Exposure to a standard culture medium alters the response of cartilage explants to injurious unconfined compression

Previous studies on chondral explants have not clearly described to what extent the degree and the distribution of cell death are dependent on the amount of free swelling seen during tissue equilibration in a standard culture medium. The current study hypothesized that increased fluid content inside equilibrated chondral explants, when subjected to injurious compression, would lead to greater matrix damage during unconfined compression. Equilibrated and non-equilibrated chondral explants were loaded to 30 MPa at a fast rate of loading ( approximately 600 MPa/s). Stress-strain curves were documented for each explant. Matrix damage was assessed by the length of surface fissures. Chondrocyte viability was also measured in the various layers of the explants. The stiffness of the equilibrated specimens was less than non-equilibrated specimens, and it correlated with the amount of fluid absorbed during equilibration. More matrix damage and associated cell death in the superficial zone were documented in equilibrated than non-equilibrated explants, and these correlated positively with fluid absorbed during equilibration. This study indicated that equilibration of chondral explants in a standard culture medium alters their response to mechanical loading in terms of stiffness, matrix damage and cell viability.     

Cellular turnover in the rat uterine cervix and its relationship to estrogen and progesterone receptor dynamics

Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERalpha and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERalpha and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERalpha were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERalpha was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERalpha and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.     

A HISTOCHEMICAL STUDY OF CALLUS INITIATION FROM CARROT TAPROOT PHLOEM CULTIVATED IN VITRO

Cylinders of carrot taproot secondary phloem were cultured on one of four media: 1) 2% sucrose + 1% agar (SA); 2) Heller's basal medium (NA); 3) NA + 10^-5 g/liter 2,4-D (H4); and 4) NA + H4 + 15% coconut milk (HW). Samples were taken from the cultured explants at 3-day intervals. A morphological study of the cultured explants revealed no differences between callus-initiating explants (cultured on HW medium) and noncallus-initiating explants (cultured on SA, NA, and H4 media) within the first 3 days of culture. All explants exhibited a typical wound response. Cell division ceased in the NA and SA explants after the sixth day in culture. Extensive cell division occurred in the subsurface layer of dividing cells in the HW explants and resulted in the formation of callus by the ninth day in culture. Histochemical staining revealed that the activity of NAD diaphorase, succinic dehydrogenase, and cytochrome oxidase were closely correlated with the wound response and with callus initiation in the cultured explants. The activity of these enzymes was high in the layer of dividing cells of all explants after 3 days of culture, but with longer periods of culture the activity of these enzymes was closely correlated with the extent of cell division. Acid phosphatase activity was associated with the dividing cell layers of all explants, but comparatively little acid phosphatase activity was observed in the NA, SA, and H4 explants as compared to the HW explants, and acid phosphatase was strongly correlated with callus initiation by the HW explants. Using the nitroso reaction, “catechol tannins” were found in the surface layers of the NA, SA, and H4 explants, while no nitroso-reaction-positive substances were detected in the HW explants during the period of callus initiation.     

Cherry fruit abscission: evidence for time of initiation and the involvement of ethylene

Initiation of abscission at the pedicel-fruit zone in the sour cherry (Prunus cerasus L. cv. Montmorency) occurs near the transition of Stage II to Stage III of fruit growth. The preinitiation phase is characterized by a high fruit removal force (FRF) and explants prepared from fruits during this period do not undergo abscission as indexed by a reduction in FRF. Ethylene does not cause a significant reduction in FRF either in attached fruit or in explants prepared during this period. By contrast, after initiation (Stage III of fruit growth), there is a marked decrease in FRF with fruit development, explants prepared from fruits during this period undergo abscission, and ethylene markedly promotes the loss in break-strength. Neither the rate of evolution nor the internal concentration of ethylene in the fruit were correlated with fruit abscission. Similar abscission responses, as indexed by FRF and sensitivity to ethylene, were observed in attached fruit and in detached fruit explants.     

Morphological and functional differentiation in epithelial cultures obtained from human skin explants

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-phase cells were found to be totally unlabelled. The grain count distributions revealed similar cell cycle subpopulations as have been shown to occur in vivo. The relationship of these subpopulations to the differentiation process is discussed.     

Development and characerization of cell lines from subhuman primates

Summary Seven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro. Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence. All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties. These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells. The research upon which this publication is based was performed pursuant to Contract No. NIH-69-100 with the Division of Biologics Standards of the National Institutes of Health.     

