Transplacental effect of urethane on DNA synthesis in mouse embryonal lung tissue during organotypical cultivation

Kinetics of DNA synthesis was estimated by autoradiography in the organ cultures of murine embryonic lung tissue. Strain A mice were taken for this experiment (intact and exposed to transplacental action of urethane--30 mg/g to a pregnant mouse, subcutaneously, once). The explants were examined 1,7, 15 and 21 days after the cultivation. Transplacental urethane inhibited DNA synthesis the first 24 hours. The label index became approximately 3 times less. The next two days it was restored and then stimulated. The maximal label index was noted in the experimental explants on the 7th cultivation day, i.e. on the 8th-10th day after the transplacental action of urethane on the lung tissue in utero. By the 15th cultivation day the label index dropped but was higher than in the intact explants; by the 21st day DNA synthesis in the intact cultures ceased completely, whereas in the experimental animals--it continued.     

Oxygen dynamics in choanosomal sponge explants

Oxygen microprofiles were measured over the boundary layer and into the tissue of 10-day-old cultivated tissue fragments (explants of 2-4 cm³) from the choanosome of the cold-water sponge Geodia barretti with oxygen-sensitive Clark-type microelectrodes. At this time of cultivation, the surface tissue and the aquiferous system of the explants is regenerating, which makes oxygen and nutrient supply by pumping activity impossible. Oxygen profiles showed a parabolic shape, indicating oxygen flux over a diffusive boundary layer and into the tissue. Oxygen was always depleted only 1 mm below the sponge surface, leaving the major part of the explants anoxic. Diffusive oxygen flux into the explant was calculated from three oxygen profiles using Fick's first law of diffusion and revealed 9 μmol O₂ cm^-3 day^-1, which is in the lower range of in situ oxygen consumption of whole sponges. The ability of G. barretti to handle continuous tissue anoxia enables choanosomal explants to survive the critical first weeks of cultivation without a functional aquiferous system, when oxygen is supplied to the sponge explant by molecular diffusion over its surface.     

Characteristics of the effect of exometabolites of the cysticerci of the cat tapeworm Hydatigera taeniaeformis on fibroblasts in organ cultures

The influence of the cestode H. taeniaeformis cysticercus exometabolites on the fibroblasts of their capsule in organ culture was studied. Half of explants were cultured in the presence of cestodes (the ratio between the parasite body weight and the cultural medium volume being 1:10 g/ml). After 2-30 days of cultivation the explants were examined by histological, histoautoradiographic and electron microscopic methods. The influence of cysticercus exometabolites on the culture resembled that of the growth-stimulating factors. The young, actively proliferating fibroblasts were mainly observed in the explants of the capsules cultured in the presence of cysticerci. Among the mature cells, fibroblasts with the predominance of the fibroclastic function were noticed rather than with the function of collagen biosynthesis. It is suggested that the change in the differentiation ways of fibroblasts may be due to some biologically active substrates of the cysticercus to be directed on slowing down the maturation of the connective tissue capsule.     

A Cellophane-Strip Technique for Culturing Tissue in Multipurpose Culture Chambers

A new technique for the cultivation of living tissues in the multipurpose culture chamber is described. This procedure employs strips of cellophane as the agent for anchoring tissue explants to the coverslip walls of the chamber and disposes of the time-honored plasma-clot technique. The primary advance embodied in this procedure lies in the fact that cells emigrating from so-cultured explants manifest themselves in a highly differentiated manner comparable to the cells of origin, whereas the outgrowth from the same types of tissue in plasma clots results in a more undifferentiated type of growth. Comparisons of outgrowths from embryonic thyroid, bone, and muscle (chicken) are photographically documented, and attention is called to certain cytochemical methods which further corroborate the differentiated quality obtained with the cellophane-strip technique.     

A cellophane-strip technique for culturing tissue in multipurpose culture chambers

A new technique for the cultivation of living tissues in the multipurpose culture chamber is described. This procedure employs strips of cellophane as the agent for anchoring tissue explants to the coverslip walls of the chamber and disposes of the time-honored plasma-clot technique. The primary advance embodied in this procedure lies in the fact that cells emigrating from so-cultured explants manifest themselves in a highly differentiated manner comparable to the cells of origin, whereas the outgrowth from the same types of tissue in plasma clots results in a more undifferentiated type of growth. Comparisons of outgrowths from embryonic thyroid, bone, and muscle (chicken) are photographically documented, and attention is called to certain cytochemical methods which further corroborate the differentiated quality obtained with the cellophane-strip technique.     

