Fabrication of 3D skeletal muscle and hydrogel structure was described previously in detail. Briefly, the cell‐matrix solution consisted of 1 × 107 cells/mL of MPCs, 30% (vol/vol) Matrigel, 4 mg/mL fibrinogen (Sigma‐Aldrich), and 0.5 U of thrombin (Sigma‐Aldrich)/mg of fibrinogen was seeded in a polydimethylsiloxane ring mould with 5 mm/6.6 mm for inner/outer diameters. After 2 h, the PM supplemented with 1 mg/mL aminocaproic acid (Sigma‐Aldrich) was added to the ring mould and changed every other day. After 7 days, compacted muscle rings were transferred into the 3D‐printed hydrogel cantilevers and cultured with the DM supplemented with 1 mg/mL of aminocaproic acid and 0.5 ng/mL of insulin‐like growth factor‐1 (Sigma‐Aldrich). The 3D hydrogel cantilever structure was designed in Solidworks (Figure S1 ) and fabricated using the digital light processing 3D printer (PICO2, Asiga) and a printing resin solution containing 20% (vol/vol) polyethylene glycol diacrylate M.W. 700 (Sigma‐Aldrich), 1 mg/mL of lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (Sigma‐Aldrich), and 0.4 mg/mL of Sunset Yellow FCF (Sigma‐Aldrich). Myofibre alignment of the 3D muscle ring was confirmed by scanning electron microscopy (FEI Quanta FEG 450).
Aminocaproic acid
Manufactured by Merck Group
Sourced in United States
Aminocaproic acid is a synthetic amino acid used as a laboratory reagent. It functions as an antifibrinolytic agent, inhibiting the breakdown of fibrin clots.
Automatically generated - may contain errors
Lab products found in correlation
Bovine fibrinogen, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Sylgard 184 elastomer base and curing agent, by Dow (2 mentions)
Thrombin, by Merck Group (2 mentions)
Triphenyltetrazolium chloride, by Merck Group (2 mentions)
Imidazole, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Fibrinogen, by Merck Group (1 mentions)
Sunset yellow fcf, by Merck Group (1 mentions)
Quanta 450 feg, by Thermo Fisher Scientific (1 mentions)
Insulin like growth factor 1, by Merck Group (1 mentions)
Lithium phenyl 2 4 6 trimethylbenzoylphosphinate, by Merck Group (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Anti runx2, by Santa Cruz Biotechnology (1 mentions)
13 protocols using aminocaproic acid
1
Fabrication of 3D Skeletal Muscle Tissue
2
Engineered Heart Tissue Culture Protocol
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EHTs were cast on polydimethylsiloxane (PDMS) microposts as previously described (26,27). Briefly, Sylgard 184 Elastomer Base and Curing Agent (Dow, 1317318) were mixed at 10:1 ratio and cured in a custom 3D printed mold for 18 h at 65C, with one flexible post and one glass rod filled stiff post per set of posts, 6 posts per array. Cured post arrays were removed from the mold and trimmed of excess PDMS. 500k Day 25 hiPSC-CMs and 100k human Hs27a stromal cells were mixed with 3 U/mL thrombin from bovine plasma (Sigma, T4648) and 5 mg/mL bovine fibrinogen (Sigma, E8630) in 100 μL EHT media [sterile filtered RPMI, B27 supplement, 5 g/L aminocaproic acid (Sigma, A2 504-256-100G), penicillin/streptomycin]. The cell slurry was added into 2% agarose wells between posts in a 24 well plate and incubated for 80 min at 37C, 5% CO2. 350 μL EHT media was added to the wells and tissues were incubated for 10 min at 37C, 5% CO2. Posts were carefully moved to a fresh 24 well plate in 2 mL EHT media, and tissues were cultured on posts for 3 weeks with media change every other day.
3
Osteogenic Differentiation of Mesenchymal Cells
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Bovine fibrinogen, thrombin, aminocaproic acid, L-Ascorbic acid and glycerol 2-phosphate were purchased from Sigma (St. Louis, MO). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA). Fetal bovine serum (FBS), HEPES buffer solution, and penicillin-streptomycin solution were from GibcoBRL (Carlsbad, CA), and ascorbic acid-free α-MEM was from WelGene (Daegu, Korea). The MicroBCA assay kit was from Pierce-Thermo (Rockford, IL). Qunat-iT PicoGreen dsDNA-assay kit was from Invitrogen (Eugene, OR). West-Zol was from Intron Biotechnology (Seoul, Korea). A Dual-GloTM Luciferase Assay System was from Promega (Madison, WI). Protease inhibitor cocktail tablets (Complete) were from Roche (Basel, Switzerland). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-fibronectin and anti-vitronectin antibodies were from Chemicon (Temecula, CA).
4
Engineered Human Cardiac Tissues
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EHTs were cast on polydimethylsiloxane (PDMS) microposts as previously described [26 (link),27 (link)] Briefly, Sylgard 184 Elastomer Base and Curing Agent (Dow, 1317318, Midland, MI, USA) were mixed at a 10:1 ratio and cured in a custom 3D-printed mold for 18 h at 65 °C, with one flexible post and one glass rod filled stiff post per set of posts, 6 posts per array. Cured post arrays were removed from the mold and trimmed of excess PDMS. A total of 500k Day 25 hiPSC-CMs and 100k human Hs27a stromal cells were mixed with 3 U/mL thrombin from bovine plasma (Sigma, T4648, St. Louis, MO, USA) and 5 mg/mL bovine fibrinogen (Sigma, E8630, St. Louis, MO, USA) in 100 μL EHT media (sterile filtered RPMI, B27 supplement, 5 g/L aminocaproic acid (Sigma, A2 504-256-100G, St. Louis, MO, USA), penicillin/streptomycin). The cell slurry was added into 2% agarose wells between posts in a 24-well plate and incubated for 80 min at 37 °C, 5% CO2. Then, 350 μL EHT media was added to the wells, and tissues were incubated for 10 min at 37 °C, 5% CO2. Posts were carefully moved to a fresh 24-well plate in 2 mL EHT media, and tissues were cultured on posts for 3 weeks with media change every other day.
5
Engineered Heart Tissues Contractility Assay
6
Detailed Biochemical Reagents Protocol
7
Enzymatic Assay for Glycerol-1-Phosphate
8
Cell Proliferation Assay with Aminocaproic Acid
9
Cell Lysis by Sonication and Soluble Extract Preparation
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Cell lysis was done by sonication. Samples were resuspended in 3 mL of lysis buffer containing 50 mM Hepes-KOH (Euromedex), 200 mM KCl (Euromedex), 1 mM EDTA (Euromedex), 10% glycerol (Roth), 1 mM benzamidine (Sigma), 5 mM aminocaproic acid (Sigma), 1 mM NAD + (Sigma). To precipitate DNA, 0.2 % of sulfate streptomycin was added and samples were centrifuged for 25 min at 13,000 x g at 4°C. The supernatant sample, corresponding to soluble extract, was frozen in liquid nitrogen and conserved at -80°C.
10
Cardiomyocyte Isolation and Culture
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All reagents used for cardiomyocyte isolation/culture and Pt black deposition were purchased from Sigma-Aldrich (St. Louis, MO): Horse serum, fetal bovine serum, penicillin-streptomycin, aminocaproic acid, ascorbic acid, insulin-transferrin-selenium solution, collagenase II, forskolin, chloroplatinic acid, lead acetate. Four-inch silicon wafers were purchased from University Wafer, and a two-part Sylgard 184 polydimethylsiloxane (PDMS) kit was purchased from Dow Corning (Midland, MI).
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