75 cm2 cell culture flask

Manufactured by Corning

Sourced in United States

The 75 cm2 cell culture flasks are a type of lab equipment used for culturing cells. They provide a consistent and controlled environment for cell growth and maintenance. The flasks offer a surface area of 75 cm2 for cell attachment and proliferation.

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43 protocols using 75 cm2 cell culture flask

Cultivation and Cryopreservation of Ovarian Cancer Cell Lines

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The human ovarian serous adenocarcinoma cell lines OV-90 (American Type Culture Collection [ATCC]^®CRL-11732 ™) and Caov-3 (ATCC^®HTB-75 ™) were obtained from ATCC, VA. The OV-90 cells were cultivated in RPMI 1640 medium, and the Caov-3 cells were cultivated in DMEM medium. Both media were supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and penicillin 100 IU/mL (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were grown in 75 cm² cell culture flasks (Costar, MA, USA) at 37 °C in a humidified atmosphere with 5% CO₂ and subcultured twice a week. Single-cell suspensions were obtained by washing the cells twice with phosphate buffered saline (PBS) (1:10 dilution of 10× stock PBS; Dulbecco’s tablets, Oxoid Limited, Thermo Fisher Scientific, Waltham, MA, USA) before incubation with trypsin (Gibco, Thermo Fisher Scientific). As a final step, cells were washed one last time, freezing media (90% FCS and 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA)) was added, and the cells were cryopreserved.

Anandan S., Thomsen L.C., Gullaksen S.E., Abdelaal T., Kleinmanns K., Skavland J., Bredholt G., Gjertsen B.T., McCormack E, & Bjørge L. (2021). Phenotypic Characterization by Mass Cytometry of the Microenvironment in Ovarian Cancer and Impact of Tumor Dissociation Methods. Cancers, 13(4), 755.

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Cultivation of SKOV-3 Ovarian Cancer Cells

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The human ovarian adenocarcinoma cell line SKOV-3 (ATCC HTB-77) was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated in Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco), 2 mM L-glutamine (Gibco) and penicillin 100 IU/ml and 100 µg/ml streptomycin (Gibco) at 37°C in a humidified atmosphere with 5% CO₂. Cells were grown in 75 cm² cell culture flasks (Costar, Cambridge, MA, USA) and subcultured twice a week. Suspensions of the cells were obtained by washing the cells twice with 10% phosphate buffered saline (PBS; Dulbecco's tablets, Oxoid Limited, Hampshire, UK) and incubating the cell cultures with Trypsin EDTA (Gibco). Thereafter, the cells were washed in growth medium, resuspended or snap-frozen for later thawing and reuse [21] (link).

Helland Ø., Popa M., Vintermyr O.K., Molven A., Gjertsen B.T., Bjørge L, & McCormack E. (2014). First In-Mouse Development and Application of a Surgically Relevant Xenograft Model of Ovarian Carcinoma. PLoS ONE, 9(3), e89527.

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Isolation and Expansion of Human MSCs

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Human BM aspirate from healthy donors (ethical approval 11-101-0303, Ethical Committee of the University Hospital of Regensburg) was diluted with DMEM supplemented with 10% FBS and 2.5 mg/ml gentamicin (Life Technologies, Darmstadt, Germany) and plated in 100 mm cell culture dishes (BD Falcon, Heidelberg, Germany). Medium was changed twice weekly and cell growth and morphology were regularly checked and documented using a Hund Willovert S microscope (Hund Wetzlar, Wetzlar, Germany). When confluence was reached, MSCs were trypsinized and further expanded in 75 cm 2 cell culture flasks (Corning Costar, Bodenheim, Germany). After 2-4 weeks (70-90% confluence), MSCs were harvested and phenotypically and functionally analyzed. The specific MSC phenotype was determined by flow cytometric analysis of cell surface proteins as described below.

Schelker R.C., Iberl S., Müller G., Hart C., Herr W, & Grassinger J. (2018). TGF-β1 and CXCL12 modulate proliferation and chemotherapy sensitivity of acute myeloid leukemia cells co-cultured with multipotent mesenchymal stromal cells. Hematology (Amsterdam, Netherlands), 23(6).

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Cultivation of Human Cervical ME-180 Cells

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Human cervical ME-180 (ATCC HTB-33) cells were maintained at 37 °C, in a humidified incubator with 5% (v/v) CO₂ in Roswell Park Memorial Institute medium (RPMI) medium 1640 (Gibco #11875–093) containing 5% heat inactivated Fetal Bovine Serum (FBS; Sigma-Aldrich, ON, Canada), 1 mM sodium pyruvate (Gibco # 11360–070) and 1x Anti-Anti (Gibco #15240–062) [10 (link)]. Cells were passaged twice weekly in 1:5 ratio in 75 cm² cell culture flasks (Corning Incorporated, Corning, USA).

