Chondroitin sulfate a

To evaluate the cell removal and preservation of ECM components in the RdECM, biochemical assays were conducted. For the assessment of DNA, glycosaminoglycans (GAGs), collagen, lyophilized native tissues, and the RdECM were digested using papain (125 μg/mL) in 0.1 M sodium phosphate with 5 mM Na2-EDTA and 5 mM cysteine-HCL at pH 6.5 for 17 h at 60 °C. As a diluent, papain solution without tissue samples was used. The DNA content was quantified using a DNA quantitation kit (DNAQF, Sigma), according to the manufacturer’s protocol. The fluorescence intensity was measured using a fluorescence spectrophotometer (Gemini XPS Microplate Reader, Molecular Device; excitation wavelength: 350 nm, emission wavelength: 460 nm). Calf thymus DNA (Sigma-Aldrich, St. Louis, MO, USA) was used to generate a standard curve for DNA. In the analysis of GAGs, the tissue-digested solution had been stained with 1,9-dimethyl methylene blue, and the absorbance at 525 nm was read. The standard curve used for estimating the number of sulfated GAGs in the samples was plotted using chondroitin sulfate A (Sigma-Aldrich). Collagen content was determined using a total collagen assay kit (Biovision, Milpitas, CA, USA). The samples were prepared according to the manufacturer’s instructions, following measurement of the absorbance at 540 nm; hydroxyproline was used to prepare the standard curve.

PBS, HBSS, DMEM/F12 (Thermo Fisher Scientific, Inc.), UW solution (Viaspan^®; Bristol-Myers Squibb, Co.), ET-Kyoto solution (Otsuka Pharmaceutical Factory, Inc.), and Optisol GS™ (Bausch & Lomb, Rochester, NY) were prepared as candidates for a basal preservation medium. The following additives were also prepared: dextran 40 (0.2, 2, 20 g/L; Wako), chondroitin sulfate A (0.01, 0.1, 1%; Sigma-Aldrich, St. Louis, MO, USA), N-acetylcysteine (0.1, 1, 10 mM; Wako), allopurinol (0.1, 1, 10 mM; Tokyo Chemical Industry Co., Ltd), glutathione (0.3, 3, 30 mM; Sigma-Aldrich), adenosine (0.05, 0.5, 5 mM; Sigma-Aldrich), hyaluronic acid (0.01, 0.1, 0.5%; Calbiochem), dibutyryl cAMP (0.2, 2, 20 mM; Enzo Life Sciences), trehalose (12, 120, 1200 mM; Hayashibara), tert-butylhydroquinone (tBHQ; 0.1, 1, 10 μM; Sigma-Aldrich), oltipraz (1, 10, 100 μM; Sigma-Aldrich), and ebselen (1, 10, 100 μM; Sigma). Each of these additives was added to HBSS individually.

Cytoadherence of infected RBCs was performed as described previously [25 (link)]. Briefly, Ibidi μ-Slide 0.2 channel slides were incubated with 100 μl chondroitin sulfate A (100 μg/ml; Sigma) in 1% BSA/PBS overnight at 37°C. Channel slides were blocked with 1% BSA/PBS for 1 h at room temperature prior to gentle washes with 37°C bicarbonate-free RPMI 1640 (Invitrogen). Mature infected cultures (3% parasitemia and 1% hematocrit) were harvested and resuspended in warm bicarbonate-free RPMI 1640. Samples were pulled through the channel at 100 μl/min using a syringe pump (Harvard Apparatus) for 10 min at 37°C to allow cytoadherence, then washed for 10 min to remove unbound cells. Adhered cells were counted at 10 points along the axis of the channel. The microscopy was performed on a DeltaVision Elite widefield imaging system (GE Healthcare).

A disposable electrode array slide (8W10E+ ECIS, Applied BioPhysics) was stabilized with F99 medium as per manufacturer’s protocol. The array surface was coated with 40 μg/cm² chondroitin sulfate A (Sigma-Aldrich) and 400 ng/cm² laminin (Sigma-Aldrich) in phosphate-buffered saline (PBS) for two hours. Five days after transduction with lentivirus (10 MOI) the cells were reseeded at 100% confluence within respective chambers of the slide array. Cells were incubated in the arrays at room temperature for one hour to facilitate even distribution of cell attachment. After seeding and preliminary cell attachment, arrays were positioned into a 16-well array station and connected to the ECIS Zθ instrument to measure electric impedance (Ω at 4000 Hz) for 4 days. Cell-cell (R_b, Ω • cm²) and cell-substrate (α, Ω^1/2 • cm) adhesion along with cell membrane capacitance (C_m, μF • cm^-2) were modeled from the electric impedance data obtained at 4000 Hz [66 (link)].