CIVIL ENGINEERING 365 ALL ABOUT CIVIL ENGINEERING


Preparation of hydrogels

PEG hydrogels crosslinked via Michael-type addition reaction were prepared as described20, mixing aqueous solutions containing thiol- and vinylsulfone- functionalized 4-arm and 8-arm PEG macromers (mol. weight 10 kDa) at various concentrations to adjust stiffness and stoichiometric ratio. Here, a v/w ratio of 7.5%, corresponding to an average shear modulus of G′ ≈ 20kPa, was used to fabricate the milliwell arrays. The solution was deposited and molded as explained above. The construct was crosslinked for 15 minutes at room temperature.

Fabrication of U-shaped hydrogel milliwell arrays

U-shaped hydrogel milliwell arrays were fabricated as described elsewhere40. Briefly, U-shaped cavities of 1.5 mm in diameter, 1.8 mm in depth spaced by 75μm were generated onto standard 4-inch silicon wafers using standard Si Bosch and soft lithography processes. PDMS (ratio 1:10) was poured onto the wafers and cured overnight at 75 °C. After crosslinking, the PDMS stamps were de-moulded and punched with various diameters. The desired stamps were mounted on custom-made holders. The final non-crosslinked hydrogel mix was deposited onto the PDMS stamp, and the holder-stamp-hydrogel construct was inserted into a custom-made PDMS ring, placed at the bottom of wells of a 24 well plate. The hydrogels were incubated at 37 °C and 5% CO2 for 15 minutes (see below). After crosslinking, aqueous buffer (e.g. 1X PBS) was pipetted into the wells and the stamps were removed carefully. The resulting microwell arrays were sterilized thoroughly in buffer under UV light and stored at 4 °C until use.

ESC culture

ESC-Crx-GFP16 were split twice on MEF and twice on gelatin before aggregate formation on milli-arrays or expanded onto MEF prior to cryo-preservation to be stored. Briefly: 6 well plates (Sigma, CLS3516 SIGMA Corning® Costar®) were coated with 0.1% of Gelatin (Sigma, G2500) for 30 minutes at 37 °C and aspirated. Fibroblasts were plated in MEF medium until confluence and inactivated 2 h at 37 °C with Mitomycin C (Sigma, M0503). The day after inactivation, ESCs were plated on inactivated feeder layer in ESC maintenance medium. After two days, when the cells reached 80% confluence, the medium was changed, cells washed in phosphate-buffered saline (PBS, Gibco, 10010-023) and trypsinized (Gibco, 25200-056). Re-suspended cells were placed in a fresh ESC maintenance medium to achieve a split ratio of 1:10 onto plates with a new feeder layer or 1:6 on gelatin. ESC maintenance medium was composed as follows: 500 ml GMEM (Gibco, 11710-035), 5.1 ml 100X non-essential amino acids (NEAA, Gibco, 11140-050) , 5.1 ml 100mM  sodium pyruvate (Gibco, 11360-039), 0.51 ml of 0.1 M beta-mercaptoethanol (2-ME, Gibco, 31350-010), 58 ml knockout serum (KSR) and 5.8 ml FCS were sterilized by filtration through a 0.2 μm bottle-top filter (Millipore Corporation), stored at 4 °C and used within 1 month5,12. 2.000 U/ml and 4.000 U/ml of LIF was freshly added to the culture medium each time at medium change on MEF and on gelatin, respectively.

Retina organoid media

Optic vesicle (OV) induction medium16

From day 0 to days 7, ESCs were kept in a medium composed of 500 ml GMEM, 5.1 ml non-essential amino acids, 5.1 ml sodium pyruvate, 0.51 ml of 0.1 M 2-ME, N2 (1%, Thermo fisher, 17502-048), B27 minus vitamin A (1%, Thermo fisher, 12587001) and 7.6 ml KSR.

Optic cup (OC) induction medium16

From day 7 to day 9 the aggregates were incubated in DMEM/F-12 with GlutaMAX (Gibco, 10565-018), and 1% of N2 supplement (Gibco, 17502-048).

Retinal maturation medium

From day 9 to day 12, aggregates were kept in DMEM/F-12 with GlutaMAX (Gibco, 10565-018), and 1% of N2, 2% B27 supplement (Gibco, 17502-044). From day 12 onwards, further maturation of the optic cups was stimulated by adding fresh Taurine (Sigma, T8691, 1 mM) to the retinal maturation medium and removing the B27 supplement. Further, from day 12 aggregates were kept in hyperoxic conditions (40% of O2, Sanyo, cat. no. MOC-175M). The optic cup morphogenesis and the following maturation were allowed to proceed inside the mother aggregate until day 24-26, when optic cups where analyzed.

