A statistical comparison of various factors on embryogenic proliferation,morphogenesis and regeneration in Lolium temulentum cell suspension colonies

Cell suspension colonies from four embryogenic Lolium temulentum lines were selected and plated individually in 25 embryoid maturation treatments which varied in various factors reported to stimulate embryogenesis or improve regeneration. Using a numerical scoring system to compare the cultures against a control, treatments were identified which increased growth, suppressed morphogenesis or encouraged premature shoot formation.No treatment significantly improved the proportion of colonies with globular or mature embryoids, but some prevented maturation and increased the proportion with translucent embryogenic proliferation. Other treatments accelerated maturation causing increased de-differentiation of embryogenic tissues. These treatments also tended to discourage the differentiation of discreet embryoids.Colonies were later transferred en masse to a regeneration medium and scored using another numerical system. Embryoid maturation conditions were then identified which increased or suppressed subsequent shoot regeneration. The two scoring systems enabled cultures of the four lines to be characterised in detail and identified somatic variation in embryogenic development, morphogenesis and de-differentiation.     

Hepatocyte culture on biodegradable polymeric substrates

The interactions of primary rat liver cells with biodegradable polymeric substrates were investigated in vitro to assess the suitability of the polymer materials for use in cell transplantation devices. The kinetics of cell adhesion to, and the growth and biochemical function of cells maintained on, films formed from poly (D,L-lactic-co-glycolic acid, 88: 12) (PLGA) or from a 50/50 (w/w) blend of PLGA and poly (L-lactic acid) (PLLA) were evaluated in comparison to two control substrates, matrigel coated or collagen-coated polystyrene petri dishes. The rate of cell adhesion to both types of polymeric substrates was similar to the rate of adhesion to the collagen control substrate, but of the two polymers, only the blend was suitable for extended culture. Hepatocytes maintained on the polymer blend films showed retention of differentiated cell function as measured by the rate of albumin secretion-the rate of albumin secretion by cells on the films was the same as the rate for cells on matrigel and reached a level in the range of reported in vivo levels (140-160 mug/10(6) cells/24 h). In contrast, albumin secretion by hepatocytes maintained on collagen-coated polystyrene culture dishes declined over five days to a level one third that of the initial level and one fifth that of cells maintained on the polymer blend films on day five. Such retention of differentiated cell function by hepatocytes in culture has previously been observed only when hepatocytes were cultured in the presence of exogenous extracellular matrix proteins or were cocultured with another cell type. In addition to retention of differentiated function, the cells maintained on the polymer blend films also displayed rates of DNA synthesis similar to controls maintained on collagen-coated polystyrene, a substrate optimal for DNA synthesis.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

Explant organ culture: A review

Organ explant culture models offer several significant advantages for studies of patho-physiologic mechanisms like cell injury, secretion, differentiation and structure development. Organs or small explants/slices can be removed in vivo and maintained in vitro for extended periods of time if careful attention is paid to the media composition, substrate selection, and atmosphere. In the case of human tissues obtained from autopsy or surgery, additional attention must be paid to the postmortem interval, temperature, hydration, and cause of death. Explant organ culture has been effectively utilized to establish outgrowth cell cultures and characterize the histiotypic relationships between the various cell types within an organ or tissue.J. Resau is a visiting scientist at the NCI-LMO-DCE in Frederick, MD 21702, U.S.A.K. Sakamoto is a visiting scientist from the Department of Surgery, Gunma University School of Medicine, Maebashi, Japan     

Role of endothelial cells in the proliferative response of cultured pulmonary vascular smooth muscle cells to reduced oxygen tension

Summary The development of pulmonary hypertension in a wide variety of human disease states and experimental animal models characterized by chronic alveolar hypoxia is mediated by two pathologic vascular processes, a) vasoconstriction and b) vasoconstruction (structural remodeling). The anatomic changes seen within the pulmonary circulation include a) increased deposition of collagen and elastin in the adventitial layer and b) aberrant pulmonary vascular smooth muscle cell proliferation and maturation in the medial segments. Despite the demonstrated ability of pharmacologic manipulation in the experimental animal to ameliorate both the structural and hemodynamic changes, the actual etiologic mechanisms are only beginning to be explored. Using the cell culture technique of co-cultivation, we have investigated the potential role of bovine pulmonary arterial endothelial cell-derived factors in mediating abnormal bovine smooth muscle cell growth under conditions of reduced oxygen tension. We have demonstrated that these cultured endothelial cells exposed in vitro to reduced levels of atmospheric oxygen concentrations of 5.0% and 2.5% O₂ for durations of 24 to 72 h produce and secrete soluble growth factor(s) which stimulate smooth muscle cell proliferation when compared to cells maintained under standard tissue culture oxygen conditions of 95% room air. This growth-stimulatory effect required the concomitant presence of serum factors (0.5% fetal bovine serum), was inhibited by heparin, was distinct from platelet-derived growth factor, and seemed to have a molecular weight greater than 14 000 Da. We conclude that reduced levels of oxygen tension in vitro can selectively induce pulmonary arterial endothelial cells to release mitogen(s) which can stimulate vascular smooth muscle replication. Furthermore, we speculate that this in vitro finding may be of importance as an etiologic mechanism to explain the accelerated smooth muscle cell growth characteristic of hypoxic pulmonary arteriopathy.     

Pokeweed mitogen inhibition of protein synthesis in cultured lymphoblastoid lines

Summary Pokeweed mitogen (PWM) and ricin are both lectins derived from plant seeds. They are glycoproteins and share the ability to agglutinate a variety of animal cells including erythrocytes. The effect of these two lectins on protein synthesis was studied in four longterm lymphoblastoid lines (8866 and GM1531, which are B cell lines; and CCRF/CEM and MOLT 4, which are T-cell lines). Ricin (50 μg/ml) completely inhibited protein synthesis by 2 hr in both B-cell and T-cell lines as measured by the uptake to [³H]leucine. The PWM appeared more specific and at a concentration of 500 μg/ml inhibited protein synthesis only in B-cell lines (8866 and GM 1531). This effect was maximal at 5 hr. To investigate the reason for the differential effect of PWM on T and B cells,¹²⁵I-labeled PWM was incubated with 8866, MOLT 4, and CCRF/CEM to see if a significant difference in binding to B cells and T cells could be demonstrated. It does not appear that the differential effect on T and B cells is due to a difference in the amount of PWM bound. On the other hand it is possible that the B cells may bind some toxic subcomponent of the PWM preparation that the T cells do not bind because of a difference in composition or arrangement of cell surface glycoproteins.     

Bombesin production by human small cell carcinoma of the lung

A series of continuous cell lines of human small cell carcinoma of the lung (SCCL) have been evaluated for the production of bombesin (BN). In early established cultures BN was detected in the medium of 9 out of 11 cell lines and in 6 out of 7 cell homogenates examined. Levels in the medium were frequently higher in cultures of later passages compared to earlier passages of the same line and low levels developed in the two previously negative cell lines. Plasma concentrations were greater than 80 pmol/l in 2 out of 27 (7%) randomly selected patients with SCCL. A culture (DMS 406) established from the tumor of a patient with the highest plasma level (1240 pmol/l) was the highest producer in vitro. The results indicate that BN, which has been demonstrated immunocytochemically to be present in normal bronchial mucosal cells, is frequently produced by SCCL in vitro but elevated plasma levels are infrequently found in patients with this neoplasm.