Partial requirements forin vitro survival of human red blood cells

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Abstract
Some of the requirements for survival of human red blood cells were studiedin vitro at 25 and 37°C for 1–2 weeks. During the first week at 25°C in Krebs-Ringer bicarbonate medium with glucose, the cells at 2–5% hematocrit (HCT) maintained normal K+, Na+, and water contents with negligible hemolysis. After six days ion gradients decreased, preceded by decline of ATP. With adenosine, ATP was maintained for 1–2 weeks. Sustainedin vitro survival of human red blood cells at 25 or 37°C requires constant pH0 and sufficient substrates to support a glycolytic carbon flux as well as a nitrogen flux via nucleotide turnover. In Earle's salts buffered with HEPES and supplemented with glucose, Eagle's essential vitamins, albumin, and antibiotics, suspensions at 0.1% HCT exhibited constant pH at 7.39±0.03 for at least two weeks at 37°C. With glucose alone, ATP declined steadily to negligible levels despite constant pH0, but 0.1mm adenine supported ATP for one week. Also, several amino acids partially prevented the decline of reduced glutathione during the first week at 37°C. These results and current knowledge of red cell metabolism suggest a new defined medium for experiments requiring long term incubations, and extend the characterization of human red cellin vitro survival to a time period not previously studied.