Infrapatellar fat pad (IPFP) tissue was obtained from patients (74 year old female and 75 year old male) undergoing total knee replacement (approved by Scripps Institutional Review Board). Mesenchymal stem cells (IPFP-MSC) were isolated using a previously described method (Grogan et al., 2020 (link)). Briefly, IPFP tissues were minced using a scalpel to create fragments (∼5 mm3), which were placed into 6-well plate wells precoated with human collagen type I (Cell Adhere, StemCell Technologies, Vancouver, Canada). For the first 12 h, the tissue fragments were maintained in a CO2 incubator at 37°C in only .5 ml MSC-medium (LONZA, Walkersville, MD) supplemented with Fibroblast Growth Factor 2 (FGF-2) (10 ng/ml; PeproTech, RockyHill, NJ). After 12 h, 1.5 ml of medium was added and the tissue fragments were cultured for 1–2 weeks until emergence of cells from the tissue. The remaining tissue fragments were discarded, and the emerging cells were detached using Accutase (Innovative Cell Technologies, Inc. San Diego, CA) and reseeded into collagen coated flasks at a density of 350,000 cells per cm2.
Msc medium
Manufactured by Lonza
Sourced in Switzerland
MSC-medium is a specialized culture medium developed for the growth and maintenance of mesenchymal stem cells (MSCs) in laboratory settings. It provides the essential nutrients and growth factors required to support the proliferation and differentiation of MSCs.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast growth factor 2 fgf2, by Thermo Fisher Scientific (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Neomycin, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
2 protocols using msc medium
1
Isolation of Infrapatellar Fat Pad Mesenchymal Stem Cells
2
Expansion of Human Mesenchymal Stem Cells
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We expanded commercially available human MSCs (Poietics; Lonza, Basel, Switzerland) as monolayers in tissue culture flasks in 5% CO2 at 37°C in the MSC medium (Lonza) for later use of experiments. To mimic the native microenvironment of NP cells, all MSC cultivations were conducted in a hypoxia working station (US Autoflow NU-4950; NuAire, Plymouth, MN, USA) with a mixture of 5% O2, 5% CO2, and 91.5% N2; this O2 concentration fulfills the requirements for a hypoxic culture that does not consume large amounts of nitrogen [25 (link)]. The basal growth medium for the following experiments was DMEM/F-12 medium containing 10% fetal bovine serum (Gibco; Life Technologies, Grand Island, NY, USA), 100 nM dexamethasone, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL neomycin (Sigma-Aldrich, St. Louis, MO, USA), and 25 mg/mL L-ascorbic acid (SigmaAldrich).
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