Research and use of human embryonic stem cells is either prohibited in many countries or strongly restricted by law or bioethical issues. A limited number of totipotent stem cells is indeed available from other sources. The development of techniques for sorting totipotent stem cells from such sources is, then, increasingly sought. Generally, it is still an open issue the possibility to sort/select totipotent/multipotent, adherent, living, and particularly human stem cells by means of a method that is relatively simple, inexpensive, which does not effect cell viability and differentiation ability, and which can be applied by bench personnel of standard specialization. Mesenchimal stem cells, hereafter referred to as MSCs, are multipotent, adherent stem cells. MSCs are present and isolated from different sources, among which bone marrow, amniotic membrane, chorial membrane, Wharton gel, cord blood and placenta, dental pulp, and lipoaspirates. Although no phenotype differences are observed in the human MSCs obtained from different sources, some preliminary work based on Transmission Electron Microscopy (hereafter referred to as TEM) characterization of MSCs from different human sources have shown that ultrastructural differences of phenotypically similar MSCs derived from different human tissues can be related to their in vitro differentiation potential. However MSCs express a panel of surface antigens that is so rich and diversified to limit the possibility to precisely distinguish and define this population just on a immuno-phenotypical basis. Here the topic of our patent pending is presented. The invention consists in a new device and methods for a tag-less sorting/enrichment of human, living totipotent stem cells from different sources. The method is able to sort cells based on their biophysical and morphological properties. The invented method realizes a flow-gravity-assisted fractionation of the cell sample. Since the flow-assisted fractionation process requires cells being in suspension, the invented method in fact realizes cell fractionation based on the cell biophysical differences acquired in dynamic conditions. The differences in cell features that are dynamically acquired during fractionation allow for cell fractionation. Because of the extremely gentle fractionation conditions, native cell features are fully restored when cells return to the adherent state after fractionation completion, and cell viability is fully preserved. The method will be hereafter referred to as Non-Equilibrium, Earth Gravity-Assisted Dynamic Fractionation (NEEGA-DF). The NEEGA-DF method is suited to employ any transport fluid for cell fractionation. In particular, the cell culture medium can be employed. This ensures optimal treatment of the cells during the fractionation process, with full maintenance of cell viability and highest cell recovery. MSCs cells isolated from different sources and characterized as described above are fractionated with NEEGA-DF method. After fractionation the cells are collected and cultured on proper surfaces to favour the re-adhesion process that is necessary to obtain their in vitro expansion before any further usage. The fractionated cells can either be immediately employed or stored for further usage.
Roda B., Reschiglian P., Zattoni A., Bagnara G.P. (2006). Device and method to sort totipotent stem cells. s.l : s.n.
Device and method to sort totipotent stem cells
RODA, BARBARA;RESCHIGLIAN, PIERLUIGI;ZATTONI, ANDREA;BAGNARA, GIAN PAOLO
2006
Abstract
Research and use of human embryonic stem cells is either prohibited in many countries or strongly restricted by law or bioethical issues. A limited number of totipotent stem cells is indeed available from other sources. The development of techniques for sorting totipotent stem cells from such sources is, then, increasingly sought. Generally, it is still an open issue the possibility to sort/select totipotent/multipotent, adherent, living, and particularly human stem cells by means of a method that is relatively simple, inexpensive, which does not effect cell viability and differentiation ability, and which can be applied by bench personnel of standard specialization. Mesenchimal stem cells, hereafter referred to as MSCs, are multipotent, adherent stem cells. MSCs are present and isolated from different sources, among which bone marrow, amniotic membrane, chorial membrane, Wharton gel, cord blood and placenta, dental pulp, and lipoaspirates. Although no phenotype differences are observed in the human MSCs obtained from different sources, some preliminary work based on Transmission Electron Microscopy (hereafter referred to as TEM) characterization of MSCs from different human sources have shown that ultrastructural differences of phenotypically similar MSCs derived from different human tissues can be related to their in vitro differentiation potential. However MSCs express a panel of surface antigens that is so rich and diversified to limit the possibility to precisely distinguish and define this population just on a immuno-phenotypical basis. Here the topic of our patent pending is presented. The invention consists in a new device and methods for a tag-less sorting/enrichment of human, living totipotent stem cells from different sources. The method is able to sort cells based on their biophysical and morphological properties. The invented method realizes a flow-gravity-assisted fractionation of the cell sample. Since the flow-assisted fractionation process requires cells being in suspension, the invented method in fact realizes cell fractionation based on the cell biophysical differences acquired in dynamic conditions. The differences in cell features that are dynamically acquired during fractionation allow for cell fractionation. Because of the extremely gentle fractionation conditions, native cell features are fully restored when cells return to the adherent state after fractionation completion, and cell viability is fully preserved. The method will be hereafter referred to as Non-Equilibrium, Earth Gravity-Assisted Dynamic Fractionation (NEEGA-DF). The NEEGA-DF method is suited to employ any transport fluid for cell fractionation. In particular, the cell culture medium can be employed. This ensures optimal treatment of the cells during the fractionation process, with full maintenance of cell viability and highest cell recovery. MSCs cells isolated from different sources and characterized as described above are fractionated with NEEGA-DF method. After fractionation the cells are collected and cultured on proper surfaces to favour the re-adhesion process that is necessary to obtain their in vitro expansion before any further usage. The fractionated cells can either be immediately employed or stored for further usage.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
