Cerebral microvascular smooth muscle in tissue culture

Summary Cerebral endothelium is being studied rather extensively in tissue culture, but no reports are available describing the tissue culture of cerebral microvascular smooth muscle. The present paper describes for the first time the isolation and culture of non-neoplastic mouse cerebral vascular smooth muscle. Microvessels from a dounce homogenate of mouse brain are plated onto plastic culture dishes in Dulbecco’s modified Eagle media plus 20% fetal bovine serum and treated briefly with collagenase. Cells migrate from vessels and proliferate sufficiently to be transferred out of primary culture in 2 to 3 wk. Light microscopy reveals generally broad, polygonal cells that grow collectively in a “hill and valley” pattern. By transmission electron microscopy the cells possess many characteristics of smooth muscle: basal laminas, clusters of pinocytotic vesicles, and bundles of thin filaments. Several ill-defined cell-to-cell junctions are also present. Isoelectric focusing and sodium dodecyl sulfate-electrophoresis of cellular proteins on polyacrylamide gels after pulse labeling cultures with [S-³⁵]methionine demonstrate that these cells actively synthesize a smooth-muscle-specific isoactin, α-actin. The identity of α-actin is confirmed by analysis of NH₂-terminal peptides after actin digestion with trypsin and subsequent peptide cleavage with thermolysin. Both their morphology and active synthesis of α-actin strongly suggest that these cells are of smooth-muscle origin. Future studies of their metabolism and interactions with endothelium and astrocytes should provide a better understanding of the cerebral microcirculation. This work was supported by Veteran’s Administration Research Funds, National Institutes of Health Grant HL 14230 (Arteriosclerosis Specialized Center of Research, sponsored by the National Heart, Lung, and Blood Institute), and Cardiovascular Research Program Project Grant from the National Institutes of Health to the University of Iowa (HL-14388). A. R. S. is a postdoctoral fellow of the National Institute of General Medical Sciences (GM-09020). P. A. R. is an established investigator of the American Heart Association.     

Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

Summary Human myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. This investigation was supported, in part, by U.S. Public Health Service Grants 5-P50-HD11149 and 5-P01-AG00306.     

A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis

Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work. Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD.     

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     

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture

Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.     

Skeletal muscle tissue engineering

The reconstruction of skeletal muscle tissue either lost by traumatic injury or tumor ablation or functional damage due to myopathies is hampered by the lack of availability of functional substitution of this native tissue. Until now, only few alternatives exist to provide functional restoration of damaged muscle tissues. Loss of muscle mass and their function can surgically managed in part using a variety of muscle transplantation or transposition techniques. These techniques represent a limited degree of success in attempts to restore the normal functioning, however they are not perfect solutions. A new alternative approach to addressing difficult tissue reconstruction is to engineer new tissues. Although those tissue engineering techniques attempting regeneration of human tissues and organs have recently entered into clinical practice, the engineering of skeletal muscle tissue ist still a scientific challenge. This article reviews some of the recent findings resulting from tissue engineering science related to the attempt of creation and regeneration of functional skeletal muscle tissue.     

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0·0%; T2 = 1·1%; T3 = 1·7%; T4 = 4·0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH‐TR reaction and strong acid‐stable mATPase activity, and a deep white one, most of the muscle mass, formed by fibres with weak NADH‐TR reaction and strong alkali‐stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid‐stable or acid‐labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1–T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters ≤8 μm was higher in the lysine treatments, and with diameters between 16 and 24 μm, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 μm and in T1–T4 were between 8 and 16 μm. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Methods for improving tissue culture of human tracheo-bronchial epithelium obtained at autopsy

Human tracheo-bronchial epithelium obtained from autopsy, surgery, and organ donation will have areas of both viable and non-viable cells. It is important in the initial establishment of epithelial explant and cell cultures that injured, non-viable mucosal epithelium not be used for the cultures. Autopsy cases selected for culture should initially be chosen on the basis of a shorter post mortem interval and cause of death in order to increase the rate of successful culture. Staining the epithelium with the vital dye, trypan blue, in combination with phase contrast microscopy of the bronchial tissues will further identify those areas of the mucosa that are enriched for viable cells. The dead, non-viable areas are trypan blue positive, while the viable areas are clear and have foci of beating, motile cilia. Treatment of the mucosal tissue with mucolytic agents to remove cell debris, dead cells, and microbes trapped in the mucus material will further improve the chances for successful culture. Human tracheo-bronchial epithelium, although non-sterile and often injured at time zero for numerous reasons, can effectively be used in in vitro pathophysiology studies.     

