Ammonium toxicity in different cell lines

The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH(4)), and renal (LLC-PK(1)) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH(4) cells and prevented the growth of LLC-PK(1) cells. The ion did not lead to the death of LLC-PK(1) cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.     

Dipropylcyclopentylxanthine triggers apoptosis in Jurkat T cells by a receptor-independent mechanism

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a xanthine analog used as selective antagonist of adenosine receptors, caused apoptosis in a human leukemia T cell line. Jurkat cells treated with DPCPX underwent apoptosis as demonstrated by flow cytometry, by DNA fragmentation and by accumulation of histones, H2A, H2B, H3 and H4, in the nucleoplasm of cells. Cell cycle and cell sorting analyses indicated an arrest of cells in G(2)/M followed by the appearance of apoptotic cells in G(1) and G(2)/M phases. The mechanism of programmed cell death does not seem to be mediated by signal transduction events at the plasma membrane since it did not involve activation of cell membrane receptors and modification of the intracellular levels of Ca(2+) or cAMP. Apoptosis by incorporation into DNA of a derivative of DPCPX is suggested in basis of the presence of radioactivity label in the DNA obtained from cells preincubated with [(3)H]DPCPX.     

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for “ex vivo” expansion of multipotent cells.     

Use of liquid nitrogen during storage in a cell and tissue bank: contamination risk and effect on the detectability of potential viral contaminants

Cryopreservation is widely used for banking cells and tissues intended for transplantation. Liquid nitrogen provides a very stable ultra-low temperature environment. Thus, it is used for longterm storage. Unlike the exhaustive microbiological monitoring of the environmental conditions during tissue processing, storage is not usually considered as a critical point of potential contamination risk in professional standards for cell and tissue banking. We have analysed the presence of microbial agents inside our nitrogen tanks. We have mainly detected environmental and water-borne bacteria and fungi. In addition, we have studied the effect of liquid nitrogen exposure on virus detectability. Only differences for hepatitis C virus RNA were observed. Measures for contamination risk reduction during storage must be mandatory in cell and tissue banking.     

Influence of volume reduction and cryopreservation methodologies on quality of thawed umbilical cord blood units for transplantation

Introduction

Although there is considerable variability in methodology among umbilical cord blood banks, their common goal is to achieve optimal product quality for transplantation. Cryopreservation is a critical issue for a long-term maintenance of cord blood viability and colony-forming capacities.

Materials and methods

We designed a prospective study to compare controlled (CRF) vs. non-controlled freezing (URF) of volume-reduced cord blood units. In addition, the influence of hydroxy ethyl starch (HES) on cryopreservation was also assayed. To assess the efficiency of protocols used, cell recoveries were measured and the presence of hematopoietic colony-forming units was quantified.

Results

In the study phase, we observed similar CB haematopoietc recoveries for CRF and URF strategies, except for TNC recovery that was better for HES volume reduced CB units in the URF group. When we analysed the data of routine processed CB units in samples from satellite cryovials, we found better BFU-E, CFU-GM, CFU-GEMM and CFU recoveries for those units processed with HES than without HES, in an URF manner.

Conclusions

URF of CB units is a cryopreservation procedure that allows similar hematopoietic progenitor recoveries than CRF with programmed devices. However, our study suggests that those banks that cryopreserve CB units in a URF manner should use HES for volume reduction. On the other hand, for CRF cryopreservation methodology volume reduction with and without HES are equally useful.     

Noise and robustness in phyllotaxis

A striking feature of vascular plants is the regular arrangement of lateral organs on the stem, known as phyllotaxis. The most common phyllotactic patterns can be described using spirals, numbers from the Fibonacci sequence and the golden angle. This rich mathematical structure, along with the experimental reproduction of phyllotactic spirals in physical systems, has led to a view of phyllotaxis focusing on regularity. However all organisms are affected by natural stochastic variability, raising questions about the effect of this variability on phyllotaxis and the achievement of such regular patterns. Here we address these questions theoretically using a dynamical system of interacting sources of inhibitory field. Previous work has shown that phyllotaxis can emerge deterministically from the self-organization of such sources and that inhibition is primarily mediated by the depletion of the plant hormone auxin through polarized transport. We incorporated stochasticity in the model and found three main classes of defects in spiral phyllotaxis--the reversal of the handedness of spirals, the concomitant initiation of organs and the occurrence of distichous angles--and we investigated whether a secondary inhibitory field filters out defects. Our results are consistent with available experimental data and yield a prediction of the main source of stochasticity during organogenesis. Our model can be related to cellular parameters and thus provides a framework for the analysis of phyllotactic mutants at both cellular and tissular levels. We propose that secondary fields associated with organogenesis, such as other biochemical signals or mechanical forces, are important for the robustness of phyllotaxis. More generally, our work sheds light on how a target pattern can be achieved within a noisy background.     

Persistence of gap junction communication during myocardial ischemia

During myocardial ischemia, severe ATP depletion induces rigor contracture followed by intracellular Ca2+ concentration ([Ca2+]i) rise and progressive impairment of gap junction (GJ)-mediated electrical coupling. Our objective was to investigate whether chemical coupling through GJ allows propagation of rigor in cardiomyocytes and whether it persists after rigor development. In end-to-end connected adult rat cardiomyocytes submitted to simulated ischemia the interval between rigor onset was 3.7 +/- 0.7 s, and subsequent [Ca2+]i rise was virtually identical in both cells, whereas in nonconnected cell pairs the interval was 71 +/- 12 s and the rate of [Ca2+]i rise was highly variable. The GJ blocker 18alpha-glycyrrhetinic acid increased the interval between rigor onset and the differences in [Ca(2+)]i between connected cells. Transfer of Lucifer yellow demonstrated GJ permeability 10 min after rigor onset in connected cell pairs, and 30 min after rigor onset in isolated rat hearts submitted to nonflow ischemia but was abolished after 2 h of ischemia. GJ-mediated communication allows propagation of rigor in ischemic myocytes and persists after rigor development despite acidosis and increased [Ca2+]i.     

Banking strategies for improving the hematopoietic stem cell content of umbilical cord blood units for transplantation

Umbilical cord blood (UCB) has become an alternative source of hematopoietic progenitors (HSC) for transplantation. Although most CB transplants have been performed in children, unrelated donor-cord blood transplants in adults have been growing steadily in recent years. HSC content of CB units influence significantly the transplantation outcome, as shown by many clinical studies. UCB banks are fundamental to support this increasing clinical activity and one of their main goals must be to store good quality units. Strategies for increasing HSC content of UCB units are reviewed and also its influence on transplantation outcome. Our bank selected the UCB units for cryopreservation on the basis of their total nucleated cells (TNC) and CD34(+) cells content. We also reviewed the results of our UCB bank program.     

Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 °C/min) and stored in the liquid phase of a nitrogen tank for 9.1 ± 1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me₂SO. After thawing in a water bath at 42 °C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.