Three-dimensional filamentous human diseased cardiac tissue model.

Fabrication of filamentous matrices

The filamentous matrices were fabricated via the TPIP system ( Figure 1A ) based on a femtosecond laser beam irradiated vertically to the photoresist, a UV-curable organic-inorganic hybrid polymer (ORMOCER®, Micro resist technology). The photoresist were spin-coated onto glass plates (25 mm in length, 3 mm in width, and 1 mm in thickness) at 4000 RPM for 100 seconds, pre-baked on a hotplate at 80°C for 2 minutes, and cured by UV light illumination for 30 minutes. Two glass plates were assembled with two 0.5 mm-thick spacers at the ends and subsequently hard baked at 140°C fo r 1.5 hour. The assembled glass scaffold was filled by uncured photoresist and placed on PC-controllable X-Y-Z motorized stages (Aerotech, ANT95-XY-MP and ANT95-50-L-Z-RH) with high precise positioning. Single fibers were fabricated along the laser beam path with a high-repetition rate femtosecond laser irradiation ( Movie S1 ). The femtosecond laser (pulse duration: ~400 fs, repetition frequency: 1 MHz, wavelength: 1045 nm, FCPA μJewel D-400, IMRA America, Inc.) was frequency-doubled to the wavelength of ~522 nm by Lithium triborate (LBO) second harmonic nonlinear crystal (Newlight photonics) and focused onto the glass plate/photoresist interface with a 5X microscope objective (M Plan Apo, N.A. = 0.14, Mitutoyo). The fibers with different diameters could be fabricated by changing the laser power and exposure time. The power of the laser beam emitted downstream of the objective lens was measured by a power meter and controlled by a half-wave plate and a polarizing beam splitter. The exposure time was set by a PC-controllable mechanical shutter. Fibers with diameters of 5 μm and 10 μm were created with powers of 2.6 mW for 0.7 s exposure and 6.4 mW for 1 s exposure respectively. Different fiber spacing within the matrices was controlled by the X-Y-Z stage with high positioning precision operated by a PC. After finishing the polymerization process, the samples were placed on a hotplate at 120°C for 30 min for post-baking. After cooling in the air for 10 min, the matrices were immersed in the developer (ORMODEV®, Micro resist technology) for 1 hour to remove uncured photoresist. To reduce the tendency of adherent between of fibers, the matrices were immersed into the 60 mg/mL asolectin (Sigma Aldrich) solution for 30 min, and then rinsed five times with 2-propanol (Sigma Aldrich) and deionized water by succession. To sterilize the sample, suspended filamentous matrices were immersed in 70% ethanol before usage.

Cardiac differentiation from iPS cells

The diseased iPS cell line (LQT3) and a healthy cell line counterpart (WT) were obtained from Dr. Conklin’s laboratory at the Gladstone Institute of Cardiovascular Research. A small molecule WNT-mediated protocol was used to derive iPS-CMs from these cell lines [ 18 ]. Briefly, iPS cells were maintained on 6-well plates coated with Matrigel (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies). On Day -3, cells were dissociated with Accutase (Invitrogen) for 5 min at 37 °C and seeded onto Mat rigel-coated cell culture plates at 25,000 cells/cm 2 in mTeSR1 containing 10 μM Y-27632 (Stemgent). 24 hours later, the medium was changed to mTeSR1 without Y-27632, and cells were maintained in mTeSR1 for two additional days. On Day 0, cells were treated with 12 μM of a GSK3 inhibitor (CHIR9902; Selleckchem) in RPMI 1640 medium containing B27 supplement without insulin (RPMI/B27-I; Life Technologies) for 24 hours. The medium was changed on Day 1 to RPMI/B27-I and incubated for 48 hours, followed by a 48-hour treatment with 5 μM of an inhibitor of Wnt production (IWP-4, Stemgent) beginning on Day 3. On Day 5, the medium was changed to RPMI/B27-I for two days, and then changed to RPMI 1640 containing B27 complete supplement (RPMI/B27-C) on Day 7.

Generation of 3D cardiac tissue

Each filamentous matrix was placed into one well of a 6-well plate. The matrices were rinsed with Dulbecco's phosphate buffered saline (DPBS, Gibco Invitrogen) three times and coated with 50 μg/mL fibronectin (Sigma Aldrich) in DPBS for 1 hour. The matrices were rinsed with DPBS three times before loading the cells. Sheets of beating CMs were dissociated using a singularization protocol, which included incubation with 1 mg/mL collagenase II (Worthington) with 40 Unit/mL DNase I (BioLabs Inc.) in Hank’s balanced salt solution (HBSS) for 1 hour and followed by 0.25% trypsin/EDTA treatment for 5 min at 37°C [ 19 ]. The cells were collected, pelleted and resuspended in EB20 media (Knockout DMEM supplemented with 20% fetal bovine serum (FBS), 2mM L-glutamine, 1X MEM non-essential amino acids (MEM-NEAA), 400 nM 2-Mercaptoethanol and 10 μM Y-27632). 500 μL cell suspension with a density of 1 million cells/mL was pipetted over each matrix. After 2 hours, another 4 mL EB20 media was added into each well to cover the whole matrix. The media was switched to RPMI/B27-C the next day to reduce the non-CMs overgrowth and changed every 2 days.

