In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation

For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl. The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine). In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline. During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur. The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6%. Addition of foetal calf serum to the basal medium favours cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%). Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium. These results are discussed with respect to the problem of cell proliferation control during limb regeneration.     

Aedes novalbopictus: establishment and characterization of larval cell lines

Two diploid cell lines were established from larval tissues of the mosquito Aedes novalbopictus. Many morphologically different types of cells were detected in primary cultures. However, only three types of cells became established in the cell lines. They were: epithelial-like cells (80–90%), fibroblast-like cells (5–10%), and giant cells (5–10%). Cell number increased about 15-fold during the first 6 days of culture and the population doubling time during the period of active growth was 18 hr. Seventy to 80% of the cells were diploid (2n = 6) and the rest were tetra-or polyploids. Cells from these two cultures so far (December, (1971)) have been subcultured 36 and 34 times, respectively. The growth pattern and general morphologic features of the cells of A. novalbopictus cultures closely resembled those of A. albopictus cultures.     

Vitrification of carnation in vitro: Changes in ethylene production,ACC level and capacity to convert ACC to ethylene

Carnation tissue was allowed to vitrify in liquid culture and ethylene production, ACC content and capacity to convert ACC to ethylene were measured in comparison to tissue developing normally on solid medium. Flask atmospheres of liquid cultures accumulated ethylene at a higher rate during the first four days. Daily ethylene production by vitrifying material decreased later. Ethylene emission by vitrifying tissues always remained above controls when subcultured daily to fresh medium. Explants and microsomal preparations from vitrifying carnations converted ACC to ethylene at a higher degree from the first day in liquid medium. ACC level markedly increased in vitrifying tissues during the first two days of liquid culture. Raising the level of ethylene in the atmosphere of solid cultures did not induce vitrification symptoms nor did use of inhibitors of ethylene biosynthesis in liquid cultures prevent the process. The role of ethylene in vitrification is reappraised.     

大岩桐花萼切块离体培养直接再生花芽的形态观察(简报)

前文发现在基本培养基上添加赤霉素和细胞分裂素能促进离体培养的大岩桐花蕾直接再生花芽[1],后又发现细胞分裂素能协同赤霉素促进离体培养大岩桐花萼切块高频率直接再生花芽[2,3],所得结果提示,这可能是一个研究赤霉素和细胞分裂素在花分化发育中作用的良好实验系统。为了完善这一实验系统,明确离体培养的大岩桐花萼切块培养物直接再生花芽的起源以及花分化的形态进程是十分必要的。为此我们对离体培养大岩桐花萼切块的形态变化在体视显微镜下进行了系统观察,并进行了石蜡切片及电镜扫描观察。进行大岩桐(Sinningia speciosa Hiern)花萼切…     

Histology of early somatic embryogenesis inHevea brasiliensis: The importance of the timing of subculturing

Somatic embryos ofHevea brasiliensis can be obtained by culturing thin sections of inner tegument of seed on two successive different media, MH1 and MH3. Histological study showed that in calli cultured on non-renewed medium MH1 for 40 days, the embryogenesis process initiated on the 20th day did not produce results owing to early degeneration of the cells involved in the embryogenic pathway. However, typical embryogenic cells formed when medium MH1 was renewed once during the first phase of culture (between day 20 and day 30). Proembryos developed when the calli were subcultured on medium MH3 10–15 days later. Embryogenic cells did not form when there was frequent renewal of medium MH1 or early subculturing on MH3 after less than 40 days of culture on MH1. Methodical histological monitoring of the development of embryogenic quality of calli thus made it possible to define the optimum culture sequences for the embryogenesis process and which are favourable for regular obtaining of proembryos.     

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32^°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.     

Medium-term preservation of mesophyll cells isolated from Asparagus officinalis L.: Development of a simple method by storage at reduced temperature

Simple conditions have been investigated allowing Asparagus officinalis L. mesophyll cells to be stored as high density suspensions with unimpaired growth capacities in cell cultures. In a classic cell culture medium, storage by itself affected the mesophyll cell population by a temperature-dependent increase of mortality. Long storage reduced the ability of living cells to enter the first mitosis when used as inoculum. This loss of mitotic capacity led to reduced growth capacities (i.e. reduced increase of the number of cells) of the cultures and was relieved by decreasing the temperature of storage. Storage at 4°C in a sucrose solution and in the usual Asparagus cell culture medium has been compared. The cell culture medium containing salts, sucrose and the hormones NAA, 6-BAP gave the best results. The effective period of cell preservation is defined as the period during which cultures could be obtained with recovery of mitotic activity and growth equaling at least 75% of the control. Even in the best conditions tested, this appeared variable and dependent upon the physiological age of the cladophylls from which the cells had been obtained. It was never less than 15 days and rose to 45 days when the cells came from young cladophylls.     

Development of highly nutritive culture media

Summary A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.     

