pyruvate (Gibco), penicillin and streptomycin (both 100 μg/ml, Sigma). To induce osteoblastic differentiation, hMSCs were cultured in osteogenic medium (DMEM 1X, supplemented with 10% calf serum, penicillin and streptomycin, 50 μg/ml L-ascorbic acid, 10 mM β-glycerophosphate, 0.1 μM dexamethasone from Sigma).

MDA-MB-231 cells (ATCC) and human mesenchymal stem cells (hMSC, Lonza) were used as cellular models to assess cell viability within the device. MDA-MB-231 cells were expanded in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were stored in a culture incubator (Fisher) maintained at 37°C and 95% humidity. Carbon dioxide was not required as Leibovitz’s L-15 media is pH buffered for use at ambient CO₂ levels. hMSCs were expanded in a commercially-available growth medium (Lonza, PT-3001). hMSCs were stored in an incubator (NuAire) maintained at 37°C, 95% humidity, and 10% CO₂ for pH buffering. Both cell types were passaged at 90% confluency using Trypsin-EDTA (Lonza) and split 1:3 in T75 flasks (Corning).
For the device exposure, MDA-MB-231 cells and hMSCs (passage 3–6) were seeded within the agarose gel disc at 10⁶ cells/ml. Low melting temperature agarose (Sigma) was dissolved in PBS to 4% w/v and stored in the incubator to prevent gelling. Once cells were resuspended to an appropriate concentration after hemocytometer counting, 0.5 ml of the cell suspension was combined 1:1 with 4% agarose to yield a final concentration of 2% and a total volume of 1 ml. The agarose-cell mixture was gelled in tissue-culture Petri dishes (Corning), where 1 ml of the mixture yielded a thickness of 3 mm. The agarose discs were allowed to sit for 20 min at room temperature to properly gel. 1.5 cm diameter cores of the gel were then removed with a bore and placed in the oxygen gradient device. The device was assembled and then 5 ml of media was added through the media port. A separate cell-seeded agarose disc was cored and placed in a static petri dish as a control.
Device exposure experiments were conducted in a humidified incubator (95% relative humidity, 37°C) to prevent media evaporation and maintain proper physiological temperature. The same incubator CO₂ concentration was used as for the expansion depending on the media formulation. After placing the assembly in the incubator, tubing from the gas cylinders was connected to the device. Flow was regulated by a rotameter in-line with the tubing, and proper connections were confirmed by attached tubing downstream of the device and bubbling through a beaker filled with water. Cells were exposed to 21% oxygen infused into both microchannels (no gradient) for 48 h.
Cell viability was assessed with a Live-Dead stain (Invitrogen) for both agarose-seeded cell types cultured in the device, as well as the controls, using the manufacturer recommended concentrations. Constructs were sectioned and incubated in the stain for 30 min. Sections were then rinsed three times in PBS. Live cells were stained with calcein-AM whereas the dead cells took up the ethidium homodimer-1 dye. Images were captured with the same microscope used for oxygen validation, but with the manufacturer-specified filters and at 4×. Live and dead cells were imaged separately depending on which filter was used. After exposure of 21% oxygen for 48 h, the agarose discs were sectioned in halves and then incubated with the dyes in a phosphate-buffered saline solution. The cross-sections of the disc were viewed under the microscope, with images taken spanning the width of the agarose. ImageJ was used to threshold and count live and dead cells separately in each of the images. The images were stitched together for a position-dependent viability assessment, and then regions were specified with a box and analyzed. Cell viability experiments were conducted three times and error bars represent the standard deviation of those three trials.