Silicone rubber membrane as a support for long-term cultivation and electron microscopic processing of nervous tissue

Silicone rubber membrane can be successfully used as a support for cultivation of nervous tissue; its processing for electron microscopy is described: The advantages of silicone rubber membrane as support, compared with glass, are. its biological inertness, the ability to withstand dry-heat sterilization at 160°C, transparency, easy processing for electron microscopy. Spatial configuration of cells and explants in specimens is preserved.     

The role of the mesenchyme in the development of the early primordium of the respiratory epithelium

Development of respiratory epithelium (RE) rudiment was studied in tissue culture after removal of mesenchyme (M) and respiratory tract (RT) in 13 days old embryos of A and C57BL mice. During long-term cultivation of intact RT, organotypic structures (branching bronchioles, alveolus-like cavities) developed. No epithelial organotypic structures developed in the presence of single M cells; explants were represented by layers of cubic epithelium. During long-term cultivation foci of atypical growth consisting of intensively proliferating basophilic cells with high nucleocytoplasmic ratio appeared in these explants. Regions of planocellular metaplasia with or without keratinization could be found in these foci. The frequency of atypical proliferates depended on the strain of donor mice and on the region of the explanted RT.     

Steady-state cultures of human skin

Pretransplantation cultivation of adult human skin has been optimized for rapid and prolonged outgrowth of epidermal cells from tissue explants using autonomic-perfusion, thin-layer culture technology (steady-state). This system fostered growth of autologous mesenchymal elements via critical control of the culture environment. The resulting cellular outgrowth maintained a balanced epithelial-dermal relationship, contained keratinocytes as well as minority epidermal cells, melanocytes and possibly Langerhans cells. Critical control of culture pH and osmolarity was found to enhance epithelial cell proliferation.     

Characterization of Calcium Deposition and Shell Matrix Protein Secretion in Primary Mantle Tissue Culture from the Marine Pearl Oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.     

Assessment of DNA methylation changes in tissue culture of Brassica napus

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rapeseed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.     

Structural aspects of the regulation of starch accumulation in stem pith explants of kale

Anatomical aspects were studied of starch deposition in primary explants of stem pith of marrow-stem kale. During cultivation on semisolid media containing glucose and growth substances starch is accumulated in the inner part of the explant but absent on its periphery. The starch localization pattern formed within originally quite homogeneous tissue seems to be caused both by gradients of substances absorbed from culture medium and by physiological gradients manifested in the explanted tissue. Strange enlarged grains were abundant in cells with high starch content.     

Influence of genotype,explant type,and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.)

The influence of explant type as well as of the type of growth regulators and concentration on callus induction and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties have a substantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the genotypes studied during the cultivation of cotyledons on Murashige-Skoog culture medium containing 2 mg/L of zeatin in combination with 0.1 mg/L of 3-indoleacetic acid. An effective protocol of indirect somatic organogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was developed.     

组织培养条件下喜树叶片细胞壁酸性降解的pH值观察

喜树叶片外植体经组织培养第13 d和23 d后进行解剖学观察,同时比较正常叶片的解剖学特征,发现在pH值为5.8的酸性培养基中,喜树叶片外植体中的海绵组织和栅栏组织等薄壁细胞相继发生明显的细胞壁降解,而表皮细胞发生较微弱的细胞壁降解现象;进行组织培养前,正常叶片的各类细胞未观察到细胞壁降解现象发生。应用BCECF-AM pH荧光探标记并采用激光共聚焦在480 nm波长下进行pH值测定发现,喜树叶片外植体经组织培养第13 d和第23 d后的海绵组织和栅栏组织等薄壁细胞部位的pH值均为5.2,但表皮细胞部位的pH值则为5.7~5.8,而正常叶片各类细胞的pH值平均为5.7。这说明, pH值为5.8的酸性培养基和喜树叶片薄壁细胞内的酸性成分自泌可能共同诱导了其细胞壁的酸性降解。     

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.     

