Clonal populations of amniotic fluid mesenchymal stem cells were obtained, isolated and characterized as previously described [18 (link)] from 3 separate patients ranging from 36–38 weeks gestation who were undergoing amniocentesis for lecithin sphingomyelin assessment. Informed consent was obtained and this protocol was approved by the Hartford Hospital IRB committee (IRB#FINC003364HU). Each patient’s AF-MSCs were cultured separately in Amniotic Fluid (AF) Medium, which consisted of MEM Alpha (Life Technologies, Grand Island, NY), 18% Chang B Medium (Irvine Scientific, Santa Ana CA), 2% Chang C Supplement (Irvine Scientific, Santa Ana CA), 1X Penicillin/Streptomycin (Life Technologies, Grand Island, NY) and 1X Glutamax (Life Technologies, Grand Island, NY). Medium was changed every other day and cells were allowed to grow to approximately 70–80% confluence prior to splitting. Cells were split at a ratio of 1:3–1:4 not exceeding passage 10 for this study. Cells had a normal karyotype (data not shown). Clonal populations obtained from these AF samples were only utilized for this study if they expressed pluripotent and mesenchymal stem cell markers and did not express hematopoietic markers. Clonal AF-MSCs isolated from each patient were cultured separately for this study, so clonal populations from different patients were not mixed or combined.
X penicillin streptomycin
Manufactured by Thermo Fisher Scientific
Product X penicillin/streptomycin is a sterile solution containing the antibiotics penicillin and streptomycin. It is commonly used in cell culture applications to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Chang c supplement, by Irvine Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Nmumg, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Williams e medium, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Mem non essential amino acids solution, by Merck Group (1 mentions)
3 protocols using x penicillin streptomycin
1
Isolation and Characterization of Clonal Amniotic Fluid Mesenchymal Stem Cells
2
Cell Culture and Genetic Manipulation
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NMuMG (ATCC CRL-1636) and HEK293T (ATCC CRL-3216) cells were obtained from ATCC. Cells were cultured in Dulbecco’s modified Eagle medium (Life Technologies), supplemented with 10% FBS and 1X penicillin/streptomycin (Life Technologies) and maintained in culture as suggested by ATCC. NMuMG cells transduced with shRNAs or sgRNA were selected using puromycin (2 μg/ml). Cells infected with VAMP2-pHluorin, VAMP2-Halo, or Akt-Halo were selected using blasticidin (10 μg/ml) or FACS.
3
Recellularization and Perfusion of Liver Scaffolds
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Livers recellularized with rat cells were perfused with Williams’ Medium E (Sigma) containing 2 mM GlutaMax-1 (Life Technologies), 0.1 mM MEM non-essential amino acids solution (Sigma), 10% fetal bovine serum (Hyclone), 10 μg/mL insulin (Sigma), 1X penicillin/streptomycin (Life Technologies), and 1 μM dexamethasone (Sigma).
Scaffolds seeded with human liver cells were perfused with Williams’ Medium E (Sigma) containing 2 mM GlutaMax-1 (Life Technologies), 0.1 mM MEM nonessential amino acids solution (Sigma), 1X ITS+1 (Sigma), 2.0 ng/mL HGF (Peprotech), 2.0 ng/mL epidermal growth factor (EGF) (Peprotech), 1X penicillin/streptomycin (Life Technologies), and 0.1 μM dexamethasone (Sigma).
Scaffolds seeded with human liver cells were perfused with Williams’ Medium E (Sigma) containing 2 mM GlutaMax-1 (Life Technologies), 0.1 mM MEM nonessential amino acids solution (Sigma), 1X ITS+1 (Sigma), 2.0 ng/mL HGF (Peprotech), 2.0 ng/mL epidermal growth factor (EGF) (Peprotech), 1X penicillin/streptomycin (Life Technologies), and 0.1 μM dexamethasone (Sigma).
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