Isolation of adult canine venous endothelium for tissue culture

Summary In order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact. This research was supported by National Heart, Lung and Blood Institute Contract NIH-NO1-HV-2054 and Grant HL23345.     

Secondary product formation in plant tissue cultures

The formation of secondary products in plant tissue culturesis reviewed. The conditions for the enhanced production of secondaryproducts, which include alkaloids, terpenoids, steroids andphenolics, can be regulated in a number of ways. For example,manipulation of secondary product formation is possible by varyingthe nutrient composition of the growth medium, light, temperatureand pH, and by the use of elicitors, permeabilisation and two-stagesystems. Molecular engineering and the use of biomass and large-scaleculture are described along with future prospects for the commercialproduction of secondary products from cell suspension cultures.     

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).     

Plant regeneration from embryo-derived tissue cultures of soybeans

Summary Routine regeneration of fertile plants from a tissue culture of soybean has been achieved. Serially propagated embryogenic cultures were initiated from immature embryos of many genotypes. Organized tissues developed only on the cotyledons of embryos. Genotypic variation in the frequency of initiation of embryogenic tissue was noted. However, embryogenic tissue cultures were generated from all genotypes tested. Embryogenic tissue was serially increased and underwent morphogenesis. Whole fertile plants were recovered. Cultures have been maintained for two years without loss of morphogenic competency. Editor's Statement This procedure for initiating embryogenic tissue cultures from commerical cultivars of soybean and the subsequent development of fertile plants establishes a framework for studying the processes of embryogenesis and embryogenyin vitro as well as providing a system for tissue culture propagation and in vitro modification of soybean. Robert B. Horsch     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.     

Lymphatic endothelial and smooth-muscle cells in tissue culture

Summary Endothelial and smooth-muscle cells from bovine mesenteric lymphatic vessels have been collected and cultured in vitro. The endothelial cells grew as a monolayer exhibiting a “cobblestone” appearance with individual cells tending to be more flattened at confluence than their blood vascular counterparts. Approximately 30% of these cells expressed Factor VIII antigen compared with bovine mesenteric artery or human umbilical-vein endothelium in which the majority of cells were positive. The lymphatic smooth-muscle cells exhibited focal areas of multilayering and were Factor VIII negative. The availability of lymphatic endothelial and smooth-muscle cells in culture will provide a new tool for the investigation of the biological properties of the lymphatic vessels and their role in homeostasis. Supported by the Medical Research Council of Canada, Grant MA-7925     

Establishment and characterization of American elm cell suspension cultures

Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A₇₀₀ and media conductivity. Combined cell growth data for all experiments within a genotype showed relatively low variances and between-genotype contrasts during repeated passages showed no significant differences. Subculturing exponentially growing cells at 8–14 day intervals, within readily measured parameters of media conductivity (4.95–4.2 mmhos) and cell concentration (≥ 1.4 A₇₀₀), consistently resulted in repeatable profiles of elm cell growth and minimized lag phase. Culture cells were essentially homogeneous after 5 subculture passages and their overall appearance was stable. We conclude that the described procedure resulted in consistent cultures suitable for elicitor treatment experiments. This revised version was published online in June 2006 with corrections to the Cover Date.     

Angiogenesis and the formation of lymphaticlike channels in cultures of thoracic duct

Summary Segments of rat thoracic duct cultured in plasma clot or in collagen gel produced microvascular and fibroblastic outgrowths. Lymphaticlike channels (LLC) with a highly attenuated endothelium, which was barely visible by light microscopy, were found in 8 out of 25 cultures (32%). Serial histologic sections revealed that the endothelium of the LLC was continuous with the intimal endothelium of the throacic duct and was therefore of lymphatic origin. In addition to the LLC, vascular channels lined by a thick endothelium with hump-shaped, cross-sectional profiles were found in 10 cultures (40%). These channels were indistinguishable from the microvessels of blood vascular origin that formed in parallel cultures of rat aorta or periductal adipose tissue and were termed hematiclike channels (HLC). Contrary to the LLC, the HLC did not originate from the lymphatic endothelium of the thoracic duct. The frequent association of the HLC with the adventitia of the thoracic duct and with the surrounding adipose tissue suggested that they probably developed from the hematic microvessels of the periductal soft tissues. This research was supported by grants from the National Cancer Institute, NIH, National Bladder Cancer Project (CA14137), and the W. W. Smith Charitable Trust.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Isoperoxidases from tobacco tissue cultures

Two anodic isoperoxidases (A₁ and A₂) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C₃ and C₄) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C₄ contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.     

Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.     

