Establishing a cell biology platform: isolation and preservation of human blood products

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

The use of human primary cells provide researchers in different areas with irrefutable more biologically relevant data than using cell lines or animal blood cells. The work was performed in the scope of the Cell Biology Services @ CEDOC, aiming to provide viable and trustful human primary cells and products. We had three main objectives: protocol optimizations for blood cell isolation, culture and cryopreservation; cost estimation and divulgation of the services. We have reviewed standard protocols and compared different strategies for blood cell isolation. The impact of those methodologies was evaluated regarding cell yield and purity, cell functional characteristics and cost. We also developed a method for serum isolation from human plasma in blood buffy coats. The resultant sera were sterile and suitable to be used in leukocyte cultures. Different protocols for T cells isolation were compared: positive versus negative immunomagnetic selection and isolation using nylon wool fiber columns. Positive selection provided the highest isolation yield (32.35%), while negatively selected cells had the highest purity (92.81%). Although nylon wool fiber column was the fastest and cheapest method, unlike the immunomagnetic methods, it did not allow complete separation of T from B lymphocytes. Positive selection of monocytes was compared using two widely used commercial kits. Miltenyi’s kit provided the highest isolation yield (25.92%), recovery rate (86.70%) and purity (95.01%). Monocytes isolated with StemCell kit presented a higher cell complexity, and when differentiated into dendritic cells (DCs), showed a more mature phenotype. Differences between both kits are probably caused by the nature of the magnetic beads, suggesting caution when choosing one or other kit, as it may have an impact on DCs’ function. Overall, although dealing with apparently straight forward methodologies, our results show that testing commercial products and optimizing protocols is very important and contribute for a better quality of products and services.