滤白残留物来源双阴性T细胞生物学活性的实验研究OA

Objective:To characterize the biological properties of double-negative T(DNT)cells isolated from leukoreduction filter residues.Methods:Leukoreduction filters containing residues from 400 mL whole blood units(n=6)were collected from a blood center.Filters were back-flushed with normal saline,and the eluate was concentrated to obtain leukoreduction filter residues.Leukocytes in the residues were counted by dual-fluorescence staining.DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation.Both cryopreserved-thawed and non-cryopreserved DNT cells derived from the residues were subjected to in vitro culture.Cells were assessed for expansion fold,viability,immunophenotype,differentiation status,and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry,with comparisons made to DNT cells derived from whole blood.Results:The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was(41.9±14.7)%.Compared to whole blood,the DNT cells starting material obtained from filter residues showed no significant difference in total T-cells content(P>0.05).However,the viability and purity of the resulting DNT cells starting materials were significantly lower(both P<0.05).After 17 days of culture,DNT cells from filter residues and whole blood showed no significant differences in expansion fold,immunophenotype,differentiation status,or cytotoxicity toward target cells(all P>0.05).However,the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells[(86.0±4.2)%vs.(92.2±1.2)%,P<0.05].After thawing(post 3 or 15 days of cryopreservation)and 17 days of culture,DNT cell starting materials from residues showed comparable immunophenotype,expansion fold,and differentiation status to their non-cryopreserved counterparts from the same source(all P>0.05).However,the viability of the 3-day cryopreservation recovery group[92.4%(91.8%,92.8%)vs.87.8%(82.0%,89.0%)]and the cytotoxicity against target cells of the 15-day cryopreservation recovery group[91.3%(89.4%,95.1%)vs.70.9%(67.3%,80.2%)]were both significantly higher than those of non-cryopreserved DNT cells(all P<0.05).Conclusions:DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion,purity,differentiation,and biological potency.Furthermore,their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells.These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf,allogeneic DNT cell therapeutics.