The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.

The cell membrane is a primary and fundamental player in most cellular processes, and fatty acids form a major structural component of cell membranes. The aim of this study was to compare the membrane fatty acid profiles of different human blood leukocytes and selected cell lines, to identify the effects of in vitro culture on fatty acid profiles, and to test medium supplements for their effect on fatty acid profiles. Different classes of leukocytes were isolated from human blood and their membrane fatty acid profiles were analysed and compared. After culturing in vitro immortalised and primary leukocytes, membrane fatty acids were analysed and compared. Finally, different lipid formulations were developed and used for supplementing leukocytes in vitro in an effort to maintain the in vivo fatty acid profile. Descriptive and analytical tests were performed to compare the obtained fatty acid profiles. Membrane fatty acid profiles of primary human CD4+ T-lymphocytes, CD8+ T-lymphocytes, B-lymphocytes and monocytes differed. Moreover, there were differences among Jurkat, Raji and THP-1 cell lines and the corresponding primary leukocyte classes, as well as between freshly prepared and in vitro cultured primary lymphocytes. A lipid supplement was able to maintain cultured Jurkat cells with a membrane fatty acid profile almost identical to that of the primary CD4+ T-lymphocytes. Finally, variations in the lipid supplement composition enabled the development of Jurkat cells with different membrane fatty acid profiles characterising different physiological or pathological human conditions. Each leukocyte class has its own specific membrane fatty acid profile in vivo. Cultured primary leukocytes and immortalized leukocytic cells display different membrane fatty acid profiles when compared to their respective in vivo counterparts. The membrane fatty acid composition of cultured cells can be restored to reflect that of the corresponding in vivo condition through use of optimised lipid supplementation. Typical physiological or pathological leukocyte membrane fatty acid profiles can be obtained by tuning in vitro fatty acid supplementation.

Cell culture conditions influence several biological and biochemical features of stem cells (SCs), including the membrane lipid profile, thus limiting the use of SCs for cell therapy approaches. The present study aims to investigate whether the in vitro culture may alter the membrane fatty acid signature of human Amniotic Epithelial Cells (hAECs). The analysis of the membrane fatty acid composition of hAECs cultured in basal medium showed a loss in polyunsaturated fatty acids (PUFA), in particular in omega-6 (ω-6) content, compared to freshly isolated hAECs. The addition to the basal culture medium of a chemically defined and animal-free tailored lipid supplement, namely Refeed^®, partially restored the membrane fatty acid signature of hAECs. Although the amelioration of the membrane composition did not prolong hAECs culture lifespan, Refeed^® influenced cell morphology, counteracted the onset of senescence, and increased the migratory capacity as well as the ability of hAECs to inhibit Peripheral Blood Mononuclear Cell (PBMC) proliferation. This study provides new information on hAEC features during culture passages and demonstrates that the maintenance of the membrane fatty acid signature preserved higher cell quality during in vitro expansion, suggesting the use of lipid supplementation for SC expansion in cell-based therapies.