Procedures established for ROAs

Milli-well plates equilibration

Milli-well plates, composed of 7 micro-cavities, were quickly washed twice in PBS and equilibrated in 140 μl of OV induction medium without supplements (see below) at 37 °C for 30 minutes.

Retinal organoid protocol in milliwell arrays

In the meanwhile, Crx-GFP mESCs16 were washed with phosphate-buffered saline (1X PBS, Gibco) and detached with trypsin (Gibco, Cat. no. 25200-056). The cells were then resuspended in Optic Vesicle (OV) induction medium at a density of 525′000 cells/mL in order to seed 3000 cells per microwell. 40 μl of the cell suspension was then added on top of the arrays, in the seeding compartment, and the cells were left to sediment for 30 minutes at 37 °C and 5% CO2. Then, 660 μL of OV induction medium was added. Aggregates form as soon as 8 h after seeding. We adjusted the amount of medium needed per well to 840 μL to match the amounts of media per organoids reported in the literature during the first 10 to 14 days of differentiation in 96 U-bottom plates (i.e. 120 μL). For this reason, after an overnight incubation, the cells formed aggregates in each microwell and 140 μL of a diluted growth factor reduced MatrigelTM solution (12%, Corning) was added in each of the 24 wells, where the final Matrigel concentration was 2%. The aggregated cells were left in OV induction medium for 7 days. At day 7, the medium was changed to OC induction medium and left until day 12. At day 12, the medium was subsequently changed to Retina Maturation medium as previously described16, until day 24-26. In this case, the medium was changed every other day. Additionally, the organoids were incubated in hyperoxic incubators from day 12 on, to promote the survival of newly born photoreceptors5,16.

Individual retinal organoids, whole array fixation and immunohisto/cytochemistry

Individual retinal organoids and whole arrays were fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature (RT) and cryoprotected by successive incubation in 10%, 20%, and 30% sucrose in PBS at least overnight at 4 °C and then embedded in yasulla (30% egg albumin and 3% gelatin in distilled water water) and snap-frozen in dry ice before sectioning. 12 μm cryosections were cut in a cryostat (Thermo Scientific Microm). The sections were stored at −80 °C until use.

12 µm sections prepared on Superfrost plus glass slides were incubated for 1 h in blocking buffer (0,1–0,3% Triton X-100, 1–10% goat, rabbit, or sheep serum, and 0.1–0.5 mg/mL of bovine serum albumin (BSA) diluted in PBS), and incubated ON at 4 °C or RT or 3 hours at RT with primary antibodies (suppl. Table 1). Sections of retina were used for immunohistochemical analysis to confirm antibody specificity. The following antibodies were used: PAX6 (Covance, rabbit, 1:300), RHODOPSIN (Santa Cruz, mouse, 1:300), OTX2 (Abcam, rabbit, 1:300), GNAT2 (Santa Cruz, rabbit, 1:200), GNAT1 (Santa Cruz, rabbit, 1:1000), CALBINDIN D28k (Labome, rabbit, 1:500), CRALBP (Abcam, mouse, 1:500). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Control sections were incubated with secondary antibodies only. Stainings were observed and pictures acquired using a confocal (LSM700, Zeiss).

Microscopy, image processing and quantification

Images were acquired with a LSM700 invert laser scanning confocal (Zeiss). Confocal microscopy was performed with similar settings for laser power, photomultiplier gain and offset, with a pinhole diameter of one Airy unit. Thin optical sections were used for channel co-localization. Images were adjusted for brightness and contrast using ImageJ (NIH; http://rsb.info.nih.gov/ij/).

RNA isolation and reverse transcription

Seven organoids collected from one ROAs and from 96 well plates at different day of differentiation, respectively day 7, 11, 15, were analysed in triplicates. RNA was extracted from the retinal organoids according to the manufacturer’s instructions using TRIzol reagent (Invitrogen-Thermofisher, Zug, Switzerland, 15596-026). RNA concentration and OD260/280 ratio were determined using a Nanodrop 1000 (Thermo-Scientific).

