Improving the stability of cultured red blood cells during storage.

Abstract

Background and objectives: Cultured red blood cells (cRBCs) have many potential applications, including in diagnostics, as drug delivery carriers or for specialized clinical use. However, cRBCs are immature reticulocytes that do not store well. After 10 days of cold storage in saline, adenine, glucose, mannitol (SAG-M), cRBCs show around 8% haemolysis compared to <0.8% for mature RBCs. This study aimed to develop a better storage medium to enhance cRBC stability and viability.

Materials and methods: cRBCs were cultured from CD34⁺ haemopoietic cells from peripheral blood and resuspended at 1.5 × 10⁹/mL in SAG-M or SAG-M with different macromolecules (10% human serum albumin, 10% Dextran-40, 10% Ficoll-70). The effect of rejuvenation before storage was also investigated. Haemolysis and morphology assessment were carried out on Days 10, 16 and 20 of storage at 4°C. In vivo assays were performed by injecting 2 × 10⁸ cRBCs into NOD.Cg-Prkdc^scidIl2rγ^tm1Wjl/SzJ mice on Days 1, 9 and 21 of storage. Clearance and maturation of cRBCs were assessed at different intervals post injection.

Results: Addition of macromolecules significantly improved cRBC stability; 10% Dextran-40 reduced haemolysis of cRBCs to <2% over 20 days of cold storage. Rejuvenation had no significant effect. Similar numbers, morphology and maturation of cRBCs were detected in the murine circulation, whether or not stored in SAG-M or SAG-M + 10% Dx-40, regardless of storage time.

Conclusion: Addition of macromolecules to SAG-M improves cRBCs storage stability and does not affect their clearance or maturation in the in vivo model. To our knowledge, this is the first report showing evidence of improved stability of cRBCs during storage.