Thakur S.D., Obradovic M., Dillon J.A., Ng S.H, & Wilson H.L. (2019). Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5′-carboxyfluorosceinsuccidyl ester. BMC Microbiology, 19, 67.

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Isolation and Culture of Rat Adipose-Derived Stem Cells

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All procedures were approved by the animal care committee of People's Hospital, Peking University (Permit Number: 201301). Sprague-Dawley rats weighing approximately 180 to 200 g were euthanized using CO₂ upon termination of the study. The procedures were performed as previously reported with some modifications [27] (link), [28] (link). Briefly, aseptically harvested rat adipose tissue was extensively washed with phosphate-buffered saline, finely minced, and digested for 60 min at 37°C with type I collagenase (Sigma, St. Louis, USA). The cell suspension was then neutralized with Dulbecco's Modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, New York, USA), and 100 U/mL penicillin/streptomycin. After filtration through a 70-µm filter and centrifugation at 1500 rpm for 5 min, the pellet was resuspended. The cell suspension was plated in 75-cm² cell culture flasks (Corning, New York, USA) and maintained at 37°C in a humidified atmosphere of 5% CO2; the medium was changed every 2 days. ADSCs were passaged at 80% confluence with 0.25% trypsin at 37°C, and all cells were used for analysis at passages three to five.

Zhang J., Zhou S., Zhou Y., Feng F., Wang Q., Zhu X., Ai H., Huang X, & Zhang X. (2014). Hepatocyte Growth Factor Gene-Modified Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation Induced Liver Damage in a Rat Model. PLoS ONE, 9(12), e114670.

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Caco-2 Cell Monolayer Establishment Protocol

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Prior to seeding, cells were trypsinized (2.5 mL) from 75 cm² cell culture flasks (Corning Inc., Corning, NY, USA) on which they had been grown (80% confluence), after washing with HBSS. Caco-2 cells (passage 54–58) were seeded onto Transwell (Corning) semi-permeable membrane supports (12 well, 1.12 cm², 0.4 μm pore size) at a density of 8×10⁴ cells/cm². Cells were maintained in DMEM containing l-glutamine (4 mM) and glucose (4.5 mg/mL) supplemented with (v/v) 10% FBS, 1% penicillin/streptomycin, 1% nonessential amino acids, amphotericin B (0.5 μg/mL), and gentamicin (20 μg/mL). Media were changed every 2–3 days and Transwells cultured at 37°C, 5% CO₂ for 21 days, after which transport studies were performed.

Dennison T.J., Smith J.C., Badhan R.K, & Mohammed A.R. (2017). Fixed-dose combination orally disintegrating tablets to treat cardiovascular disease: formulation, in vitro characterization and physiologically based pharmacokinetic modeling to assess bioavailability. Drug Design, Development and Therapy, 11, 811-826.

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SH-SY5Y Cell Differentiation Protocol

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SH-SY5Y cells were cultured as previously described (Attoff et al. 2016 (link)). Briefly, they were cultured in MEM supplemented with 10% fetal bovine serum (Gibco, 31330095), 1% non-essential amino acid solution (Gibco, 11140035), 2 mM L-glutamine (Gibco, 25030024), 100 μg/ml streptomycin, and 100 U/ml penicillin (Gibco, 15140122). For maintenance culture, SH-SY5Y cells were seeded at 27,000 cells/cm² in 75 cm² cell culture flasks (Corning). The cells were passaged once a week using TrypLE Express Enzyme (Gibco). SH-SY5Y cells were differentiated into a neuronal-like phenotype by exchanging the maintenance medium with differentiation medium consisting of DMEM/F12 (Gibco, 31330095) supplemented with 1 mM L-glutamine (Gibco, 25030024), 100 μg streptomycin/mL, 100 U penicillin/mL, 1 × N2-supplement (Gibco, 17502048) and 1 µM all-trans retinoic acid (RA, Sigma, R2625) 24 h after seeding. The cells were incubated in 100% humidity at 37 °C in air with 5% CO₂.

