Cell cultures from marine mammals

Cell cultures grown from the gray seal Halichoerus grypus and the dolphin Lagenorhynchus obliquidens support the replication of several human viruses. Further evidence indicates the gray seal kidney cell culture has transformed.     

Cell cultures over nanoneedle fields

In this paper a new nanostructured support for the culture of cells is presented. The support consists of fields of sharp and high-aspect-ratio nanoneedles. The support is obtained through a specifically developed process that allows controlling the nanoneedles’s densities and height. The nanoneedles are typically 10 μm high with tip diameters under 200 nm. Cell viability on this support was evaluated through long-term cells cultures. The narrow interface between the cells’ membrane and the nanoneedles has been carefully observed to conclude on the perforation of the cells’ membrane thanks to the sharp nanoneedles. Such a nanostructured chip, allowing specific interaction, opens the door to a large number of exciting and valuable applications such as nanoporation for transfection or internal cell potential recording.     

Cell size in aging monolayer cultures

Summary Changes in the size of the area covered by individual cultured WI-38 cells as the cultures age have been studied by using a new microphotographic paper cutout technique. This method is nondestructive and nonintrusive and avoids a number of artifacts which can occur in the measurement of suspended cells. The measurements reveal that the decreased cell yield of late passage cultures-reflects not only the appearance of a subpopulation of larger cells but also the failure of the cells to utilize all the growth surface available to them. This work was supported in part by USPHS research grant AG-00378 and by a fellowship, AG-05019, from the National Institute on Aging.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Cell size in aging monolayer cultures.

Changes in the size of the area covered by individual cultured WI-38 cells as the cultures age have been studied by using a new microphotographic paper cutout technique. This method is nondestructive and noninstrusive and avoids a number of artifacts which can occur in the measurement of suspended cells. The measurements reveal that the decreased cell yield of late passage cultures reflects not only the appearance of a subpopulation of larger cells but also the failure of the cells to utilize all the growth surface available to them.     

Cell cultures in microsystems: Biocompatibility aspects

Bio‐Micro‐Electro‐Mechanical Systems (BioMEMS) are a new tool in life sciences, supporting cell biology research by providing reproducible and miniaturized experimental platforms. In order to cultivate cells in such systems, appropriate microenvironmental conditions are required. Due to the multitude and variety of microbioreactors and cultivated cell types available, standardized cell handling methods and comprehensive biocompatibility data are sparse. The bioreactor developed at Ilmenau University of Technology features BioMEMS consisting of silicon, glass, and polymers, supplied by peripheral components. To verify the system's suitability for cell cultivation, it was necessary to prove whether materials and surfaces are biocompatible. Custom‐tailored biocompatibility test procedures along with adequate cell seeding and handling methods had to be developed. According to this, proper positive and negative control samples had to be identified. The cultivation procedures were carried out using osteoblast‐like murine fibroblasts (MC3T3‐E1) and primary human osteoblasts (hOB). We could provide evidence that cultivation of these cells in our BioMEMS is feasible. In this context the relevant materials and the system's structure can be regarded as to be biocompatible. We could show that cell seeding and handling methods possess a strong impact on growth, development, and cellular activity of cell cultures in BioMEMS. Statistical biocompatibility data for the materials used is given. Biotechnol. Bioeng. 2011; 108:687–693. © 2010 Wiley Periodicals, Inc.     

Cell cycle analysis of primary sponge cell cultures

Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2 d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth.     

Cell cultures as models of cardiac mechanoelectric feedback

Although stretch-activated currents have been extensively studied in isolated cells and intact heart in the context of mechanoelectric feedback (MEF) in the heart, quantitative data regarding other mechanical parameters such as pressure, shear, bending, etc, are still lacking at the multicellular level. Cultured cardiac cell monolayers have been used increasingly in the past decade as an in vitro model for the studies of fundamental mechanisms that underlie normal and pathological electrophysiology at the tissue level. Optical mapping makes possible multisite recording and analysis of action potentials and wavefront propagation, suitable for monitoring the electrophysiological activity of the cardiac cell monolayer under a wide variety of controlled mechanical conditions. In this paper, we review methodologies that have been developed or could be used to mechanically perturb cell monolayers, and present some new results on the acute effects of pressure, shear stress and anisotropic strain on cultured neonatal rat ventricular myocyte (NRVM) monolayers.     

