Establishment of an in vitro choroid complex system for vascular response screening – Scientific Reports

Animal

Mice (C57BL/6 J and BALB/c) were obtained from CLEA Japan (Tokyo, Japan). All procedures involving animals were conducted in accordance with the guidelines of the Ethics Committee on Animal Research of Keio University School of Medicine (#16,017), the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the International Standards of Animal Care and Use, Animal Research: Reporting in Vivo Experiments.

Cell culture

Mouse choroidal fibroblasts (msCFs) were isolated from C57BL/6 J mouse eyes using a modified method based on the procedures outlined by Djigo AD et al.¹⁷ and Benedicto et al.⁴⁵ Briefly, the RPE/choroid tissue was isolated from 6–8-week-old mouse eyes. The RPE was removed from the 0.25% trypsin/EDTA-treated tissue through vigorous pipetting. To make a single-cell suspension, the choroid tissue was incubated with 6.25 mg/ml collagenase A (038–24,561, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 6.25 mg/ml dispase II (17,105,041, Thermo Fisher Scientific, Sunnyvale, CA, USA), and 62.5 μg/ml DNase (EN0521, Thermo Fisher Scientific, Sunnyvale, CA, USA) solution at 37 °C for 15 min, followed by incubation in 0.25% trypsin/EDTA at 37 °C for 5 min. Endothelial cells (ECs) were removed from this dissociated cell suspension using Dynabeads CD31 (11155D, Invitrogen, Logan, UT, USA) following the manufacturer’s instructions. The suspension was cultured in Dulbecco’s Modified Eagle Medium with high glucose (08,457–55, Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin (P/S) at 37 °C in a 5% CO₂ incubator. For the choroid model, msCFs with passage 4–10, which outgrew the other cells, were utilized.

Human induced pluripotent stem cell (hiPSC)-derived ECs (iCell Endothelial Cells 11,713, FUJIFILM Cellular Dynamics, Inc., Madison, WI, USA) and RPE (iCell Retinal Pigment Epithelial 01,279, FUJIFILM Cellular Dynamics, Inc., Madison, WI, USA) cells were obtained from FUJIFILM Cellular Dynamics, Inc. (Madison, WI, USA). hiPSC-ECs were cultured on 30 µg/ml fibronectin-coated plates in VascuLife VEGF Medium (LL-0003, Lifeline Cell Technology, Walkersville, MD, USA) with iCell Endothelial Cells Medium Supplement (M1019, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37 °C in a 5% CO₂ incubator. hiPSC-RPE cells were cultured on plates coated with 2.5 µg/ml vitronectin in MEM alpha (12,571,063, Thermo Fisher Scientific, Sunnyvale, CA, USA) supplemented with 1% N-2 Supplement (17,502,048, Thermo Fisher Scientific, Sunnyvale, CA, USA), 55 nM hydrocortisone (H6909, Sigma-Aldrich, St Louis, MO, USA), 250 µg/ml taurine (T0625, Sigma-Aldrich, St Louis, MO, USA), 14 pg/ml triiodo-L-thyronine (T5516, Sigma-Aldrich, St Louis, MO, USA), 5% FBS, and 1% P/S at 37 °C in a 5% CO₂ incubator. Both hiPSCs with passage 3–5 were used in this study.

Construction of the choroid in vitro model

hiPSC-RPE were cultured on a vitronectin-coated 24-well plate for two weeks and then used to construct the choroid model (Supplementary Fig. S2). hiPSC-ECs or cells containing a 1:1 mixture of hiPSC-ECs and msCFs were seeded into a fibronectin-coated transwell insert. The insert was placed in a 24-well plate with or without RPE cells, and the choroidal models were cultured with the hiPSC-EC medium for one week at 37 °C in a 5% CO₂ incubator while replacing the medium every two days.

Cryosection staining

For cryosectioning, 8-week-old BALB/c mouse eyes were enucleated and fixed in 4% paraformaldehyde (PFA) solution for 3 days. The constructed choroid model was then removed from the transwell insert and fixed in 4% PFA solution for 1 h. After rinsing with PBS, the samples were sequentially incubated in 10%, 20%, and 30% sucrose solutions for 1 h each and subsequently embedded in Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA) for 1 h before being frozen in liquid nitrogen. Cryosectioned slides, 6 µm thick, were obtained using a Leica CM3050S Cryostat (Leica Microsystems, Reichert Jung, Germany) and air-dried. The slides were counterstained with hematoxylin and eosin (H&E), and images were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan).