Establishment and characterization of a diploid cell line from the tick, Dermacentor parumapertus Neumann (Acarina: Ixodidae)

Establishment of a continuous cell line (RML-14) from embryonic tissues of the tick Dermacentor parumapertus Neumann is reported. The culture medium employed consisted of a combination (2:1) of Eagle's and L-15 (Leibovitz) media supplemented with 20% fetal bovine serum, 10% tryptose phosphate broth, and 0.1% bovine plasma albumin. At the 8th passage, 99% of dividing cells had the female chromosome complement, among which more than 70% had a diploid chromosome number of 22. At the 13th passage, cell population showed approximately a 3-fold increase during the first 8 days of culture. As of December 1976, had been subcultured 40 times.     

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures.

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.     

Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

The nature of substrate-attached materials in human fibroblast cultures: localization of cell and fetal calf serum components.

Human fibroblasts have been used as an in vitro model to examine the morphology and origin of substrate-attached materials. In cultures of subconfluent cells, no ‘tracks’ or ‘pools’ of material could be detected on substrata by anodic oxide interferometry or electron microscopy. However, a continuous layer of densely staining material was present on Falcon plastic tissue culture dishes never exposed to cells or culture medium. Exposure of substrata to culture medium caused the adsorption of fetal calf serum (FCS) components onto the substratum within a few minutes. Although antigenic FCS components remained on the substrata for several days, they were seldom adsorbed to the cells. The hypothesis was formulated that adhesion was mediated by FCS components on the substrata, but not by cellular materials deposited extracellularly. Support for this hypothesis was obtained by studying serum-dependent differences in cell adhesion. Fibroblasts subcultured in the presence of FCS components were usually separated from the substratum by a distance of at least 30 Å. In the absence of FCS components, the cells were more closely adherent, in the range at which the near van der Walls forces were effective. Fibroblasts subcultured in the absence of serum components could be removed readily from the substratum, leaving lsfootprints’ of cell surface material behind. Although this material has been prepared similarly to ‘microexudates’ from other types of cultured cells, its relationship to those microexudates has not been determined.     

Group locomotion of PtK1 cells

PtK1 cells were found to form groups of variable size which locomoted in unison. A continuous spectrum of net displacements during two days of observation ranged from no displacement to about 150 μm/day. The majority of single cells migrated less than 50 μm/day. The phenomenon demonstrates in tissue culture the existence of a form of cellular migration which appears intermediary between single cell locomotion and the deformations of extended sheets of cells [8] in the sense that more than one single cell but less than an entire sheet displace themselves. It also suggests coordination of locomotion between individual cells.     

Hepatopancreas cell cultures from mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3 d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.     

Cellular activities associated with the transition of chick embryo fibroblasts from stationary to proliferation state

Chick embryo fibroblasts on the 5th day of culture in proteinfree medium were stimulated to accelerated growth by supplementation of the medium with ATP (50 mumol/l, insulin (0.16 I.U./ml) or chick serum (5% v/v). Kinetics of the entry of cells into the S phase and later into the logarythmic phase of growth were found to be different in cultures treated with these three factors in spite that the final saturation densities reached after 30 days of culture were similar. No direct correlation between cell spreading and the cell transition from the stationary to the proliferation state was found. The proliferating cells showed the higher rate of locomotion than in stationary cultures. The initial protein-free medium, supporting the long survival of chick embryo fibroblasts and their susceptibility to growth accelerating factors, was further simplified by replacement of ADA buffer with EDTA (0.4 mmol/l).     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.     

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-β1 with time in culture

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-β1 (TGF-β1) when treated within 1–2 days after plating. The purpose of the present studies was to examine the effects of TGFβ1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGFβ1 decreased dramatically as the cultures aged. The IC₅₀ for inhibition of colony forming efficiency was 0.18 pM when TGFβ1 was added 24 h after cell plating. When TGFβ1 treatment was begun on day 5 of culture, the IC₅₀ was 3–4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGFβ1 was begun on day 19, the IC₅₀ was 65 pM or > 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGFβ1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGFβ1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGFβ1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGFβ1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGFβ1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-α, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGFβ1 by about twofold at both early and late times in culture. This indicates that the changes in TGFβ1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events. © 1992 Wiley-Liss, Inc.     

Choline acetyl transferase activity in chick embryo neuroretinas during development in ovo and in monolayer cultures

The specific activity of the enzyme choline acetyl transferase (CAT) in chick neuroretinas was investigated during in ovo development and in monolayer cultures. The enzyme activity was barely detectable on the 6th day of incubation but increased markedly between the 7th and 11th days. The activity increased sharply between the 15th and 17th days and then slowly until hatching. When cell suspensions from 6- to 7-day neuroretinas were cultured as monolayers, CAT specific activity increased rapidly. After 4–5 days in culture, the activity of the enzyme was identical to that found in the neuroretina on the 11th day of incubation. Cells from 9-day neuroretinas also differentiate in monolayer cultures, but with a more irregular pattern. These data show that cholinergic neurons from chick embryo neuroretina differentiate in monolayer cultures without a lag and at the same rate as in vivo.