Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates.

It has been hypothesized that, in addition to freezing injury, some damage to platelets may result from the cell packing that occurs during removal of the cryoprotectant. This study examined DMSO removal by fluid exchange across hollow-fiber (HF) filters as an alternative to centrifugation. The DMSO solution with or without cell suspension was passed once through the filter. The optimum exchange during unloading of DMSO was determined by varying the flow rates in the external and internal compartments of the HF filter. Initially, buffered solutions of a 5% DMSO solution in the absence of platelets were pumped into the fibers and exchanged against PBS. The residual DMSO was determined by osmometry. The exchange of DMSO across the membrane was flow dependent and also influenced by the chemical nature of the HF fibers. No protocol using a reasonable rate flow through the fibers removed more than 95% of the DMSO in a single pass. The optimum protocol was achieved with polysynthane fibers with an internal flow rate of approximately 20 mi/min and an external flow rate of 100 ml/min. Subsequently, frozen/thawed platelet concentrates in DMSO were washed using centrifugation and compared to the HF filtration method. Platelet quality was assayed by flow cytometry, cell count, morphology and osmotic stress test. Both filtration and centrifugal washing techniques resulted in comparable morphological scores and numbers of discoid cells. When agents reducing platelet activation were added, platelet quality was improved after washing by either technique. The lower platelet osmotic response with HF filtration than with centrifugation while using activation inhibitors was attributed to the remaining amount of the inhibitors. All other parameters tested were similar. The expression of CD62P was equivalent with both techniques, and centrifugation did not activate platelets more than filtration contrary to what was originally anticipated. In conclusion, platelet quality was comparable after washing by either technique but hollow fiber filtration does remove cryoprotectant more rapidly than does centrifugation.

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.

[Positive and negative regulation of replication in hybrid cells]. / Pozitivnaia i negativnaia reguliatsiia replikatsii v gibridnykh kletkakh.

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.

Differential regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells with various proliferative potentials.

The regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells of various proliferative potential was studied. The following regularities were found: 1) Both immortalized and non-immortalized cells can efficiently reactivate DNA synthesis in erythrocyte nuclei. 2) Erythrocytes drastically inhibit the entry of non-malignant culture cell nuclei into the S-period, not acting upon DNA synthesis. 3) The inhibitory action is characteristic of erythrocytes from different stages of chicken ontogenesis (from 5-day-old embryos to the adult bird). 4) Malignant cells are completely refractory to the inhibitory action of erythrocytes. The ability of erythrocytes to inhibit the onset of replication in heterokaryons may be connected with the mechanisms of maintaining these terminally differentiated cells in a non-proliferating state.

[The nature of a proliferation block in differentiated cells with heterokaryons as a model: various types of absence of proliferation in cells in terminal differentiation]. / Issledovanie prirody bloka proliferatsii v differentsirovannykh kletkakh na modeli geterokarionov: razlichnye tipy otsutstviia proliferatsii v kletkakh pri terminal'noi differentsirovke.

Heterokaryons obtained by fusion of proliferating and terminally differentiated cells were studied. The data obtained suggest that mechanisms of proliferation arrest are different in macrophages on one hand and nucleate erythrocytes and polymorph leukocytes on the other. Macrophages appeared to be devoid of factors preventing replication in nontransformed and spontaneously immortalized cells. Inhibition of proliferation was probably due to certain modifications of macrophage genome which arise during differentiation and can be compensated by the effect of "immortalizing" oncogenes. On the contrary, nucleate erythrocytes and polymorphs evidently contain some factors mediating negative control of proliferation. For reactivation of DNA synthesis in these cell types after fusion with other cells the latter did not have to be immortalized. After cell fusion macrophages specifically inhibit DNA synthesis in cells containing active oncogenes.