During reverse transcription, to denature any secondary structures in the RNA template, 1 µg of total RNA, and 1 µl of random hexamer primers (Promega, Madison, USA; 0.5 mg/ml) were incubated at 70 °C for 10 min and immediately cooled on ice. For the reverse transcription reaction, 4 µl AMV-RT buffer (Promega, Madison, USA), 2 µl dNTP mix (Promega, Madison, USA; 10 mM), 1 µl RNAse Inhibitor (RNAsin; Promega, Madison, USA), and 1 µl AMV reverse transcriptase (AMV-RT; Promega, Madison, USA) were added to a 20 µl final volume reaction and incubated at 42 °C for 1 h, followed by 10 min incubation at 95 °C.

Quantitative real-time RT-PCR (qPCR) analysis

Primers were designed using the Primer 3 (Simgene.com) software, spanning an exon–exon junction where applicable (see Supplementary Table 1 for mouse qPCR primer list). Primers were synthesized by Microsynth (Microsynth AG, Balgach, Switzerland). For qPCR, we used a 1:30 dilution of the synthesized cDNA.

qPCR was carried out using LightCycler® 480 SYBR Green I Master (Roche, Zug, Switzerland, 04887352001) with the following protocol: 95 °C 600 s; 45 × : 95 °C 10 s, 60 °C 10 s, 72 °C 10 s; 95 °C 10 s, 65 °C 60 s, 97 °C 1 s, 37 °C 30 s. The reaction mixtures were set up according to the manufacturer’s recommendations (Roche, LightCycler® 480, Zug, Switzerland). Primers were synthesized by Microsynth (Microsynth AG, Balgach, Switzerland). Amplification products were resolved on a 1.5% agarose gel to check the band. All PCR experiments were performed at least 3 times on at least 3 independent cell samples. All expression values were normalized to the value of Erl8 gene. Relative normalized expression Basic ∆∆C -Method and the standard error of mean (s.e.m) were calculated using Roche integrated software.

Electron microscopy (EM)

Retinal organoids were incised using a microsurgical knife (Oasis Medical, Inc. cat no. PE3015) before fixation in 2.5% glutaraldehyde – 2% paraformaldehyde in 1X PBS at 4 °C overnight. The organoids were then washed three times in Cacodylate buffer (0.1 M, pH 7.4) for five minutes and post-fixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide in Cacodylate buffer (0.1 M, pH 7.4) at room temperature (RT) for 40 minutes. The solution was then changed to 1% osmium tetroxide in Cacodylate buffer (0.1 M, pH 7.4) and the samples were further incubated for an additional 40 minutes at RT. Then, the organoids were washed two times in distilled water for five minutes, stained with 1% uranyl acetate (in H2O) and washed again two times in distilled water for five minutes. The samples were then dehydrated in graded alcohol series of 15 minutes each with the following alcohol contents: 50%, 70%, 96% and 100%. Finally, we embedded each sample separately in Durcupan by rotating their respective recipients continuously and adding increasing amounts of resin to the sample initially in 100% EtOH. After rotation, the samples were incubated overnight in a ratio of 1:1 of Durcupan 100% to EtOH 100%. The next day each organoid was incubated in 100% Durcupan for the entire day. The samples were then sectioned, embedded on coated glass slides and incubated in the over at 65 °C overnight. Serial sections were performed and the sections were imaged using a transmission electron microscope (FEI Tecnai Spirit, 120 kV).

Computational characterization of diffusion on the ROAs

Using COMSOL (COMSOL Multiphysics®), two computational reaction-diffusion models were designed to mimic seven spheres, having the same negative reaction rate, on a hydrogel microwell substrate, characterized by a slower diffusion coefficient than the medium (10X slower), into 840 μL of medium. We simplified the system by assuming that the medium is a single species and that its maximum concentration is normalized. In addition, the organoids, represented as spheres of a constant size, were considered to have a constant medium consumption rate over the time span of interest. We chose to simplify the model as much as possible to only analyze the effect of geometry on the local depletion of a normalized species.

Statistics and counting

Statistical analyses are based on at least four biological replications and three technical replicates. At least 84 organoids were analyzed per experiment. The percentage of cell type counted in the organoids was related to the retina-like structure and not to the whole aggregate/organoid. For one retinal organoid slice, the retinal ganglion cell layer and the outer nuclear layer served to delineate the area to count. All means are presented with ± SEM (standard error of the mean). Statistical significance was assessed using Graphpad Prism 5 software, and applying one-way ANOVA with Tukey’s correction. In figures, *P < 0.05, **P < 0.01, and ***P < 0.001.



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