Selective adhesion of embryonal carcinoma cells and differentiated cells by Ca2+-dependent sites

The specificity of adhesion between embryonal carcinoma cells and fibroblastic cells of various origins was studied. Embryonal carcinoma cells have intercellular adhesion sites requiring Ca²⁺ (CDS). These sites were found to be sensitive to proteases but resistant to them in the presence of Ca²⁺. CDS with a similar protease sensitivity is present in fibroblastic cells. When embryonal carcinoma cells of different lines were mixed, they adhered to each other nonselectively by CDS. Nonselective adhesion by CDS occurred also between fibroblastic cells of various lines. When embryonal carcinoma and fibroblastic cells were mixed, they preferentially adhered to homotypic cells. Fab fragments of antibodies raised against F9 cells (a nullipotent line of embryonal carcinoma) inhibited the adhesion between embryonal carcinoma cells but not between fibroblastic cells. This inhibitory activity of Fab was absorbed with embryonal carcinoma cells with CDS, but not with fibroblastic cells with CDS or embryonal carcinoma cells from which CDS was experimentally removed. SDS-polyacrylamide gel electrophoresis of radioiodinated cell surface proteins showed that the presence of a 140K-dalton component correlated with the presence of CDS in embryonal carcinoma cells, while the presence of a 150K-dalton component correlated with the presence of CDS in fibroblastic cells. These results suggest that CDS in embryonal carcinoma and fibroblastic cells comprise distinct molecules.     

Variations in 5'-nucleotidase activity in established mesenchymal cell lines from syngeneic A/J mice

5'-Nucleotidase activity was analyzed in four different mesenchymal cell lines (F, m, e and SP) established from syngeneic A/J mice. The 5'-nucleotidase activity of fibroblasts was lower in transformed cells (F and m) than in nontransformed cells (e). An increase in cell contact during confluence or during high cell density increased 5'-nucleotidase activity, and a decrease in cell contact caused a decrease in 5'-nucleotidase activity in both fibroblastic (F, m and e) and reticulum (SP) cell lines. These results are evidence that 5'-nucleotidase activity in mesenchymal cells is influenced by intercellular contact as well as transformation.     

Cellular morphology of human malignant melanoma in primary culture

Summary Early monolayer outgrowths of cells from human cutaneous malignant melanomas mostly derived from metastatic lesions were examined microscopically. Cells resembling the two dendritic types of melanoma previously described in the established lines could readily be recognized. Of 22 specimens, 14 consisted of cells with a triangular dendritic morphology, four had both triangular and elongated dendritic morphology, and one had a cuboidal morphology. The remaining three specimens showed only fibroblastic outgrowths. It is concluded that cells with a triangular dendritic morphology are either the most common type of the secondary cutaneous melanomas, or alternately the most adaptable to the present culture conditions. An association of a more favorable prognosis with the homogeneous triangular dendritic cell type is noted. This study was supported in part by grants from The Medical Research Council of Canada and The Ontario Cancer Treatment and Research Foundation.     

Mineral uptake in tobacco leaf discs during different developmental stages of shoot organogenesis

Relationships between mineral uptake and tobacco shoot organogenesis were investigated during three morphogenic phases: phase 1, days 0-10, pre-meristem formation; phase 2, days 10-20, meristem initiation and formation; and phase 3, days 20-35, growth and differentiation of induced meristems into leafy shoots. The mineral content of both shoot-forming (SF) and non-shoot-forming (NSF) media was examined over the 35-day culture period. Both SF and NSF explants rapidly consumed iron during phase 1. Nitrate uptake in SF explants was high and independent of explant growth during phases 1 and 2, but greatest and strongly correlated with growth during phase 3. Phosphorus uptake was highest in SF explants during phases 2 and 3, and correlated with explant growth. Uptake of potassium, calcium and sulphur was strongly associated with explant growth during phase 3 whereas magnesium uptake was only poorly correlated with growth. Results from this study indicate that particular minerals may have an important role in regulating development as well as generally supporting growth.     

Ultrastructure of Junín Virus in Mouse Whole Brain and Mouse Brain Tissue Cultures

Comparative ultrastructural studies were performed on the development of Junín virus in mouse brain and in cerebellum explants and brain monolayers of the same animal. In mouse brain, neurons and astrocytes released virus particles by a budding mechanism identical to that previously described for this virus. In the neurons, the viral multiplication took place in the perikarion as well as in the cytoplasmic processes, including areas near synapses. Viral particles were observed emerging from pericapillary neurons and astrocytes. In the explants, the budding also occurred in neurons and astrocytes. In the monolayers, however, the virus originated in astrocytes and cells of fibroblastic appearance, which were the two cell types that developed in this substrate. These results indicate that the characteristics of the development of Junín virus in mouse brain are faithfully reproduced in cerebellum explants from the same animal, thus allowing some extrapolation of data from one system to the other. The explant proved to be a better model than the monolayer, not only because it reproduced the structural complexity of nervous tissue better, but also because it contains neurons and astrocytes, i.e., the two cell types that release the virus in the in vivo system.     

