cRBC are highly similar to donor RBC. (A) Ghosts from cRBC and peripheral blood RBC were lysed and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel was stained with Coomassie and depicts the most abundant proteins in RBC membranes. (B) Deformability of cRBC was measured under shear stress by the Automated Rheoscope and Cell Analyzer, which elongates cells and measures length over width as deformability parameter. Progressively maturating reticulocytes were isolated from peripheral blood (in order of maturation: R1, RNA^highCD71^high; R2, blue squares; RNA^highCD71^low, R3, blue circles; RNA^highCD71⁻, blue triangles; R4: RNA^lowCD71⁻, blue diamonds; as described previously²⁴ ) were compared with fully mature RBC (red curves) and filtered cRBC from normal culture dishes (thick green line) or the G-Rex bioreactor (thick blue line). Right bar graph represents the quantification of >1000 cells per culture condition. (C-D) Expression of hemoglobin variants was determined by HPLC in cRBC in culture dishes before filtering (C) or G-Rex after filtering (D). Hemoglobin variants are indicated; exact retention time is indicated for each peak. (E) Oxygen association and dissociation curve for peripheral blood RBC (teal and red) and cRBC cultured in a G-Rex bioreactor (blue and green). The percentage oxygenated hemoglobin is measured at a gradient of oxygen tension given in Torr (upper line x-axes) and kPa (lower line x-axes).