Incubation In Tissue Culture Medium Research Articles

Change in vascular smooth muscle response to 5-HT due to short- or long-term endothelial denudation of the bovine digital vein

Several chronic progressive vascular diseases, such as laminitis, show vasocontractile dysfunction that might evolve into reperfusion injury and/or vessel structural remodelling, which may be traced back to aberrant endothelial function. In the present study, the vasomotor responses of bovine digital veins (BDVs) to 5-hydroxytryptamine (5-HT) were investigated in blood vessels, with and without endothelium present, and in samples deprived of endothelium before or after overnight incubation in tissue culture medium, to evaluate the effects of short- and long-term endothelial damage on vascular smooth muscle (VSM) reactivity. No significant effects were observed in the blood vessels tested immediately after the removal of endothelium. In contrast, a significant increase in VSM reactivity to 5-HT was seen in vessels incubated without endothelium. This long-term change in smooth muscle reactivity was prevented by exposure to the nitric oxide (NO) donor nitroprusside (P < 0.01), suggesting that the long-term lack of inhibitory control exerted by endothelium-derived NO is involved in increased VSM reactivity. The RhoA/ROCK pathway inhibitor fasudil reduced VSM hyper-contractility to ~65% (P < 0.001), the superoxide dismutase-mimetic tempol normalised the vascular response and the non-selective COX-inhibitor indomethacin exerted a moderate inhibitory effect (P < 0.05). Thus, over-activation of the RhoA/ROCK pathway and production of reactive oxygen species could account for VSM hyper-reactivity, triggered by long-term endothelium-deprivation in BDVs, suggesting that these biochemical mechanisms are potential targets for controlling the progressive vasocontractile dysfunction of digital veins in animals affected with laminitis.

Use of an in vitro model in tissue engineering to study wound repair and differentiation of blastema tissue from rabbit pinna.

Rabbit ear wound repair is an accepted model for studies of tissue regeneration, leading to scar less wound repair. It is believed that a specific tissue, blastema, is responsible for such interesting capacity of tissue regeneration. To test this idea further and to elucidate the cellular events happening during the ear wound repair, we designed some controlled experiments in vitro. Small pieces of the ear were punched and washed immediately with normal saline. The tissues were then cultured in the Dulbecco's Modified Eagle(')s Medium, supplemented with fetal bovine serum in control group. As a treatment vitamin A and C was used to evaluate the differentiation potency of the tissue. These tissues were fixed, sectioned, stained, and microscopically studied. Micrographs of electron microscopy provided evidences revealing dedifferentiation of certain cells inside the punched tissues after incubation in tissue culture medium. The histological studies revealed that cells of the tissue (i) can undergo cellular proliferation, (ii) differentiate to epithelial, condrogenic, and osteogenic tissues, and (iii) regenerate the wounds. These results could be used for interpretation of the possible events happening during tissue engineering and wound repair in vitro. An important goal of this study is to create a tissue engineering and tissue banking model, so that in the future it could be used in further blastema tissue studies at different levels.

Efficacy of common surgical compounds in preventing articular chondrocyte death from desiccation.

Purpose of this study was to identify potential substances that prevent desiccation of chondrocytes. Macroscopically normal bovine cartilage explants (n = 80) were exposed to room air, or covered with surgical lubricant, Lactated ringer (LR) or Seprafilm (Genzyme Biosurgery, Cambridge, MA) for 0, 30, 60 or 120 min. The viability of superficial chondrocytes was measured after 48 h of incubation in tissue culture media at 37 °C by Live/Dead staining. Chondrotoxicity was measured as the extent of cell death below the articular surface. Statistical analysis was performed with a two-way analysis of variance on the data set and a subsequent Tukey's post hoc test. Chondrocyte death correlated positively with the length of exposure, regardless of the treatment (p < 0.0001). The extent of superficial chondrocyte death was minimally lower in the LR (89.1 ± 2.6 %, 80.8 ± 1.2 %) and surgical lube (84.3 ± 1.8 %, 75.9 ± 2.7 %) groups than the control (82 ± 5.7 %, 65.6 ± 13.3 %) and Seprafilm group (77.6 ± 3.9 %, 63.3 ± 6.9 %) (p < 0.001) at the first two time points, with no significant difference between the latter groups. After 60 and 120 min, surgical lube resulted in less chondrocyte death than all other groups (70.4 ± 6.8 % and 60.9 ± 5.9 %, all p < 0.0001). The data suggest that depending on the expected length of exposure of the articular cartilage surface, different compounds appear to be protective. For exposures exceeding 60 min, surgical lubricant demonstrated the highest protective potential. Results from this study indicate that protecting exposed articular surfaces with surgical lubricant for orthopaedic procedures lasting more than 1 h lead to decreased chondrocyte death and suggest improved cartilage functional outcomes postoperatively.

