Handley Maxwell G, Medina Rodolfo A, Paul Rowena L, Blower Philip J, Southworth Richard
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.
Nucl Med Commun. 2013 Oct;34(10):1015-22. doi: 10.1097/MNM.0b013e328363f25e.
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).
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.
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.
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.
我们设计了一种低成本、可重复使用的培养系统，该系统可使细胞在完全可控的大气条件下进行贴壁培养或悬浮培养，用于放射性示踪剂的表征。在此，我们首次对成年大鼠心室肌细胞（ARVMs）中缺氧依赖性铜双乙酰双（N4 - 甲基硫代半卡巴腙）（Cu - ATSM）的积累进行定量，证明了该系统的实用性。
将ARVMs接种于9 cm培养皿中过夜贴壁（2×10个细胞/培养皿），或用于悬浮培养，置于培养箱内，用含氧量正常（95%或21% O₂/5% CO₂）或缺氧的气体（95% N₂/5% CO₂）进行平衡。给予100 kBq的Cu - ATSM，细胞孵育30或60分钟。然后收获细胞，计数并分级以确定细胞内铜的生物分布。
1小时后，在含氧条件下贴壁培养的ARVMs中，细胞内铜的平均滞留量为23.9±2.5 mBq/细胞（95% O₂），在21% O₂条件下增加到27.3±5.1 mBq/细胞（P<0.05），在0% O₂条件下增加到36.1±3.1 mBq/细胞（P<0.05）。当ARVMs进行悬浮培养时，常氧 - 缺氧对比不太明显，但仍具有统计学意义[30分钟后为63.2±14.1 vs. 53.4±10.9% mBq/细胞（P<0.05）]。60%的示踪剂积累在细胞质中，并且尽管在缺氧期间细胞内总滞留量增加，但在任何特定细胞区室中均未出现富集。
该装置能够在完全可控的大气条件下对细胞进行放射性示踪剂摄取研究，我们首次证明心室肌细胞中Cu - ATSM的缺氧依赖性摄取就证明了这一点。它非常适合筛选、验证和表征新型缺氧选择性放射性示踪剂。