Commercialization of plant tissue culture in India

Commercial application of plant tissue culture started in USA with micropropagation of orchids in 1970s. It has seen tremendous expansion globally from 1985 to 1990 in the number of production units as well as the number of plants produced. With an estimated global market of 15 billion US dollars per annum for tissue cultured products, even with exponential expansion in the industry, the demand far exceeds production, leaving enough scope for expansion. This industry appears to be undergoing a pause in growth presently in developed countries as it is finding difficult to remain cost–effective. In US, only half the production capacity is being utilized currently due to high labour costs. In developing countries, with lower wage scales, plants are being produced at much cheaper rates. Indian micropropagation industry, though a late starter by almost a decade, compared to its western counterparts, has expanded exponentially from 5 million annual capacity in 1988 to 190 million in 1996. The facilities now created are at par with the best in leading countries like the Netherlands and USA. To remain in profitable business and to earn the much needed foreign exchange, Indian units need to judiciously mix steady revenue generating items with unique speciality items based on demand in domestic and international markets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth of human muscle in tissue culture. An improved technique.

A method is described for the culture of normal and diseased human muscle cells. Cell outgrowth was obtained from 63/63 biopsies, and cells differentiated to form myotubes in 57/63 biopsies. The culture technique used readily permitted the growth of both normal and diseased human muscle cells.     

植物组织培养外植体褐变的研究进展

外植体的褐变是组织培养过程中的主要障碍。目前一般认为影响褐变的因素主要有外植体材料、培养基和培养条件。为了防止褐变的发生,主要可以从外植体和培养条件、进行细胞筛选和材料的预处理、抑制剂或吸附剂的加入等方面入手。酚、酚酶的区域性分布假说、自由基伤害假说、保护酶系统假说是褐变机理方面较为成熟的3种假说。     

Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering

The human recombinant collagen I α1 chain monomer (rh‐gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az‐rh‐gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az‐rh‐gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az‐rh‐gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az‐rh‐gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose‐dependently. It was also possible to immobilize Az‐rh‐gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen‐binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering. Biotechnol. Bioeng. 2011;108: 2468–2476. © 2011 Wiley Periodicals, Inc.     

Long-term storage of tissue samples for cell culture

Summary The establishment of cultured cell lines from skin biopsies stored at −196°C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately. This project was supported by the South Carolina Department of Mental Retardation and the Self Foundation.     

Impact of the tissue factor pathway inhibitor gene on apoptosis in human vascular smooth muscle cells

Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation pathway. Recent studies indicated that TFPI induces apoptosis in vascular smooth-muscle cells (VSMCs) in animals. The present study investigated whether the TFPI gene could also induce apoptosis in human vascular smooth-muscle cells (hVSMCs). Such cells were isolated from human umbilical arteries and subsequently transfected with pIRES-TFPI plasmid (2 μg/mL). MTT assaying and cell counting were applied to measure cell viability and proliferation, RT-PCR was utilized to analyze TFPI gene expression in the cells. Apoptosis was analyzed by fluorescence activated cell sorting (FACS). Several key proteins involved in apoptosis were examined through Western blotting. It was shown that TFPI gene transfer led to its increased cellular expression, with a subsequent reduction in hVSMC proliferation. Further investigation demonstrated that TFPI gene expression resulted in lesser amounts of procaspase-3, procaspase-8 and procascase-9, and an increased release of mitochondrial cytochrome c (cyt-c) into cytoplasm, thereby implying the involvement of both extrinsic and intrinsic pathways in TFPI gene-induced apoptosis in hVSMCs.     

太白米组织培养的研究

太白米地下鳞茎在MS附加不同浓度KT、BA、IAA、NAA、2,4－D的培养基上,可诱导产生愈伤组织,其中在MS附加NAA 0.5mg/L,KT0.1mg/L的培养基上愈伤组织诱导率最高,可达65％,且生长快;培养在MS附加2,4－D 1.0mg/L,KT 0.1mg/L培养基上的鳞茎可经不定根直接发育成新的鳞茎,由此建立了太白米的鳞茎再生体系,鳞茎再生率达2．17倍。     

盾叶薯蓣组织培养技术的优化

以盾叶薯蓣的根状茎、茎段、叶柄、幼叶为材料,进行愈伤组织诱导、分化及再生植株形成的研究。结果表明：盾叶薯蓣不同外植体均能诱导出愈伤组织,其中茎段愈伤组织的诱导率最高;不同激素配比的培养基对愈伤组织的形成有很大的影响：以LS为基本培养基,2,4-D浓度为4．0mg／L、6-BA浓度为1．0mg／L的激素配比诱导率最高,达62．5％;以改良MS为基本培养基,2,4-D浓度为2．0mg／L、6-BA浓度为0．5mg／L的激素配比诱导率最高,达71．4％。筛选到优化的分化培养基为改良MS附加2．0mg／L的6-BA和0．5mg／L的Vc,且能直接诱导出根,并形成完整植株。