Incubation in tissue culture media allows isolated rabbit proximal tubules to regain in-vivo-like transport function: response of HCO3-absorption to norepinephrine.

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.

Genes of chicken MHC regulate the adherence activity of blood monocytes in Rous sarcomas progressing and regressing lines

The influence of the chicken major histocompatibility (B) complex (MHC) on the adherence potential of monocyte-derived macrophages was examined using the congenic chicken lines CB and CC. These lines represent well-defined genetic models for the study of resistance (CB) or susceptibility (CC) to the progressive growth of Rous sarcomas. Using a monoclonal antibody specific for chicken monocytes/macrophages, CB and CC chickens were shown by flow cytometry analyses to have similar proportions of peripheral blood monocytes. However, when the glass-adherence potential of these cells was compared during incubation in tissue culture medium over 24, 48 and 72 h at 40°C, significant differences were seen between cells from these two inbred lines. After 24 and 48 h, glass-adherence by CB cells was 2–3 fold higher than that of CC cells. After 72 h this difference decreased to 1.5 fold. At 24 and 48 h, the adherent CB macrophages also appeared about 1.5 times larger than those of CC chickens. Genetic analysis using F 1 hybrids (CB×CC) showed that this trait is regulated by a dominant gene that segregates with the B12 haplotype in the backcross generation F 1×CC. From the results obtained with the recombinant congenic lines CB.R1 and CC.R1, we conclude that the gene regulating adherence potential is localized within the B-F/L region of the chicken MHC. About 50% of adherent cells were able to phagocytose opsonised FITC-labelled Zymosan particles. The level of nitric oxide production in vitro by CB and CC macrophages was equal. The importance of cells of the mononuclear phagocyte system for the response to Rous sarcoma virus (RSV) infection was studied in CB chickens using the anti-macrophage agents silica, carrageenan, and Cl 2MDP, encapsulated in liposomes. In those chickens treated with silica and carrageenan, we observed progressive growth of RSV-induced tumors. The graft-versus-host reactivity of peripheral blood lymphocytes (PBL) of treated chickens was comparable to controls. In vitro nitric oxide production by macrophages from silica-treated chickens was higher than by macrophages from untreated controls.

Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule. II. Change of Na-HCO3 cotransport stoichiometry and of response to acetazolamide.

In the preceding publication we reported that some transport properties of proximal tubules perfused in vitro differ from those of tubules perfused in vivo, and that the in vivo function can be largely recuperated by improved metabolic substrate supply and stimulation with norepinephrine (NE). Since we have previously observed that the basolateral Na-HCO3-cotransporter operates with an overall stoichiometric ratio of q approximately 3 HCO3- :1 Na+ in vivo, but with q approximately 2 HCO3- :1 Na+ in vitro and that it responds differently in both cases to acetazolamide (ACZ), we have now tested whether the cotransporter can regain its in vivo function in vitro if the incubation conditions are improved. Cell membrane potentials (Vb) and cell pH (pHi) were measured with microelectrodes and microfluorimetric techniques on isolated S2 segments of rabbit proximal tubule and the instantaneous Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration was determined. (DeltaVb)HCO3 and (deltaVb)Na averaged 13.1 +/- 0.9 mV (SEM) and 6.9 +/- 0.5 mV in D-glucose-containing control HCO3-Ringer solution and decreased respectively to 10.1 +/- 0.5 mV and 3.8 +/- 0.2 mV (n = 8) after incubation in tissue culture medium and NE (10(-5) mmol/l). These data imply that q increased from 1.9 to 2.7. Concomitantly the tubules became susceptible to ACZ (1 mmol/l), which reduced (deltaVb)HCO3 in control conditions only to 94.6 +/- 1.2% but under improved incubation conditions to 64.5 +/- 2.4%. As verified in voltage divider measurements the latter reduction was not caused by activation of a basolateral K+ conductance. The results indicate that improved incubation conditions can at least partially revert cotransport function towards that of the in vivo state. The effect of ACZ may be explained if in the improved state 1 CO32- + 1 HCO3- + 1 Na+ are cotransported, in which case inhibition of carbonic anhydrase (CA) may cause a CO32-/pH disequilibrium to develop in the basal labyrinth which impedes the cotransport. Under conventional incubation conditions, however, when only 2 HCO3- + 1 Na+ are cotransported no such disequilibrium should develop irrespective of whether CA is active or inhibited.