Induction of alpha- and beta-crystallin synthesis in organ cultures of the adenohypophyseal anlage of chickens as affected by 5-iododeoxyuridine and 5-bromodeoxyuridine

The effects of 5-iododeoxyuridine and 5-bromodeoxyuridine on differentiation of the cells of adenohypophysis rudiment from 3, 4, and 5 day old chick embryos were studied in the in vitro organ culture. On the 7th day of cultivation most explants from 3 and 4 day old embryos formed lentoids and individual cells with the lens phenotype among the adenohypophysis tissue. Alpha-, beta- and delta-crystalline were immunochemically detected in them. When cultivating explants from 5 day old embryos, no lentoids formed. But the immunochemical study of serial sections made it possible to detect in individual explants single alpha-crystalline-containing cells. There is a period in the development of chick adenohypophysis, which lasts five days of incubation and during which the adenohypophysis rudiment retained its capacity for lens differentiation despite the fact that it is already determined in the adenohypophysis direction.     

Effect of thymus polypeptide fractions on the development of rat thymus and spleen in organ culture

Peptides of the thymus--vilon, thymogen and thymalin, alone or in combination with concanavalin A, were used to investigate their effect on organotypic culture of thymus and spleen explants from 1- and 21-day old rats. Vilon, thymogen and thymalin in concentrations of 2 and 10 ng/ml and 5 ng/ml, resp., exerted stimulating effects in thymus and spleen tissue cultures from 21-day old rats as compared to the control explants. Vilon and thymogen showed inhibiting effect in the thymus tissue cultures from 1-day old rats as compared to the control explants. However, the peptides together with concanavalin A in concentration of 10 mkg/ml resulted in decreasing the action of concanavalin A alone. The polypeptide fractions of thymus and their synthetic analogs play different roles in the regulation of thymus and spleen development in rats of different age.     

巴戟天组织培养和快速繁殖研究

以巴戟天顶芽及嫩茎节段为外植体,以MS为基本培养基,通过不同的激素种类和浓度配比,建立巴戟天组培快繁体系。结果表明,外植体表面消毒以70%酒精预处理60s,再用0.1%HgCl2浸泡10min,效果较好,茎节为外植体优于顶芽。培养基MS+BA1.0mg/L+IBA0.05mg/L利于诱导出芽,可用于初代培养;MS+BA1.0mg/L+IBA0.2mg/L利于形成丛生芽,用于继代增殖,繁殖系数6.0/50d;1/2MS+IBA0.4~0.8mg/L适宜诱导生根获得再生植株,生根率100%;生根苗移栽于排水良好的火土或砂土中,成活率90%。     

Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland.

Mouse mammary gland contains choline kinase activity that can be stimulated by polyamines. Developmental studies show that the activity of choline kinase in mammary gland is low in both virgin and nonpregnant primiparous animals but increases severalfold during pregnancy and reaches a maximal level during the lactation period. Similar increases in enzyme activity are observed by cultivation of tissue explants in the presence of insulin, cortisol, and prolactin, a combination of hormones which induces the ultrastructural and biochemical changes associated with the development of mammary gland during pregnancy and lactation. The increase in enzyme activity in cultured explants is dependent only on the actions of both insulin and cortisol and parallels the formation of rough endoplasmic reticulum, which is effected by the same combination of hormones. The hormonal stimulation of choline kinase activity appears to involve the action of spermidine, a polyamine which accumulates in the cells under the influence of cortisol and mimicks the effect of cortisol on milk-protein synthesis in cultured explants.     

Amino acid transport and protein synthesis in induced ectoderm of amphibian embryos

Summary Isolated gastrula ectoderm ofTriturus alpestris orAmbystoma mexicanum was induced by the vegetalizing factor. Protein synthesis in the induced and uninduced control explants was measured by double labelling with³H-and¹⁴C-amino acids after different periods of cultivation. Slight differences were observed in the pattern of nuclear proteins after 12 h of cultivation and in the pattern of cytoplasmic proteins after 48 h of cultivation.The uptake of leucine started to increase in induced explants after 48 h of cultivation and after 96 h was about 50 times greater than in uninduced control explants. The uptake is reduced under partially anaerobic conditions. Ouabain inhibits the uptake by about 50%.     

A Novel Method for Coral Explant Culture and Micropropagation

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5?C2.5?mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.