|Cell line/type||Skeletal myofibers|
|Product||hSKM Growth Medium|
From: Oleaga, C., Bernabini, C., Smith, A. S., Srinivasan, B., Jackson, M., McLamb, W., ... & Berry, B. (2016). Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs. Scientific reports, 6, 20030.
In this study, a functional human model is used to evaluate multi-organ toxicity in a 4-organ system under continuous flow conditions in a serum-free defined medium. The cell types and corresponding media used in this study are described seperately in the FCS-free Database. The other cell types are:
Human Skeletal Myofibers were obtained as a gift from H. Vandenburg, Brown University without identifiers and prepared by isolation and differentiation steps previously described by this group, with small modifications. Biopsies were performed according to procedures approved by the Institutional Clinical Review Board of the Miriam Hospital. For each culture, human skeletal muscle (hSKM) SCs/progenitors were plated 22 days (approximately) before the co-culture on N-1(3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) modified 15 mm diameter coverslips (30 cells/mm2) in hSKM Growth Medium (Lonza), and fed afterwards every 2 days by replacing the medium. On day 7, myoblast fusion into postmitotic myofibers was induced by incubation with differentiation medium 164. The cells were fed every 2 days by changing half of the medium. Four days later, the cells were fed with NBActive4 (Brain Bits) every 2 days by changing half of the medium, for a minimum of 11 days.
The defined serum-free medium base composition used to maintain the functionality of the 4 organ culture system for up to 14 days was previously published and was modified by the addition of an antibiotic/antimycotic cocktail at 1X (Life Technologies, 15240-062) and the elimination of the G5 supplement. The base medium was composed of 1X Neurobasal (Life Technologies, 21103-049), 1X B27 (Life Technologies, 17504-044), 1X Antibiotic and antimycotic (Life Technologies, 15240-062) and 1X Glutamax (Life Technologies, 35050-061). It was supplemented with 10 ng/mL Glial-derived Neurotrophic Factor (GDNF) (Cell Sciences, CRG400B), 20 ng/mL Brain-derived Neurotrophic Factor (BDNF) (Cell Sciences, CRB600B), 5 ng/mL Ciliary-derived Neurotrophic Factor (CNTF) (Cell Sciences, CRC400A), 20 ng/mL Neurotrophin-3 and 4 (NT3 and NT4) (Cell Sciences, CRN500B and CRN501B), 100 ng/mL Vitronectin (Sigma, V8379-50UG), 10 ng/mL Insulin-like Growth Factor-I (IGF-1) (PeproTech, 100-11), 100 ng/mL Agrin (R&D, 550-AG-100), 1 μ M Adenosine 3′ , 5′ -cyclic monophosphate (cAMP) (Sigma, A9501), 4 μ g/mL laminin (Life Technologies 23017-015), 50 ng/mL Sonic Hedgehog, N-terminal peptide (Shh) (R&D, 1845-SH-025) and 0.1 μ M Retinoic acid (Sigma, R2625). All the compounds purchased as a powder were dissolved in water, except for retinoic acid which was dissolved in DMEM 1X.https://www.nature.com/articles/srep20030
|Source||Literature - modified commercial product|
|Chemically defined > No||No|
Oleaga et al (2016) - Oleaga, C., Bernabini, C., Smith, A. S., Srinivasan, B., Jackson, M., McLamb, W., ... & Berry, B. (2016). Multi-Organ toxicity demonstration in a functional human in vitro system composed of four organs. Scientific reports, 6, 20030