FORMATION OF BONE TISSUE IN CULTURE FROM ISOLATED BONE CELLS

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.     

FINE STRUCTURE OF SMOOTH MUSCLE CELLS GROWN IN TISSUE CULTURE

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150–250 A in diameter) and thin (30–80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80–110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.     

STAINING AND ACID PHOSPHATASE REACTIONS OF SPHEROSOMES IN PLANT TISSUE CULTURE CELLS

Spherosomes in plant tissue culture cells from normal sunflower stems and sunflower crown gall tumors reacted similarly to several nonfluorescent and fluorescent lipid dyes. Sudan IV and black B were good selective spherosome stains. The lipid fluorochromes, Nile blue and 3, 4-benzpyrene were excellent selective spherosome stains and visualized the smallest particles more readily than did Sudan IV. Spherosomes could not be seen in tissues stained with Sudan IV or 3,4-benzpyrene after ether-alcohol extraction. Acid phosphatase was detected on the spherosomes in both normal and tumor tissues using the lead sulfide precipitation and the post-incubation coupling procedures.     

DIVALENT CATION-ACTIVATED ECTO-NUCLEOSIDE TRIPHOSPHATASE ACTIVITY OF NERVOUS SYSTEM CELLS IN TISSUE CULTURE

Abstract— Intact neuroblastoma and glial cells in monolayer culture hydrolysed ATP added to their medium. Evidence is presented that ATP is cleaved outside of the permeability barrier of the plasma membrane and the product is liberated in the extracellular medium, i.e. the enzyme is an ecto-enzyme. Divalent cations such as Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ activate the enzyme. In neuroblastoma cells, Ca²⁺ is the preferential cation for activation; Mg²⁺ in glial cells. Substrate specificity was very low when different nucleoside-5'-triphosphates were examined. Competition studies have revealed that all of the nucleoside triphosphates are hydrolysed by the same enzyme: divalent cation-activated ecto-nucleoside-5'-triphosphate phosphohydrolase.
Developmental pattern of the enzyme in several lines was established. The role of enzyme in the transport of divalent cations across the plasma membrane and/or in the physical properties of the membrane is suggested.     

AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.     

STUDIES ON THE INTRACELLULAR DIGESTIVE PROCESS IN MAMMALIAN TISSUE CULTURE CELLS

DNA-protein coacervates containing colloidal gold particles were readily phagocytized by strain L fibroblasts. During the subsequent digestion process, the gold particles served as markers which permitted the demonstration of the evolution of digestive vacuoles to multivesicular bodies and finally to dense bodies. Acid phosphatase and esterolytic activity was present in these structures. The hydrolytic enzymes were apparently brought to the phagocytotic vacuoles in small vesicles originating in the Golgi region. These vesicles entered the vacuoles prior to the digestion of the coacervates and the appearance of positive cytochemical reactions. The cytoplasmic dense bodies frequently merged with the phagocytotic vacuoles. This was demonstrated by prelabeling the dense bodies with colloidal iron prior to phagocytosis of the coacervates. In addition, evidence is presented for the interrelationship of the phagocytotic and autophagic pathways.     

诸葛菜组织培养中的器官形成

植物组织和细胞培养技术已在农作物的改良及园艺植物的快速繁殖方面得到广泛的应用。在这方面,十字花科植物正在受到相当多的注意。新近,为了建立十字花科植物的细胞转化系统,我们试验了多种十字花科植物,发现诸葛菜的叶和叶柄等外植体具有极强的器官分化能力,现将结果报告如下。本文取材植物诸葛菜,又名二月兰(Oryc-hophragmus violaceus/Moricandia sonchifolia),     

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E₁ by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G₁-stage of the cell cycle. 2. Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP. 3. Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other. 4. Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells. 5. A working hypothesis for the malignancy of nerve cells has been proposed. This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells. 6. A new experimental therapeutic model for the treatment of neuroblastoma is proposed. This involves the administration of sodium butyrate followed by the injection of l -dihydroxyphenylalanine (l-dopa) and prostaglandin E₁ in the presence of cyclic AMP phosphodiesterase inhibitor. 7. Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells. Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells. In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells.     

CYTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE MITOTIC APPARATUS OF HELA AND SARCOMA 180 TISSUE CULTURE CELLS

ATPase was localized in distinct regions of the mitotic apparatus of HeLa and Sarcoma 180 tissue culture cells. ATPase was demonstrated in the metaphase spindle of HeLa and Sarcoma 180 cells fixed in cold buffered 2 per cent formalin (pH 6.5 to 6.8) containing 2 x 10^-3 M CaCl₂. A high concentration of ATPase was frequently observed at the poles of the spindle. ATPase was also demonstrated in the interzonal region of both cell types during anaphase. The narrowing of the band of ATPase activity localized in the interzonal region during telophase indicates that ATPase activity is associated with the central spindle. In polar views of Sarcoma 180 cells fixed in cold, unbuffered, 2 per cent formalin, ATPase was frequently localized in granules in the region of the inner circumference of the ring of chromosomes formed at metaphase. ATPase in the mitotic apparatus of HeLa and Sarcoma 180 cells was shown not to be due to non-specific alkaline phosphatase. Mitotic apparatus ATPase in Sarcoma 180 cells was suppressed by an —SH inhibitor.     

FORMATION OF LIGNIN IN TISSUE CULTURE OF PINUS STROBUS

The formation of shikimic acid and lignin from glucose in thecambium tissue was investigated. Glucose-1-¹⁴C, shikimic acid-G-¹⁴C, sodium acetate-1-¹⁴C and sodium acetate-2-¹⁴C were administeredto the tissue culture of strob pine. Glucose was well incorporatedinto shikimic acid, but acetic acid was less effective. Shikimicacid was very efficient as a precursor of aromatic nucleus andglucose was also converted efficiently to lignin. The extentof incorporation of acetic acid, however, was considerably low.A possibility was discussed that in the cultured tissue ligninand its precursor were synthesized from glucose via the shikimicacid pathway. (Received May 14, 1960; )     

IN VITRO CULTURE OF SOMATIC WHEAT CALLUS TISSUE

Sterile culture of excised wheat tissue, Triticum vulgare Vill., was attempted on 20 different media. Most tissue explants did not grow. Endosperm tissue did not grow on any of the media. Callus tissue from intercalary meristems grew for several weeks but gradually became necrotic. Callus tissue derived from the cotyledonary node grew well and produced branching and enlarging root systems when placed on different media. Shoot primordia were not observed. Callus tissue derived from 13 varieties of wheat was cultured. These unique somatic tissues are interesting experimental materials for a study of the developmental biology of wheat.     

ATPase ACTIVITY OF NEUROBLASTOMA CELLS IN CULTURE

—Neuroblastoma cells were cultured in the presence of fetal serum. When serum was omitted from the growth medium and also when dibutyryl cyclic AMP or insulin were added without omitting serum, there was a growth of neuron-like processes which was in most cases accompanied by an increase in Na⁺-K⁺ -ATPase activity, whereas Mg²⁺-ATPase activity increased to a lesser extent. The increase in ATPase activities was related to cell density. When the ATPase activities were measured in the presence of 0·25 m -sucrose or 1% deoxycholate the specific activities were decreased. There was no loss of activity during storage at –20°C for several days when 20% glycerol was added to the medium.     