Complement-derived Anaphylatoxins in Human Donor Corneas Treated With Excimer Laser

An inflammatory response produced by excimer laser photorefractive keratectomy (PRK) may be associated with the subsequent corneal haze and regressions in refractive error observed after treatment. Complement-derived anaphylatoxins, potent mediators of inflammation, may have a role in postoperative healing. Twenty right human donor corneas underwent a 6-D excimer laser PRK treatment. The corresponding left donor corneas served as the controls. After incubation in tissue culture media for 6 hours and elution in phosphate-buffered saline with EDTA for 24 hours, complement-derived anaphylatoxins C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay. Compared with control corneas, the excimer PRK corneas failed to demonstrate a significant increase in C3a, C4a, or C5a levels (P > .05). These results suggest that the excimer laser at this dose does not activate significant complement in the cornea.

Effect of Lidocaine on Natural Killer Activity: Rapid Inhibition of Lysis

We have previously found that NK cell activity is present in rat lung lavage and inhibited by lidocaine. The purpose of this report is to characterize further the inhibition by lidocaine on NK activity. Peripheral blood lymphocytes from normal human volunteers were assayed in a four-hour 51chromium release assay in the presence of varying concentrations of lidocaine during or prior to the lytic assay. During the assay, increasing concentrations of lidocaine inhibited NK activity. This effect was not related to pH and at high concentrations was unrelated to methylparaben found in topical lidocaine. Preincubation of effector cells for one hour with different concentrations of lidocaine produced similar results which were temperature dependent and related to incubation time. Preincubation of effector cells in lidocaine (0.5g%) for one to ten minute intervals at 37 degrees C resulted in 60 to 92% inhibition, respectively. The lidocaine did not alter effector cell viability nor did it affect the phenotypes of the effector cells as determined by flow cytometry. Lidocaine's inhibition of NK activity remained the same for cells which were further purified by plastic adherence, nylon wool filtration and percoll density centrifugation. The effect was not reversed by a 16 hour incubation in tissue culture medium. In a single cell assay, we determined that the inhibition was at the level of cell lysis.

Structural changes in hair cells after incubation in tissue culture medium

A great deal of work is presently being pursued with in vitro preparations of isolated single hair cells or isolated segments of the cochlear sensory surface. In this study we evaluate the appearance of hair cells along the neonatal chick basilar papilla that have been freshly dissected and placed in culture medium for between one and six hours. Quantitative measures of the apical hair cell surface revealed that the number of cytoplasmic protrusions was minimal and that surface area was well preserved in the one and three hour samples. By 6 h, the proportion of hair cells showing cytoplasmic protrusions significantly increased, as did the surface area of the hair cells. In this preparation, it appears that physiologic studies can be conducted on cells with a healthy appearance for at least 3 h after sacrifice.

In vitro studies on the control of human myometrial gap junctions

In this study human myometrial tissues were examined for the presence of gap junctions by quantitative electron microscopy before and after incubation in tissue culture media with and without indomethacin. The area of gap junctions was very low in tissues from pregnant women at term but not labor, before incubation. After 24 and 48 h incubation without any treatment, segments of some of the same tissues developed many gap junctions and other tissues contained few junctions. Prostaglandin E (PGE), prostaglandin F (PGF) and prostaglandin F metabolite (PGF metabolite) levels in the media at various times were measured by radioimmunoassay. The prostaglandins increased progressively during the incubation period. Treatment of tissues with indomethacin decreased prostaglandin levels in the media and increased the numbers of gap junctions in those control tissues that developed few junctions over the same incubation interval. We conclude that the capacity of human myometrial tissues to develop gap junctions in vitro may depend upon a maturational stage in preparation for labor. Furthermore, our results suggest that products of the cyclo-oxygenase or lipoxygenase pathways may control the presence of gap junctions in the human myometrium and that changes in synthesis in these patterns may occur as part of the maturational process.

