Cardiac tissues were transferred from the operating theater in Dulbecco's phosphate-buffered saline (DPBS) on ice. The ventricular tissue block was carefully cleaned to remove all non-myocardial portions in a 60 mm culture dish in the tissue culture hood and cut into 1 mm blocks, transferred to a 25 cm2 TPP tissue culture flask (MidSci, St Louis, MO), washed thrice with DPBS, twice with FM-b (ScienCell) containing penicillin/streptomycin, spread evenly into 20-30 blocks per flask and cultured in 5 ml FM-2 media (ScienCell) containing 5% fetal bovine serum (FBS) and penicillin/streptomycin. The flask was inverted, with the bottom (surface with tissue) up, in the incubator (37°C; 21% O2; 5% CO2) and blocks were allowed to adhere for about 4 h. After 2-3 h, the flasks were turned to the normal orientation. The media was changed every 2 days. In 2 weeks, cardiac fibroblasts migrated from the ventricular tissue explants and reached 70% confluency. The cells were trypsinized and transferred to 150 cm2 TPP tissue culture flasks (MidSci) with FM-2 media (ScienCell) containing 5% FBS and penicillin/streptomycin and grown to 70% confluency. These initial cultures were split, and passages 2 and 3 were stored in liquid nitrogen until experiments were conducted.
Fm 2 media
Manufactured by ScienCell
FM-2 media is a serum-free, low-protein formulation designed for the culture of various cell types. It supports the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Tpp tissue culture flask, by MidSci (2 mentions)
Sircol soluble collagen assay kit, by Biocolor (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Regm singlequots, by Lonza (1 mentions)
Fgs 2, by ScienCell (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by Merck Group (1 mentions)
Growth supplement, by Lonza (1 mentions)
Stellate cell growth supplement, by ScienCell (1 mentions)
Recombinant human tgf β1, by BioLegend (1 mentions)
Penicillin streptomycin, by ScienCell (1 mentions)
4 protocols using fm 2 media
1
Cardiac Fibroblast Isolation and Culture
2
Isolation of Human Ventricular Fibroblasts
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Left ventricular fibroblasts were isolated from human cardiac tissues as reported earlier.16 Cardiac tissues were transferred in an ice‐cold Dulbecco's phosphate‐buffered saline. The ventricular tissue block was cleaned off all non‐myocardial portions in a 60 mm culture dish in laminar flow hood and cut into 1 mm blocks, transferred to a 25 cm2 TPP tissue culture flask (MidSci, St. Louis, MO), washed thrice with Dulbeccos phosphate‐buffered saline, twice with FM‐b (ScienCell Inc., Carlsbad, CA) containing penicillin/streptomycin, spread evenly into 20–30 blocks per flask, and cultured in 5 mL FM‐2 media (ScienCell Inc.) with 5% foetal bovine serum and penicillin/streptomycin. The flask was inverted, with the bottom (surface with tissue) up, in the incubator (37°C; 21% O2; 5% CO2), and blocks were allowed to adhere for ~4 h. After 2–3 h, the flasks were turned to the normal orientation. After changing media every 2 days, in 2 weeks, fibroblasts migrated from the tissue explants and reached 70% confluency. The cells were trypsinized and transferred to 150 cm2 TPP tissue culture flasks (MidSci) with FM‐2 media (ScienCell Inc.) containing 5% foetal bovine serum and penicillin/streptomycin and grown to 70% confluency. These initial cultures were split, and Passages 2 and 3 were stored in liquid nitrogen until experiments were conducted.
3
Collagen Deposition in Ventricular Fibroblasts
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Ventricular fibroblasts (passage 3 or 4) from both failing and non-failing hearts were plated separately at 104 cells/cm2 in T-25 flasks in FM-2 media (ScienCell) containing 5% FBS. After 24 h, the media was replaced with fresh media with or without YM-58483 (10 µM), a specific blocker of ICRAC channel (Ohga et al., 2008 (link); Yoshino et al., 2007 (link); Zitt et al., 2004 (link)). After 48 h, the soluble collagen deposited in the ECM was quantified colorimetrically using Sircol™ soluble collagen assay kit (Biocolor) and normalized to number of cells for analysis. The assay was performed in all the samples at the same time and repeated at least twice.
4
Culturing Primary Human Cell Lines
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Normal human lung fibroblasts (Lonza, #CC-2512, Lot # 0000343490, passage 3) were cultured in fibroblast basal medium (Lonza, #CC-3131) with growth supplements (Lonza, #CC-4126). Human primary cardiac fibroblasts (Sciencell, #6300, Lot #5433, passage 4) were cultured in FM2 media (Sciencell, #2331), supplemented with FBS (Sciencell, #0025), FGS-2 (Sciencell, #2382), and Penicillin-Streptomycin (Life Technologies, #15070-063). Human primary renal proximal tubule epithelial cells (Lonza, #CC-2553, Lot#0000362300, passage 3) were cultured in renal epithelial cell growth basal medium (Lonza, #CC-3191) supplemented with REGM SingleQuots (Lonza, #CC-4127). Primary human hepatic stellate cells (Sciencell, #5300, Lot#10279, passage 4) were cultured in Stellate Cell Medium (Sciencell, #5301) supplemented with FBS (Sciencell, #0010), stellate cell growth supplement (Sciencell, #5352), and Penicillin-Streptomycin solution (Sciencell, #0503). All cells were Incubated at 37°C in the presence of 5% CO2. For TGFβ treatment, cells were plated and serum-starved for overnight. The next day, 5ng/ml of recombinant human TGFβ1 (BioLegend, #580702) was added to the culture medium and cells were incubated at 37°C for the indicated time. For inhibitor treatment, cells were treated with recombinant human TGFβ1 in the presence of vehicle DMSO (Sigma, #D2650) or compounds at various concentrations.
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