Department Research, Sanquin Blood Supply, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands.
Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, De Boelelaan 1117, 1081HV, Amsterdam, The Netherlands.
Sci Rep. 2024 Jul 6;14(1):15592. doi: 10.1038/s41598-024-66440-z.
The production of cultured red blood cells (cRBC) for transfusion purposes requires large scale cultures and downstream processes to purify enucleated cRBC. The membrane composition, and cholesterol content in particular, are important during proliferation of (pro)erythroblasts and for cRBC quality. Therefore, we tested the requirement for cholesterol in the culture medium during expansion and differentiation of erythroid cultures with respect to proliferation, enucleation and purification by filtration. The low cholesterol level (22 µg/dl) in serum free medium was sufficient to expand (pro)erythroblast cultures. Addition of 2.0 or 5.0 mg/dL of free cholesterol at the start of differentiation induction inhibited enucleation compared to the default condition containing 3.3 mg/dl total cholesterol derived from the addition of Omniplasma to serum free medium. Addition of 5.0 mg/dl cholesterol at day 5 of differentiation did not affect the enucleation process but significantly increased recovery of enucleated cRBC following filtration over leukodepletion filters. The addition of cholesterol at day 5 increased the osmotic resistance of cRBC. In conclusion, cholesterol supplementation after the onset of enucleation improved the robustness of cRBC and increased the yield of enucleated cRBC in the purification process.
为了输血目的而生产培养的红细胞（cRBC）需要大规模培养和下游工艺来纯化去核 cRBC。在（pro）成红细胞的增殖过程中和 cRBC 质量方面，膜组成，特别是胆固醇含量很重要。因此，我们测试了在红系培养物的扩增和分化过程中胆固醇在培养基中的需求，以实现增殖、去核和过滤纯化。无血清培养基中的低胆固醇水平（22μg/dl）足以扩增（pro）成红细胞培养物。与默认条件（含 3.3mg/dl 总胆固醇，源自添加 Omniplasma 至无血清培养基）相比，在分化诱导开始时添加 2.0 或 5.0mg/dl 的游离胆固醇会抑制去核。在分化的第 5 天添加 5.0mg/dl 胆固醇不会影响去核过程，但在用白细胞去除过滤器过滤后显著增加去核 cRBC 的回收率。胆固醇的添加增加了 cRBC 的渗透压抗性。总之，在去核开始后添加胆固醇可提高 cRBC 的稳健性，并增加纯化过程中去核 cRBC 的产量。