Allergens from short ragweed leaf tissue cultures

Summary Short ragweed (Ambrosia elatior L.) leaf cells were grown as callus and suspension cultures. Immunodiffusion tests against anti-pollen crude extract and anti-antigen E sera did not detect antigen E in the tissue cultures. However, two allergenically active fractions were isolated from the tissue cultures by ammonium sulfate precipitation, diethylaminoethyl-cellulose and Sephadex G-100 chromatography. Two fractions isolated (Sephadex G-100, fractions II and III) were electrophoretically homogeneous, carried net charges similar to that of antigen E in polyacrylamide disc electrophoresis at pH 8.9 and had molecular weights of 10,000 to 15,000 and 30,000 to 40,000, respectively. These fractions were highly skin reactive in individuals sensitive to short ragweed pollen. This paper was abstracted from a thesis submitted by A. Shafiee to the University of Minnesota in partial fulfillment of the Doctor of Philosophy degree requirements.     

Beneficial effects of activated charcoal on plant tissue and organ cultures

Summary Addition of activated charcoal to the medium for plant tissue cultures improves growth by adsorbing toxic metabolites. This research was supported in part by the National Science Council, Republic of China     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

Direct infection of embryogenic tissue cultures of haploidBrassica napus with resting spores ofPlasmodiophora brassicae

Summary A direct infection of cultured tissue with resting spores of the obligate endoparasitePlasmodiophora brassicae has been made possible by using stem embryo cultures of haploid rape. The fungus develops and completes its lifecycle in the cultured cells. The results are discussed in connection with the possibility of using this system to select for resistance to the pathogen.Supported by the Deutsche Forschungsgemeinschaft     

Growth of human vascular endothelial cells on various types of microcarriers

Various types of microcarriers were tested as growth substrate for the cultivation of either endothelial cells from human umbilical cord veins or of EA. hy926, an immortalized cell line of endothelial origin. Cell growth was tested on microcarriers in tissue culture flasks and spinner flasks. Solid (Cytodex type I, II, III, Gelibeads, Mica) and macroporous (Polyhipe, CultiSpher GL, PolyporE type I) microcarriers were tested. For the solid carriers the best results were obtained with Mica and for the macroporous carriers with CultiSpher GL.Abbreviations DAPI 4,6-diamidino-2-phenylindole-di-hydrochloride - DEAE diethylaminoethyl - EC vascular endothelial cells - FGF fibroblast growth factor - HUVEC vascular endothelial cells from human umbilical cord veins - IF 11 mixture of Iscove's MDM and F12 basal media - NCS newborn calf serum - PBS phosphate buffered saline - TE 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS     

Serum-free media for cultures of primitive and mature hematopoietic cells

The in vitro culture of human hematopoietic cells has many research and therapeutic applications. Traditionally, human hematopoietic cultures have been conducted using serum-containing media. The disadvantages inherent in the use of serum could be eliminated by the use of serum-free media. In this review, we summarize and discuss the current status of serum-free media for both mature and immature human hematopoietic cells. The mature hematopoietic cells discussed are of lymphoid (e.g., lymphokine activated killer cells and tumor infiltrating lymphocytes) and myeloid origin (e.g., monocytes/macrophages). The cultures of immature hematopoietic cells discussed are clonogenic and long-term cultures. In addition, we briefly review the types of human hematopoietic cells, their clinical applications, and the basic strategies and components used to formulate serum-free media, Finally, we outline future requirements and directions in the development of serum-free media for primitive hematopoietic cells.     

Studies on growth and steroid formation in tissue cultures of Holarrhena antidysenterica

Callus cultures derived from the hypocotyl of germinated seedlings of the plant, Holarrhena antidysenterica showed an inherent lack of organ-forming ability when grown under the influence of a wide range of exogenous growth factors. Several sterols were isolated from the callus, of which, the predominant ones were identified as cholesterol, 24-methylenecholesterol, 28-isofucosterol, sitosterol and stigmasterol.     

Cell and tissue cultures of Catharanthus roseus: A literature survey

The literature concerning the regulation and the biosynthesis of secondary metabolites in cell and tissue cultures of Catharanthus roseus is reviewed. The aim of this review is to summarise the progress achieved since the previous review of this subject from 1988 to December 1993. Several factors influencing the production of indole alkaloids are discussed. Special attention is given to large-scale cultivation methods. Some economic considerations on the production of ajmalicine are also discussed.     

Isolation and characterization of two isoperoxidases from tobacco tissue cultures

Two isoperoxidases (A_f and C_n) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C_n and A_f have MWs of ca 30 000 and 54 000, respectively. A_f has ca 5.1% carbohydrate, but none could be detected in C_n. Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K_ms for guaiacol are 4 and 13.3 mM for Af and C_n, respectively, while both isoperoxidases have a pH optimum at 6.5. C_n, is dissimilar to other isoperoxidases from tobacco tissue cultures, but A_f is very similar to isoperoxidase A₃ from W-38 tobacco tissue culture.