TRANSFORMATION OF MONOCYTES IN TISSUE CULTURE INTO MACROPHAGES, EPITHELIOID CELLS, AND MULTINUCLEATED GIANT CELLS : An Electron Microscope Study

The sequential transformation of chicken monocytes into macrophages, epithelioid cells, and multinucleated giant cells in vitro was studied by electron microscopy after fixation and embedment in situ. The following changes occur. In the nucleus, margination of chromatin, evident in monocytes, decreases in later forms. Nucleoli become more complex and nuclear pores increase in number. In cytoplasm, a progressive increase in volume of the ectoplasm and endoplasm occurs in culture. Lysosomes increase in number and size prior to phagocytosis. During phagocytosis (most active from 1 to 3 days of culture) lysosome depletion occurs. Lysosomes are present in greatest number and show maximal structural variation in the epithelioid and young giant cells. Aging giant cells lose lysosomes. All stages possess variably large quantities of rough-surfaced endoplasmic reticulum and free ribosomes. The Golgi apparatus, small in monocytes, increases in size and complexity. Massive accumulations of lysosomes within the Golgi apparatus of macrophages and epithelioid cells suggest that lysosomes originate there. In giant cells, multiple Golgi regions occur, often ringing the nuclei. Monocytes and macrophages have few mitochondria. Mitochondria of epithelioid cells are larger, more numerous, and may have discontinuous outer membranes. Mitochondria are most numerous in giant cells where they increase with age and become polymorphous. Cytoplasmic filaments are approximately 50 to 60 A in diameter and of indeterminate length. They occur both singly and in bundles which touch cytoplasmic vesicles and mitochondria. Few filaments occur in monocytes and macrophages. A large increase in the number of filaments occurs in epithelioid cells, where filaments (90 to 100 A) surround the cytocentrum as a distinctive annular bundle often branching into the cytoplasm. The greatest concentration of filaments occurs in aged giant cells. Pseudopodia are always present. They are short and filiform in monocytes and giant cells, and broad, with abundant micropinocytotic vesicles, in macrophages and epithelioid cells. At every stage, the cell membrane contains dense cuplike structures. These may represent the membranous residue of lysosomes which have discharged to the outside, analogous to merocrine secretion. Contiguous epithelioid cells display elaborate cytoplasmic interdigitation. In places, the plasma membranes break down and epithelioid cells fuse to form giant cells.     

MUCOPOLYSACCHARIDES PRODUCED IN TISSUE CULTURE

1. A method of mass tissue culture has been devised by which, in a relatively short period of time, samples large enough for chemical isolation of mucopolysaccharides can be obtained. 2. Chemical isolation of acid mucopolysaccharides from mass cultures of human fetal skin, human fetal bone, bovine fetal skin, and rat subcutaneous tissue has been carried out. It has been found that the fibroblasts of each of these tissues produce in tissue culture more than one mucopolysaccharide, namely, hyaluronic acid, and a chondroitin sulfate. 3. The chondroitin sulfate produced by fibroblasts of the above tissues in tissue culture was not fully sulfated. The possible significance of this finding is discussed.     

DEVELOPMENT OF CHLOROPLAST FINE STRUCTURE IN ASPEN TISSUE CULTURE

Chloroplast ontogeny has been examined in 42-day etiolated triploid aspen callus (Populus tremuloides Michx.) subjected to two different light conditions. White and low-intensity red illumination showed little differences in their stimulatory effects on plastid development, the red light-irradiated plastids developing only slightly more slowly. Asynchronous plastid development was noted in both lighting systems. Etioplasts contained an interconnected tubular net, phytoferritin aggregates, electron-transparent vesicles which seem to invaginate from the inner plastid membrane, membrane-bound homogeneous spheroids and starch grains. Irradiation caused various morphological changes within the proplastids; the tubular complex became transformed into the more ordered prolamellar body-like structure from which radiated membrane-bound sacs filled with electron-dense material. These sacs, characterized as thylakoid precursors, were transformed into a thylakoidal system typical of mature chloroplasts. This ontogenetic scheme represents an additional pathway for the development of photosynthetic lamellae. Other light-induced changes in the developing plastid include disappearance of phytoferritin particles and homogeneous spheroids, decrease in starch content, and appearance of osmiophilic droplets.