Cell wall proteomics of sugarcane cell suspension cultures

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl₂ and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second‐generation ethanol production.     

Cell cultures from cryopreserved human lung tissue.

To assess gene induction in primary human fibroblasts, we have developed a method for cryopreservation of lung biopsies in liquid nitrogen. Fresh biopsies (n = 10) were chopped into 5 x 5 mm pieces and transferred into an ice-cold freezing medium. Biopsies were kept on ice for 15 min, followed by further cooling of the tissue to -70 degrees C. With this method, lung biopsies were preserved for more than 1 year before they were used for generating cell cultures. There was no significant difference in the biological responsiveness of fibroblasts generated from immediately cultured lung biopsies compared with those from cryopreserved tissue. The doubling rate of fibroblasts from fresh tissue was 23.6 +/- 1.1 hr; compared to 23.5 +/- 1.5 hr for fibroblasts generated from cryopreserved tissue. PDGF-BB enhanced de novo synthesis of DNA 100 times, in both the immediately cultured fibroblasts and those generated from cryopreserved biopsies. Macrophages, dendritic cells and endothelial cells could also be recovered from cryopreserved lung tissue. This method permits long-term storage of lung tissue and the possibility of establishing primary cell lines from the same tissue at later times without appreciable changes in their cellular biological characteristics.     

Cell identification in primary cell cultures from skin

Summary Primary cell cultures can readily be obtained from human skin using the explant method. With special care it is possible to obtain primary cultures consisting of epidermal keratinocytes without fibroblast contamination. By means of differences in their growth patterns and retention of specific differentiative functions in vitro, keratinocytes and fibroblasts can easily be distinguished. The high degree of confidence in establishing cell identity permits meaningful experimental use of this system. The method of enzymatic separation of epidermis from dermis, followed by culture of cells from the dissociated epidermal tissue, provides both epithelial and dendritic cells. The former are probably keratinocytes, whereas the latter are definitely melanocytes. The possibility of eventual fibroblast overgrowth, using this latter method, has not yet been ruled out with certainty. Presented at the Workshop on Primary Cell Culture, November 1–3, 1973, Convenor Dr. Warren I. Schaeffer, University of Vermont, at the W. Alton Jones Cell Science Center, Lake Placid, N. Y. The editorial assistance of Dr. and Mrs. Joseph Leighton in preparing for press the five papers from that workshop is gratefully acknowledged. This work was supported by grants from the National Cancer Institute, 1 PO 1 CA 11536, and the National Institute of Arthritis and Metabolic Diseases, 1 PO 1 AM 15515.     

Cell surface gamma-glutamyl transpeptidase in live cultures.

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.     

Cell cultures in the biocompatibility study of synthetic materials

In vitro cytotoxicity (Neutral Red uptake, Kenacid Blue and MTT) and cytocompatibility (cell adhesion and proliferation) tests were applied to the biocompatibility study of a series of poly(ester-ether-ester) block copolymers of potential interest as biomaterials. Our results indicate that the copolymer extracts after 72 hours incubation with a 3T3 mouse fibroblast cell line do not induce significant toxic effects. Furthermore, human umbilical vein endothelial cells seeded on thin copolymer films show a normal pattern of growth. We conclude that thein vitro tests used are a valid instrument to evaluate the potential toxic action of synthetic materials on different cell compartments and that the tested materials seem to be promising for future applications in the field of biomedical devices.     

Cell cultures from the symbiotic soft coral Sinularia flexibilis

The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed.     

Cell cultures of Rauwolfia sellowii: growth and alkaloid production

Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell cycle analysis of hybridoma cultures: study by cytofluorimetry

An hybridoma cell line cultivated in flasks has been used as a model to study the loss of cell viability in high density cell cultures. Cell cycle analysis by cytofluorimetry has shown that a new hypochromosomic cell population appeared as soon as the viability began to decrease. However there is no evidence that this new population is constituted of dead cells.     

Cell cultures derived from embryos and melanoma of poeciliid fish

In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.     

Cell cycle status of stromal cells in long-term haematopoietic cultures

This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.