|Product||hSCSC 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 Motoneurons, obtained from NeuralStem, were differentiated as described from human spinal cord stem cell line (hSCSC) previously isolated, with slight modifications. Briefly, approximately 35 days before the co-culture, 0.5 × 106 hSCSCs were plated in a T25 flask in Growth medium for 7 days until they were close to confluence. They were then harvested by trypsinization and plated into a PDL/fibronectin-coated 60 mm paranox cell culture dish (Nunc, Cat #174888) at a density of 2.5 million cells/dish and differentiated 4 days in the priming medium followed by 6 days in differentiation medium. Then the cells were replated onto DETA-coated 15 mm diameter round glass coverslips at a density of 250 cells/mm2 and cultured in the defined serum-free medium for another 16–18 days before being transferred into the in vitro system.
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 published62 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