Epithelial outgrowth from suspension cultures of human prostatic tissue

Summary The primary objective of this study was to obtain pure cultures of prostatic epithelium. Encapsulation by epithelial cells and hypocellularity in stroma occurred when explants of prostatic tissue were maintained in suspension cultures. Twenty per cent fetal bovine serum incorporated into the medium provided optimal conditions for encapsulation and preservation of epithelial cell viability and architecture. Horse serum at the same concentration was less effective. When encapsulated explants were allowed to attach to the substrate, 10 and 20% horse serum favored growth of epithelial cells while fetal bovine serum also stimulated fibroblastic growth. Mechanisms for the induction of hypocellularity, encapsulation, squamous metaplasia and central necrosis in explants were studied. Relationship between the type and concentration of serum and the nature and extent of outgrowth are discussed. This work was supported in part by the American Cancer Society Grants DT-20 and IN-5N, U. S. Public Health Service Grant RR-05357, the Parke Davis Fund, and the John U. White Fund for Urological Research.     

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Development in vitro of epithelial-cell monolayers derived from fetal rat pancreas.

Purified epithelial-cell monolayers were generated in vitro from explants of fetal rat pancreas. The extent of the development of the epithelial monolayer, as determined by planimetric analysis, was enhanced by the application of two methodological procedures: (a) preincubation of fetal pancreas in situ at 27 degrees C for 5 hr prior to dissection and explantation; and (b) incubation of the explants in medium containing a high concentration (50% to 70%) of fetal bovine serum. By utilizing such culture conditions, sheets of contiguous epithelial cells, with little or no peripheral fibroblastic contamination, were maintained for 9 days. Whereas the majority of cells within the monolayer had morphological characteristics of pancreatic ductal cells, endocrine cells were identified by the specific immunocytochemical localization of insulin and glucagon. In addition, insulin could be detected in the incubation medium throughout the course of experiment. The simplicity of this preparation offers some advantages over other techniques including reduced chance of contamination and reduced cellular damage or death. It provides a model for future studies directed toward developing individual cell strains derived from pancreatic epithelial cells.     

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.     

In vitro chondrogenesis and cell viability

Anterior somites cultured with (NSA) or without (SA) notochord, and posterior somites cultured with (NSP) or without notochord (SP) were compared with respect to changes in their DNA content, their potential to synthesize the active sulfate principle phosphoadenosine phosphosulfate (PAPS), and their ability to accumulate ³⁵S-sulfate.Chondrogenesis was observed in the NSA, NSP, and SP explants, but was rarely noted in the SA explants. A decrease in DNA content during the initial 48 hr of culture was common to all explants. After this initial decrease, DNA content increased most in those explants forming cartilage. The synthesis of PAPS by cell-free extracts of each type of somite explant also decreased during the initial period of culture. Only extracts of those explants undergoing chondrogenesis showed increases in PAPS synthesis with continued culture. Each type of somite explant accumulated ³⁵S-sulfate into chondroitin sulfate during the first hours of culture. The non-chondrogenic SA explants accumulated little ³⁵S-sulfate during the period of culture. At varying times after 24 hr the chondrifying explants (NSA, SP, and NSP) initiated an increased rate of accumulation of ³⁵S-sulfate.Cartilage nodules, increases in DNA content, PAPS synthesis and ³⁵S-sulfate accumulation occurred within the same 24 hr period, during the 2nd day in NSP explants, the 3rd day in NSA explants, and between the 3rd and 4th day for SP explants. A hypothesis of in vitro somite chondrogenesis based on differential cell viability is presented.     

Effect of a short-term disturbance of the cell contacts on the mesodermal differentiation of clawed toad embryos

The differentiation of the presumptive mesoderm explants from different sectors of the early gastrula marginal zone was compared with that of the identical explants placed just after explantation into a medium free of divalent cations for 30 s. The development of the treated dorsal explants differed from that of control explants by the presence of well differentiated forebrain structures and the development of the explants from more ventral zones by a decrease in the occurrence of blood cells and nondifferentiated endoderm and an increase in occurrence of epithelioid structures, which form frequently deep invaginations. The shape of the treated explants was more organized and differentiated than that of the control explants. A conclusion has been reached that the development of epithelioid and forebrain structures in the explants is stimulated after a short-term disturbance of cell contacts.     

Fetal rat pancreas in organ culture: effects of serum on the development of the endocrine cells

Eighteen-day-old rat fetal pancreas was grown in organ culture for four days on medium consisting of tissue culture Medium 199 and varying concentrations of chicken serum. The glucagon and somatostatin concentration of the explants was decreased when the serum concentration of the medium was reduced from 50 to 10%. There was a further reduction in these hormones when the explants were cultured on Medium 199 alone. Explant insulin content was reduced only when serum was omitted from the medium. A "serum factor" tripeptide was not able to substitute for this serum requirement. Heat-inactivation of the serum resulted in a significant increase in medium insulin content and an increase in both the insulin and glucagon contents of the explants. This increase in hormone content was directly correlated with increases in the beta and alpha cell volumes of the explants. There was no change in the somatostatin content or delta cell volume of the explants grown on heat-inactivated medium. It is suggested that the serum is an important component of the culture media and is apparently required in high concentration for the development of the islet cells in vitro. The islet cell types differ in their requirement for serum. The alpha and delta a higher concentration than do the beta cells.