Reversible Oxidant-Induced Increases in Albumin Transfer Across Cultured Endothelium: Alterations in Cell Shape and Calcium Homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant-induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine-xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.

Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant- induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine- xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.

Demonstration of inhibitory factors affecting cell-mediated immunity in patients with Hodgkin's disease

In the present investigations attempts have been made to study factor(s) involved in impairment of cellular immunity in Hodgkin's disease (HD). Peripheral blood lymphocytes from 42 untreated HD patients in differnent stages of the disease showed depressed PHA responsiveness as compared to 23 mormal healthy individuals. Peripheral blood lymphocytes of 7 13 HD patients showed partial restoration of ability to respond to PHA after overnight incubation in tissue culture media, supplemented either with human AB serum or fetal calf serum (FCS). The restoration appeared to be more common in cultures incubated in media supplemented with FCS than with AB serum. Supernatants of lymphocyte cultures from HD patients and their sera showed significant inhibition of PHA induced transformation of lymphocytes obtained from healthy donors. In contrast, normal lymphocyte culture supernatants and sera were less inhibitory to PHA induced transformation of normal lymphocytes. Cultures of HD lymphocytes, which failed to restore PHA responsiveness after overnight incubation, also showed inhibitory factors in supernatants.

Technetium-99 m-labeled mouse-spleen leukocytes: Radiolabeling parameters

Bouse-spleen leukocytes were radiolabeled with 99m Tc under neutral pH conditions. A low concentration of stannous chloride was used to reduce the sodium pertechnetate to a cell-binding lower-valence state. Cell radiolabeling was measured by radioactivity retention on glass-fiber filters. This procedure is quick and allows the assay of small aliquots of the radiolabeling reaction mixture. A stable cell radiolabel of 24.4% ± 5.2% was obtained which remained attached to the cell up to 27.5 h as measured by in vitro incubation in tissue culture medium. The viability of the cells after 99m Tc-cell-labeling remains as high as the control. The radiolabeling efficiency of the cells with 99m Tc was dependent on the concentration of cells and stannous chloride, but was independent of Na 99m TcO 4 concentration.

Receptors for IgM are expressed on acute lymphoblastic leukemic cells having T-cell characteristics

Six acute lymphoblastic leukemia (ALL) patients were studied whose lymphoblasts formed rosettes with sheep erythrocytes to determine if they expressed a receptor for IgM, which is a characteristics of normal T cells; chronic lymphocytic leukemia (CLL) cases carrying B-cell markers were compared as controls. In all six cases of ALL, but not CLL, IgM was expressed after overnight incubation in tissue culture medium. It is therefore considered that the IgM receptor can be used as an additional reliable marker for classifying leukemic disorders.

Exodus of 42K+ and 86Rb+ from rat thymic and human blood lymphocytes exposed to phytohemagglutinin.

We have found that PHA produces an alteration in the lymphocyte membrane which allows 86Rb+ or 42K+ in prelabeled lymphocytes to exchange for cations present in washing solutions. These observations suggested that PHA might induce an increase in the exodus of intracellular potassium during incubation in physiologic media. We, therefore, examined 86Rb+ and 42K+ efflux from rat and human lymphocytes during incubation in tissue culture medium. The rate constant for efflux, Ke, was significantly increased by PHA. 86Rb+ efflux was increased by 27% in rat thymic lymphocytes and by 78% in human blood lymphocytes following PHA treatment.

EVIDENCE FOR THE INDUCTION OF URIDINE AND DEOXY-URIDINE PHOSPHORYLASES OF REGENERATING RAT LIVER IN VITRO.

Increases in the specific activities of undine and deoxyuridine phosphorylases of slices of regenerating rat liver were found 4 hours after incubation in tissue-culture medium containing uridine or 6-azauridine. These increases were not found when the tissue-culture medium contained either 8-azaguanine or puromycin, or when it lacked amino acids. Although both uridine and 6-azauridine were more effective in increasing the specific activity of uridine phosphorylase than that of deoxyuridine phosphorylase, azauridine was more effective than uridine in increasing the specific activities of both enzymes.In time studies, in which slices of regenerating rat liver were incubated in tissue-culture medium containing optimal concentrations of uridine, the specific activities of the two enzymes reached maximum levels at 3–4 hours. Puromycin prevented these increases.