S8045

The exoskeletons of Trachyphyllia geoffroyi were sectioned into 0.2–1.0 mm sections and treated with sodium hypochlorite solution (10%, RT, 10 min). Sigma-Aldrich 425044, Burlington, MA, USA) and NaOH solution (1 M, 5 min, RT, Sigma Aldrich S8045) were used to digest any adhering organic matter. The fragments were then transferred to a H₂O₂ solution (30% v:v aq, 10 min, RT, Romical, Be’er Sheva, Israel). Afterward, the fragments were rinsed with distilled water, air-dried, and ground using a Smart Dentin Grinder (KometaBio, Cresskill, NJ, USA). Following that, grains sized under 40 mm were sieved.

Cellulose nanofibers were functionalized with RGD peptides via EDC/NHS coupling. EDC (Sigma‐Aldrich E6383) and NHS (Sigma‐Aldrich 130672) were dissolved in 2‐(N‐morpholino)ethanesulfonic acid (MES, Sigma Aldrich M0164) buffer containing NaCl (Merk 1064040500) 0.9% at pH 6. To activate the carboxylate groups, equimolar solutions of EDC and NHS were added to the gel in a mole ratio 1:1:1 to the existing carboxylate concentration and stirred 15 min at room temperature. A volume of (GRGDSPC) RGD peptides (GenScript) (final concentration = 2 mm) was added to the hydrogel and stirred for 2 h at room temperature. The pH of the hydrogel was adjusted to 7 by adding NaOH (Sigma‐Aldrich S8045) (1 m) measured by pH strips. Glycine (Sigma‐Aldrich 50046) (250 mm) and 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES, Sigma‐Aldrich H7523) (25 mm) were dissolved in the hydrogel. A volume of 100 µL was used to measure the osmolality in a K‐7400S osmometer (Knauer). The pH was adjusted to 7 by addition of NaOH (1 m), measured with pH strips. The hydrogel was sterilized under ultraviolet radiation exposure during 20 min prior to use. For functionalization, hydrogels were mixed with IGF‐1 and laminin‐1, at 5 ng mL⁻¹ and 0.5 mg mL⁻¹ final concentration, respectively.

Cells grown on 96-well plates were washed twice with DPBS and decalcified with HCl (30721, Sigma, 0.6 mol/L) for 30 min at room temperature. The Ca content of the HCl supernatants was determined by using a QuantiChrome Calcium Assay Kit (DICA-500, Gentaur, Kampenhout, Belgium). Following decalcification, cells were washed twice with DPBS and solubilized with a solution of NaOH (S8045, Sigma, 0.1 mol/L) and sodium dodecyl sulfate (11667289001, Sigma, 0.1%), and protein content of samples were measured using the BCA protein assay kit (23225, Pierce Biotechnology, Rockford, IL, United States). The Ca content of the cells was normalized to protein content and expressed as mg/mg protein. The observer who performed all the Ca measurements was blinded to the group assignment.

Decalcification solution was prepared by mixing 20% w/v ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich 324503) with H₂O. Sodium hydroxide (Sigma-Aldrich S8045) was added to adjust the pH to 7.0.

Isolated DC endometrial tissue stock was flash-frozen in a mortar with liquid N₂, milled manually, and lyophilized (Lyoquest-85, Telstar, Valencia’s Polytechnic University) over 96 h at 20 Pa. The resulting endometrial lyophilized powder was digested and neutralized using a modified protocol (Brown et al., 2017 (link)). Briefly, 1% (w/v) lyophilized powder was suspended in 0.01 M HCl (H1758, Sigma-Aldrich) with 0.1% pepsin (P7000, Sigma-Aldrich) and digested for 48 h under agitation. The solution was left on ice and neutralized with 10% (v/v) 0.1 M NaOH (S8045, Sigma-Aldrich), 11.11% (v/v) 10X PBS (P5493, Sigma-Aldrich), and 1X PBS was used to reach the desire concentration. The resulting EndoECM solution was stored at −80°C.
This process was also performed with isolated DC myometrial and No-DC endometrial tissue stocks to create myometrial extracellular matrix (MyoECM) and No-DC endometrial matrix (No-DC Endo). MyoECM and No-DC Endo were used as controls for subsequent proteomic analyses.

The ME49 strain of Toxoplasma gondii (ATCC^® 50611™, second haplogroup) was maintained as tachyzoites in parasite culture medium, which contains DMEM with 3% HIFBS (heat-inactivated FBS; 1 h at 56 °C). Infected cells were incubated at 37 °C and 5% CO₂. Bradyzoites were induced in vitro using the high (pH 8.2) pH shock method [50 (link)]. Bradyzoite differentiation medium contained high glucose DMEM powder (D5648, Sigma–Aldrich), 50 mM HEPES (H3375, Sigma–Aldrich), and 1% FBS. Media were adjusted to pH 8.2 with freshly made 1 M NaOH (S8045, Sigma–Aldrich) and sterilized by filtration. The medium was replaced every 1–2 days with fresh differentiation medium, which reduces the likelihood of bradyzoites reverting to tachyzoites. After 7 days, differentiated bradyzoites were confirmed with immunofluorescence assays using rabbit MAG1 antibodies as bradyzoite-specific markers and rabbit SAG1 antibodies as tachyzoite-specific markers (see Section 4.4 and Section 4.5). Six parasitized monolayers were harvested after 7 days by scraping, and bradyzoites were purified by passage through a 27 gauge needle, washed in phosphate-buffered saline (PBS) and filtered through a 0.45 μm filter. Parasites were enumerated, and pellets were resuspended in phenozol for RNA extraction using a Total RNA Mini and Clean-Up RNA Concentrator (A&A Biotechnology, Gdańsk, Poland).

To analyse the ECM remodelling ability of PSCs (control or ATRA treated), Collagen-I (BD Bioscences, 354249, stock concentration 9.37 mg ml⁻¹) and Matrigel (BD Bioscences, 354234, stock concentration 9 mg ml⁻¹) mixture gels were prepared with one part 10 × DMEM (Sigma, D2429) and one part FBS (Gibco, 10500), yielding to a final concentration of 4.5 mg ml⁻¹ Collagen-I and 2 mg ml⁻¹ Matrigel. The gel mixture was neutralized with 1 M NaOH (Sigma, S8045), then 5 × 10⁵ cells were embedded in gels in culture media. A measure of 80 μl gel volume was added per well of a 96-well plate, which was pretreated with 2% BSA (Sigma, A8022) for 1 h, washed with PBS and air dried for 10 min. Gels were set 1 h at 37 °C, and then incubated with culture media for 3 days at 37 °C. For SHG microscopy, gels were prepared as explained above. After 3 days of incubation at 37 °C, gels were fixed with 4% paraformaldehyde (Sigma, P6148) in PBS for 1 h at 37 °C, and then washed with PBS and permeabilised with 0.3%Triton X-100 (Sigma, T8787) in PBS for 30 min. After that, gels were blocked with 1% BSA–0.1% Triton X-100 in PBS for 1 h. Gels were washed with PBS and stained with Alexa Fluor 546-conjugated Phalloidin at 1/300 dilution in 1% BSA in PBS for 30 min. Finally, gels were washed two times with PBS.