Immunostaining and microscopy

Cells were characterized using immunostaining and fluorescent microscopy. Samples were fixed with 4% (vol/vol) paraformaldehyde (PFA) for 15 min, permeabilized with 0.2% Triton-X-100 for 5 min, and blocked with 2% BSA, 4% goat serum and 0.1% Triton-X-100 for 30 min. The samples were then incubated with primary antibodies for 2 hours and secondary antibodies for 1.5 hour. DAPI was used to stain cell nuclei in monolayer cell culture and To-Pro-3 was used for filamentous matrices, because the fiber material was auto-fluorescent under UV excitation. For bright-field and epi-fluorescent microscopy, the images were taken using a Nikon Eclipse TS100F microscope with SPOT Flex camera. For confocal microscopy, the images were taken with a Zeiss LSM710 laser-scanning microscope the in Biological Imaging Facility at UC Berkeley. The antibodies used in this study are listed in Table S1 .

Flow cytometry analysis

The pluripotency of iPS cells and efficiency of cardiac differentiation was evaluated using flow cytometry. The iPS cells were dissociated with Accutase and stained using a human pluripotent stem cell multicolor flow cytometry kit against Oct4, SSEA4, and Sox2 (R&D system). Human iPS-CMs were dissociated using the singularization protocol described above, fixed with PFA for 15 min, and incubated with primary antibody (mouse monoclonal cardiac Troponin T, Thermo Scientific) and secondary antibody (Alexa488, Life Technologies) for 30 min each. The labeled cells were analyzed by Guava easyCyte™ Flow Cytometer (EMD Millipore) in the Stem Cell Shared Facility at UC Berkeley.

RT-qPCR analysis

The expression level of cardiac specific genes was analyzed using RT-qPCR at Day 12. Adherent cells were washed with DPBS and homogenized with 1 mL TRIzol LS reagent (Invitrogen). Total RNA was collected and purified using RNeasy Mini Kit (Qiagen). The total RNA concentration was quantified using a Nanodrop and integrity was determined using the Agilent BioAnalyzer. Conversion of total RNA to cDNA was carried out using SuperScript III Reverse Transcriptase (Invitrogen) with random primers. qPCR was performed on the Applied Biosystems StepOnePlus instrument with customized target arrays in 96-well format (SA Biosciences/Qiagen), using 10 ng cDNA per reaction and SYBR Green ROX MasterMix (Qiagen). The data was analyzed using 2 −ΔCt method relative to level of the housekeeping gene. The genes used in this study are listed in Table S2 .

Assessment of tissue formation

The formation of 3D cardiac tissue was assessed using fluorescent images from confocal microscopy. The cells penetrating the top layer and spreading on the middle layer were counted by nuclei as N CM , and the total number of fibers within the image was counted as N F . Thus, the tissue formation was quantified as cells per fiber ( N CM / N F ). Assessment of iPS-CM alignment The CM alignment along the fiber direction was assessed with respect to the cell’s elongation and orientation in fluorescent images from confocal microscopy. The cell nuclei were measured with transverse diameter ( D t ) and conjugate diameter ( D c ). The nuclei index representing the cell’s elongation was calculated using the ratio of D c / D t . The cell’s orientation was measured with the acute angle between the fiber direction and nuclei transverse diameter as θ (0° ≤ θ ≤ 90°) . The alignment index relative to fiber long axis was calculated by cosθ. Assessment of iPS-CM electrophysiology The CM electrophysiology was assessed with an MEA system (MED64, Alpha MED Scientific Inc). The iPS-CMs on Day 15 were dissociated using the singularization protocol described above and replated on an MEA chip with a density of 1 million cells/mL. The cells were cultured using RPMI/B27-C for 7 days before starting the measurement. During the recording, the cells were maintained at 37°C in a sterile environment. The el ectrical waveform representing the field potential of CMs can export the electrophysiological parameters, including beating frequency (BF), field potential duration (FPD) and field potential amplitude (FPA). Assessment of iPS-CM contractility The CM contractility was assessed based on the recorded video and motion tracking software. The video was taken at 30 f/s and exported as image stacks for motion tracking analysis. The in-house developed motion tracking software, based on block matching algorithms [ 20 ], was optimized for tracking CM beating events. This software can identify the contraction and relaxation events during the entire CM beating sequence and export the contractility parameters, including BF, maximal contraction velocity (MCV), maximal relaxation velocity (MRV), and beating duration (BD).

Fiber stiffness measurement

The fiber stiffness is represented as the elastic modulus ( E f ) of the fiber, which can be measured by atomic force microscopy (AFM, XE-100, Park Systems) with tip-less AFM cantilevers (TL-CONT-SPL and TL-FM-SPK, Nanosensors). The shape of fiber is assumed to be a cylinder with circular cross-section, and accordingly the elastic modulus of the fiber with can be calculated by: (1) E f = 64 K c d c l 3 3 π D 4 ( d f − d c ) where l is the fiber length, D is the fiber diameter, d c is the displacement of the tip-less AFM cantilever, and d f is the relative deflection of the fiber. The spring constants ( K c ) of tip-less AFM cantilevers were determined using the thermal tune method [ 21 ] and calculated using AFM software (XEI, Park system).

Statistical analysis

Data were presented as mean ± SEM. For single comparisons, one-tailed Student’s t-test was used. For multiple comparisons, one-way ANOVA was used with Bonferroni post hoc test. p