Tranexamic acid

The iPSC-RPE cells were obtained from LAgen Laboratories (Rochester, MN) and cultured on top of a fibrin hydrogel as previously described.¹⁵ (link) Cells were cultured for a minimum of 30 days in RPEM media (LAgen Laboratories) supplemented with 2.5 mg/mL tranexamic acid (Sigma-Aldrich, St Louis, MO), B27 supplement (ThermoFisher Scientific, Waltham, MA) and antibiotic–antimycotic (ThermoFisher Scientific). For use, implants were aseptically processed to create 1.5 × 5.0-mm implants, loaded into the newer surgical tips before use.
Implantation of MRPE611 was performed in live domestic pigs as described above using the modified surgery technique and a second-generation implantation tip (n = 4). After implantation, perfluorocarbon liquid was used to flatten the retina above the implant to visualize the implant. Owing to the xenogeneic nature of implanting human cells into a pig model, the animals were sacrificed immediately at the conclusion of the surgery as a nonsurvival procedure.

Stock solutions of tranexamic acid (TXA, 500 mM in water), thioglycolic acid (TGA, 1 M in water), methyl thioglycolate (MTG, 1 M in ethanol), and 4-bromobenzyl mercaptan (BBM, 1 M in ethanol), all purchased from Sigma Aldrich, were prepared and sterilized by filtration. Cultures of A. atramentaria in MPM were fed every 24 h, starting at 72 h and finishing at 192 h. A variety of nucleophile amounts were assayed for each feeding experiment, with the daily addition of a final concentration of 1, 2, 5, 10, 20, and 50 mM tested for TXA, TGA, and MTG, and 1, 10, and 50 mM for BBM. Extraction of metabolites and LC-MS analysis was carried out as described for LC-MS “Method 2” section.

Tissue engineering of the bioartificial muscles (BAMs) was described in detail in previous work (Gholobova et al., 2015 (link); Gholobova et al., 2018 (link); Thorrez et al., 2018 (link); Gholobova et al., 2019 (link); Gholobova et al., 2020 (link)). Briefly, myogenic cells were isolated, cultured, and harvested as described. After cell expansion, for each BAM, 2 million cells were mixed with 500 µl of human thrombin (4 U/mL, Stago, #HT1002a). To create the 3D constructs, the cell-thrombin mixture was added to 500 µl of fibrinogen (2 mg/ml, Merck Chemicals, #341576) into 25-mm silicone molds containing two metal pins spaced 20 mm, serving as attachment points. Following a 2-h incubation at 37°C during which the fibrin solidified, BAMs were cultured for the first 2 days in growth medium supplemented with the fibrinolysis inhibitors aprotinin (92.5 μg/ml, Carl Roth, #A1624) and tranexamic acid (400 μM, Sigma, #857653). The medium was switched to differentiation medium with fibrinolysis inhibitors (92.5 μg/ml aprotinin and 400 µM tranexamic acid) from day 3 until day 8 to induce myoblast fusion. After day 8, BAMs were cultured further in growth medium containing fibrinolysis inhibitors until the end of the experiment. The cell culture medium was refreshed every two days.