For immunohistochemistry, the cryosectioned slides were washed for 5 min with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and then blocked with 1% bovine serum albumin solution in PBS for 1 h at room temperature. Primary antibodies were applied in the blocking buffer at 4 ℃ overnight, followed by PBS washing three times (5 min per wash). Subsequently, the slides were incubated with secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI, Dojindo, Kumamoto, Japan) in PBS, washed again with PBS, and mounted on glass slides using a mounting medium. Finally, the samples were observed under a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan). The primary antibodies were Armenian hamster anti-CD31 (ab119341, Abcam, Cambridge, MA, USA), rat anti-endomucin (MAB2624, Millipore, Billerica, MA, USA), rabbit anti-PDGFRβ (3169S, Cell Signaling Technology, Denvers, MA, USA), mouse anti-alpha smooth muscle actin (14–9760-82, Invitrogen, Logan, UT, USA), rabbit anti-lumican (ab168348, Abcam, Cambridge, MA, USA), rat anti-perlecan (sc-33707, Santacruz, Dallas, TX, USA), rabbit anti-collagen I (SAB4500362, Sigma-Aldrich, St Louis, MO, USA), rabbit anti-collagen III (ab184993, Abcam, Cambridge, MA, USA), and rabbit anti-collagen IV (ab6586, Abcam, Cambridge, MA, USA). The secondary antibodies were Alexa Fluor 488-conjugated anti-Armenian hamster (ab173003, Abcam, Cambridge, MA, USA), Alexa Fluor 488-conjugated anti-rabbit (A21206, Invitrogen, Logan, UT, USA), Alexa Fluor 488-conjugated anti-rat (A21208, Invitrogen, Logan, UT, USA), Alexa Fluor 555-conjugated anti-mouse (A31570, Invitrogen, Logan, UT, USA), and Alexa Fluor 555-conjugated anti-rabbit (A31572, Invitrogen, Logan, UT, USA).

Quantification of choroidal vessel formation

For the evaluation of vessel formation, the vessels in choroid tissues isolated from 8-week-old BALB/c mouse eye and the constructed choroid models were immunostained with CD31 antibody. Briefly, the samples were fixed for 45 min at room temperature in 4% PFA solution and washed with PBS. To facilitate clear observation of choroid vessels by removing RPE pigments, the choroid tissues were treated with a melanin bleach kit (24,883–1, Polysciences, Warrington, PA, USA), following the manufacturer’s instructions. Subsequently, the samples were blocked in a 1% bovine serum albumin solution in PBS containing 0.5% Triton X-100 for 1 h. The primary antibody (Armenian hamster anti-CD31, ab119341, Abcam, Cambridge, MA, USA) was applied in blocking buffer at 4 °C for two overnights, followed by three washes in PBST and incubation with the secondary antibody (Alexa Fluor 488-conjugated anti-Armenian hamster, ab173003, Abcam, Cambridge, MA, USA). Finally, the samples were washed in PBST and mounted using a mounting medium. Stained images of samples were acquired using a confocal laser-scanning microscope (Olympus FV3000, Tokyo, Japan). The CC layer of the mouse choroid within 500 to 1000 μm around the optic nerve was captured. Quantitative analyses of vessel area, total number of junctions, vessel length, and lacunarity of obtained images were analyzed by AngioTool software (National Institutes of Health, National Cancer Institute)⁴⁶. The average value of 4 to 5 images obtained from one sample was treated as one value.

Sprouting assay

Sprouting assays were performed as described by Shao Z et al.²² 8-week-old C57BL/6 J mouse eyes were enucleated, and unwanted tissues were removed, which leaves only the RPE, choroid, and sclera. The central regions of the choroid tissue, along with RPE and sclera, was then cut into approximately 1 mm × 1 mm fragments. The mouse choroid fragments and the constructed choroid models (E and EF), removed from the transwell insert, were embedded in 100 µL of Matrigel matrix (354,234, Corning, NY, USA). The mixture was incubated at 37 °C in a 5% CO₂ incubator for 10 min, followed by adding the hiPSC-EC medium. After 5 days of culture, images of the samples were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan). The areas of sprouting were quantified using ImageJ software (National Institute of Health).

Quantitative PCR (qPCR)

Total RNA was extracted from the constructed choroid models (EF and EF/R) and 6–8-week-old C57BL/6 J mouse choroid tissues using TRI reagent (TR118, MRC Global, Cincinnati, OH, USA) following the manufacturer’s protocol. Subsequently, reverse transcription was performed to obtain cDNA using ReverTra Ace qPCR RT Master Mix (FSQ-301, TOYOBO, Osaka, Japan). qPCR was performed using THUNDERBIRD SYBR qPCR Mix (QPS-201, TOYOBO, Osaka, Japan), and PCR amplification was performed using Applied Biosystems 7500 Fast PCR System (Applied Biosystems, Waltham, MA, USA). The primer sequences used in this study are listed in Table 1. To quantify differential gene expression, the ΔΔCT method was employed, and the results were normalized to the reference gene (β-actin).

Drug treatment

The α1-adrenergic blocker, bunazosin hydrochloride (B689585; Toronto Research Chemicals Inc., ON, Canada), known as a vasodilator⁴⁷, and the α1-adrenergic receptor agonist, phenylephrine hydrochloride (P0398, TCI chemicals, Tokyo, Japan), known as a vasoconstrictor²⁴, were individually dissolved in PBS to a concentration of 10 mM. Each solution was then adjusted to 10 μM in the hiPSC-EC medium. The medium was replaced in the constructed model (EF/R) with a medium supplemented with each reagent. After 5 h of incubation, the expected effect of each vasodilator or vasoconstrictor on the constructed model was evaluated using the method for quantification of choroidal vessel formation as described above.

Statistical analysis

The results of this study are presented as the mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA, followed by a post-hoc Tukey’s test. Statistical significance was determined when the p-value was less than 0.05.

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• Source: https://www.nature.com/articles/s41598-024-67069-8