The explant cultures were prepared from ITB and ACL tissues. Surrounding the connective tissue, in the case of ACL, the synovial membranes were also removed. The pure ligament tissue was cut into 2–3 mm2 slices and incubated in T-25 culture flasks with a growth medium (Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12, 1:1) containing 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 2.5 µg/mL amphotericin B, 1% nonessential amino acids (all from Biochrom AG, Berlin, Germany), and 25 µg/mL ascorbic acid (Sigma-Aldrich) at 37 °C and 5% CO2. After 1–2 weeks, the ITB and ACL fibroblasts started to migrate from the tissue slices (Graphical abstract). Subsequently, fibroblasts were harvested using 0.05% trypsin/0.02% EDTA (Biochrom AG) and expanded in T-75 and T-175 culture flasks (CellPlus, Sarstedt AG, Nümbrecht, Germany) (Figure 1 E,F) for further characterization, spheroid, and scaffold cultures. The explants were cultured for 8–10 weeks. For analyzing the marker protein expression profile by immunocytochemistry, ITB as well as ACL fibroblasts derived from three different donors each were seeded on poly-l -lysin-coated cover slides at 1 × 104 cells/cm2. For the gene expression analysis, three human ITB and three human ACL samples obtained from different donors were cultured in a monolayer (passages 3–4).
Trypsin 0.02 edta
Manufactured by Harvard Bioscience
Sourced in Germany
Trypsin/0.02% EDTA is a cell dissociation reagent used to detach adherent cells from cell culture surfaces. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which work together to break down the extracellular matrix and cell-cell adhesions, allowing cells to be harvested for further experiments or passage.
Automatically generated - may contain errors
Lab products found in correlation
Non essential amino acids (neaa), by Harvard Bioscience (2 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (2 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Amphotericin b, by Harvard Bioscience (1 mentions)
Cellplus, by Sarstedt (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Pcs 201 012, by American Type Culture Collection (1 mentions)
C3h10t1 2 clone 8, by American Type Culture Collection (1 mentions)
Penicillin streptomycin pen strep, by PromoCell (1 mentions)
5 protocols using trypsin 0.02 edta
1
Isolation and Characterization of Ligament Fibroblasts
2
Overexpressing ABCG2 in MDCKII Cells
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All studies were performed with Madin-Darby canine kidney II cells (MDCKII) that overexpressed the bovine ABCG2 efflux transporter, as developed by Wassermann et al. 2013 [22 (link)]. Cells were cultivated in MEM medium with Earle’s Salts (2.2 g/L NaHCO3, stable glutamine; Biochrom, Berlin, Germany), supplemented with 10% (v/v) fetal calf serum (Life Technology, Karlsruhe, Germany), 1% (v/v) non-essential amino acids (Biochrom, Berlin, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin (Biochrom, Berlin, Germany), and grown at 37°C and 5% CO2. Cells were sub-cultured using 0.05% trypsin/0.02% EDTA (Biochrom, Berlin, Germany) every 3 to 4 days, up to a total of 14 passages.
3
Murine Embryonic Fibroblast Culture
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C3H10T1/2 embryonic fibroblasts (Clone 8) were purchased from ATCC (CCL-226, LGC
Standards GmbH Wesel, Germany) and grown in Dulbecco’s modified Eagle’s medium
(DMEM, Biochrom AG, Germany) supplemented with 10% fetal bovine serum (FBS,
Biochrom AG) and 1% antibiotic–antimycotic solution (AAS, Promocell, Germany) at
37°C in a humidified 5% CO2/95% air atmosphere. Prior to reaching
confluence, the cells were harvested from the culture flasks by treatment with
0.25% trypsin/0.02% EDTA (Biochrom AG) followed by subsequent centrifugation and
re-suspending in DMEM at a concentration of 20,000 cells mL−1.
Standards GmbH Wesel, Germany) and grown in Dulbecco’s modified Eagle’s medium
(DMEM, Biochrom AG, Germany) supplemented with 10% fetal bovine serum (FBS,
Biochrom AG) and 1% antibiotic–antimycotic solution (AAS, Promocell, Germany) at
37°C in a humidified 5% CO2/95% air atmosphere. Prior to reaching
confluence, the cells were harvested from the culture flasks by treatment with
0.25% trypsin/0.02% EDTA (Biochrom AG) followed by subsequent centrifugation and
re-suspending in DMEM at a concentration of 20,000 cells mL−1.
4
Colon, Breast, Melanoma and Endothelial Cell Culture
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HT-29 (ACC-299), HCT-116 (ACC-581) and DLD-1 (ACC-278) colon carcinoma, MCF-7 (ACC-115) breast carcinoma, KB-V1 (ACC-149), 518A2 (Department of Radiotherapy and Radiobiology, University Hospital Vienna) melanoma and Ea.Hy926 (ATCC no. CRL-2922) HUVEC derived endothelial hybrid cells line were grown in Dulbecco’s Modified Eagle Medium (DMEM; Biochrom), high glucose supplemented with 10% (v/v) fetal bovine serum (FBS; Biochrom.) and 1% (v/v) Antibiotic-Antimycotic solution (anti-anti; Thermo Scientific). HDFa human dermal fibroblasts (ATCC: PCS-201-012™) were grown in DMEM supplemented with 10% FBS, 1% anti-anti and 2 mM glutamine. The cells were incubated at 37 °C, 5% CO2, 95% humidified atmosphere. By repeated addition of topotecan or vinblastine at the maximum tolerated dose to the cell medium of MCF-7 and KB-V1 cells, the cells were rendered multidrug-resistant, indicated as MCF-7Topo and KB-V1Vbl, respectively. They were serially passaged following trypsinization by using 0.05% trypsin/0.02% EDTA (Biochrom). Mycoplasma contamination was routinely monitored, and only mycoplasma–free cultures were used.
5
Cryopreserved Mouse Mesenchymal Stem Cell Culture
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Cryopreserved embryonic mouse mesenchymal stem cells (C3H10T1/2, clone 8, ATCC, USA)
were thawed and grown in Eagle's Basal medium (EBM ) supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, Earle's salts, 10% FBS and 1% penicillin/streptomycin (pen/strep, Promocell, Germany) at 37 °C in a humidified 5% CO 2/ 95% air atmosphere. Cells of almost confluent cultures were washed once with sterile PBS pH 7.4 followed by treatment with 0.25% trypsin/0.02% EDTA (Biochrom, Berlin, Germany) at 37 °C for a maximum of 5 min to detach the cells. Trypsin was neutralized with EBM containing 10% FBS, and the cells were resuspended in EBM after centrifugation at 250 g for 5 min. Afterward, the cells were seeded on plain and PEM-modified round glass coverslips at a density of 5 × 10 4 cells/mL.
were thawed and grown in Eagle's Basal medium (EBM ) supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, Earle's salts, 10% FBS and 1% penicillin/streptomycin (pen/strep, Promocell, Germany) at 37 °C in a humidified 5% CO 2/ 95% air atmosphere. Cells of almost confluent cultures were washed once with sterile PBS pH 7.4 followed by treatment with 0.25% trypsin/0.02% EDTA (Biochrom, Berlin, Germany) at 37 °C for a maximum of 5 min to detach the cells. Trypsin was neutralized with EBM containing 10% FBS, and the cells were resuspended in EBM after centrifugation at 250 g for 5 min. Afterward, the cells were seeded on plain and PEM-modified round glass coverslips at a density of 5 × 10 4 cells/mL.
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