Optimising methods for the 'in vitro' production of red blood cells from human peripheral blood

Abstract

Despite a wide therapeutic use of blood transfusions, various limitations are well known in the field; these are a burden especially on patients with chronic haemoglobinopathies since they require constant and lifelong blood transfusions. In an attempt to eliminate these problems, commercial media were developed for the culturing of red cells with the aim of one day replacing blood needed from donors. Although appealing, the use of commercial culture media is not economical. Therefore, if an affordable in-house culture media can be designed and optimized to be comparable to existing media, further research may eventually make way for the possibility that patients be transfused with in vitro synthesized red cells. In the proposed study, this hypothesis will be tested using haematopoietic stem cells from healthy individuals. By culturing and comparing the proliferation and eventual differentiation of these cells into erythrocytes on the two different media chosen (in-house culture media and commercially available media (SFEM)), it can be assessed whether the feasible medium designed is appropriate and optimized for the growth of red cells. Cells growing on the in-house media did not survive for long; this is likely because optimisation requires time, trial, and error. Incubation of both types of culture media were carried out in normal and hypoxic culture conditions. This was done in response to research that shows that when utilizing conditions similar to the microenvironment of the bone marrow, erythropoiesis will be more successful. The CD marker expression for CD34, CD45, and CD71 were compared in normal and hypoxic conditions by calculating the fold change difference for the three target genes. For example, CD34+ and CD45 were both overexpressed in normal conditions on day 4 with values of 2.437 and 5.279/3.701, respectively. qPCR data from normal and hypoxic conditions was analysed using an Independent-Samples Kruskal-Wallis Test and null hypothesis. The distribution of all target genes in both conditions was the same across categories of the day: in normal conditions, CD34 p=0.229, CD45 p=0.150 and CD71 p=0.215, and in hypoxic conditions, CD34 p=0.395, CD45 p=0.199 and CD71 p=0.343. Additionally, related samples two-way analysis of variance by ranks and pairwise comparisons were carried out to further analyse the data obtained.