title: Demonstration of the retention of 64Cu-ATSM in cardiac myocytes using a novel incubation chamber for screening hypoxia-dependent radiotracers.
abstract: OBJECTIVE
  We have designed a low-cost, reusable incubation system that allows cells to be cultured in either plated or suspension culture under complete atmospheric control for radiotracer characterization. We demonstrate its utility here in the first quantification of the hypoxia-dependent accumulation of Cu-diacetyl bis(N4-methylthiosemicarbazone) (Cu-ATSM) in adult rat ventricular myocytes (ARVMs).
  
  
  METHODS
  ARVMs were allowed to adhere overnight in 9 cm culture plates (2×10 cells/dish) or were used in suspension culture, placed inside the chamber and equilibrated with either oxic (95 or 21% O₂/5% CO₂) or anoxic gas (95% N₂/5% CO₂). Cu-ATSM of 100 kBq was administered, and the cells were incubated for 30 or 60 min. Cells were then harvested, counted and fractionated to determine intracellular Cu biodistribution.
  
  
  RESULTS
  After 1 h, the average cellular Cu retention in plated ARVMs under oxygenated conditions was 23.9 ± 2.5 mBq/cell (95% O₂), increasing to 27.3 ± 5.1 under 21% O₂ (P<0.05) and to 36.1 ± 3.1 under 0% O₂ (P<0.05). When ARVMs were cultured in suspension, normoxic-hypoxic contrast was less marked but still significant [63.2 ± 14.1 vs. 53.4 ± 10.9% mBq/cell after 30 min (P<0.05)]. Sixty percent of tracer accumulated in the cytosol, and, although total cellular retention increased during hypoxia, there was no enrichment in any particular cellular compartment.
  
  
  CONCLUSIONS
  This apparatus allows the conduction of radiotracer uptake studies in cells under complete atmospheric control, as evidenced by our first demonstration of the hypoxia-dependent uptake of Cu-ATSM in ventricular myocytes. It is ideally suited for screening, validating and characterizing novel hypoxia-selective radiotracers.
authors.affiliation: Department of Imaging Chemistry and Biology, Division of Imaging Sciences and Biomedical Engineering, King's College London, BHF Centre of Research Excellence, King's Health Partners, St Thomas' Hospital, London, UK.
authors.forename: Maxwell G
authors.initials: MG
authors.lastname: Handley
authors.forename: Rodolfo A
authors.initials: RA
authors.lastname: Medina
authors.forename: Rowena L
authors.initials: RL
authors.lastname: Paul
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Blower
authors.forename: Richard
authors.initials: R
authors.lastname: Southworth
mesh_terms: D000818:Animals; D001692:Biological Transport; D015687:Cell Hypoxia; D002470:Cell Survival; D056831:Coordination Complexes; D003301:Copper Radioisotopes; D003470:Culture Media / Q000737:chemistry; D006352:Heart Ventricles / Q000166:cytology; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D009942:Organometallic Compounds / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011849:Radioactive Tracers; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013882:Thiosemicarbazones / Q000378:metabolism*
chemical_list: D056831:Coordination Complexes; D003301:Copper Radioisotopes; D003470:Culture Media; D009942:Organometallic Compounds; D011849:Radioactive Tracers; D013882:Thiosemicarbazones; C089548:copper (II) diacetyl-di(N(4)-methylthiosemicarbazone); D010100:Oxygen