Loser D., Grillberger K., Hinojosa M.G., Blum J., Haufe Y., Danker T., Johansson Y., Möller C., Nicke A., Bennekou S.H., Gardner I., Bauch C., Walker P., Forsby A., Ecker G.F., Kraushaar U, & Leist M. (2021). Acute effects of the imidacloprid metabolite desnitro-imidacloprid on human nACh receptors relevant for neuronal signaling. Archives of Toxicology, 95(12), 3695-3716.

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Myotube Differentiation and Exosome Isolation

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The C2C12 cells were seeded in 75 cm² cell culture flasks (1.0 × 10⁶ cells/flasks) (Corning, Corning, NY, USA) exosome-free 10% FBS DMEM and grown for 48 h. Then, the cellular supernatant was collected. The C2C12 cells were plated in 12-well plates (Corning, 3513), seeded with 8.0 × 10⁴ cells per well in DMEM supplemented with 10% FBS and 1% P/S. By adding 2% horse serum (HS, Gibco) after reaching 80% confluency, C2C12 cells became myotubes for 4 days. The supernatant was collected by contacting the cells with 2% exosome-free HS DMEM for 48 h.

Qin M., Xing L., Wu J., Wen S., Luo J., Chen T., Fan Y., Zhu J., Yang L., Liu J., Xiong J., Chen X., Zhu C., Wang S., Wang L., Shu G., Jiang Q., Zhang Y., Sun J, & Xi Q. (2023). Skeletal Muscle-Derived Exosomal miR-146a-5p Inhibits Adipogenesis by Mediating Muscle-Fat Axis and Targeting GDF5-PPARγ Signaling. International Journal of Molecular Sciences, 24(5), 4561.

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Calu-3 Cell Culture for MUC5AC

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Calu-3 cells ^{23 (link)} were obtained from the ATCC (Rockville, MD) and maintained in unlabeled (¹²C) mEMEM in a humidified atmosphere with 5% CO₂ at 37°C. Cells were plated in 75 cm² cell culture flasks (Corning) and growth medium was replaced every 48h. Upon reaching ~80% confluence, cells were passaged using a 0.25% trypsin solution in EDTA (Sigma T4049). Cells from passages 11–18 were then used to generate MUC5AC at the air-liquid interface of polarized cell cultures. To do so, cells were collected and resuspended in ¹³C-labeled mEMEM at a concentration of 1×10⁶ cells/mL. 1.5 mL aliquots were then seeded into the apical chamber of each 24mm insert of a Transwell microtiter plate (Corning 3450), and 2.5 mL of ¹³C mEMEM was added to the basolateral chamber. Cells reached ~90% confluency between 3–7 days, at which point the apical medium was removed to establish an air-liquid interface. Apical medium was removed daily until seepage from the basolateral side had stopped. Polarized cells were maintained for additional 21–28 days in ¹³C mEMEM with medium replacement every 48h prior to mucin collection. Unlabeled control cultures were grown in parallel using ¹²C mEMEM at all stages.

Evert C., Loesekann T., Bhat G., Shajahan A., Sonon R., Azadi P, & Hunter R.C. (2019). Generation of 13C-labeled MUC5AC mucin oligosaccharides for stable isotope probing of host-associated microbial communities. ACS infectious diseases, 5(3), 385-393.

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Monocyte/Macrophage Cytotoxicity Assay

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We used a phorbol myristate acetate (PMA, Sigma-Aldrich) induced human monocyte/macrophage (THP-1) leukemia cell line for this study. Cells were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS and supplemented with penicillin G (50 U/mL) and streptomycin sulfate (50 μg/mL). Cells were grown and maintained in 75 cm² cell culture flasks (Corning) at 37 °C in 5% CO₂ in a humidified incubator. When confluent, THP-1 cells were centrifuged and seeded in 96-well plates (VWR Scientific) at 5 × 10⁵ cells/mL (total volume 200 μL/well), in the presence of 20 ng/mL (MA, Sigma-Aldrich) in order to differentiate them into mature macrophage-like cells. Twenty-four hours after seeding, 100 μL of the medium was removed, and cells were treated with different ENM doses in the 1–50 μg/mL range (0, 1, 5, 10, 30, 50) or an equivalent amount of medium alone.
For the purposes of this paper, which were to investigate the impact of differences in particle size distribution measurements and dosimetry estimates on cytotoxicity end points, we used cell viability (MTT) data.

Pal A.K., Aalaei I., Gadde S., Gaines P., Schmidt D., Demokritou P, & Bello D. (2014). High Resolution Characterization of Engineered Nanomaterial Dispersions in Complex Media Using Tunable Resistive Pulse Sensing Technology. ACS Nano, 8(9), 9003-9015.

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