Explant organ culture: A review

Organ explant culture models offer several significant advantages for studies of patho-physiologic mechanisms like cell injury, secretion, differentiation and structure development. Organs or small explants/slices can be removed in vivo and maintained in vitro for extended periods of time if careful attention is paid to the media composition, substrate selection, and atmosphere. In the case of human tissues obtained from autopsy or surgery, additional attention must be paid to the postmortem interval, temperature, hydration, and cause of death. Explant organ culture has been effectively utilized to establish outgrowth cell cultures and characterize the histiotypic relationships between the various cell types within an organ or tissue.J. Resau is a visiting scientist at the NCI-LMO-DCE in Frederick, MD 21702, U.S.A.K. Sakamoto is a visiting scientist from the Department of Surgery, Gunma University School of Medicine, Maebashi, Japan     

Passive release of monoclonal antibodies from hybridoma cells

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed.Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted.Abbreviations MCAB monoclonal antibodies - PBS phosphate buffered saline - RT room temperature - SDS sodium dodecyl sulphate     

Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels

Summary A new low shear stress microcarrier culture system has been developed at NASA’s Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.     

Insect fungal symbionts: A promising source of detoxifying enzymes

Summary Many species of insects cultivate, inoculate, or contain symbiotic fungi. Insects feed on plant materials that contain plant-produced defensive toxins, or are exposed to insecticides or other pesticides when they become economically important pests. Therefore, it is likely that the symbiotic fungi are also exposed to these toxins and may actually contribute to detoxification of these compounds. Fungi associated with bark beetles, ambrosia beetles, termites, leaf-cutting ants, long-horned beetles, wood wasps, and drug store beetles can variously metabolize/detoxify tannins, lignins, terpenes, esters, chlorinated hydrocarbons, and other toxins. The fungi (Attamyces) cultivated by the ants and the yeast (Symbiotaphrina) contained in the cigarette beetle gut appear to have broad-spectrum detoxifying abilities. The present limiting factor for using many of these fungi for large scale detoxification of, for example, contaminated soils or agricultural commodities is their slow growth rate, but conventional strain selection techniques or biotechnological approaches should overcome this problem.Presented at the Symposium on Fungal Detoxification at the 48th Annual Meeting of the Society for Industrial Microbiology, Philadelphia, PA, August 4–9, 1991.     

玉米未成熟胚乳愈伤组织和器官的发生及其倍性变化的初步研究

本试验在附加和不附加外源激素的MS培养基上,均得到了玉米未成熟胚乳愈伤组织。愈伤组织在附加外源激素的MS培养基上达到器官分化,获得了发育正常的和许多畸形的胚乳植株。所得到的愈伤组织细胞和植株根尖细胞染色体数目和倍性是不稳定的,二者有相同的趋向,其中有整倍体的细胞(2n=10,20,30,40,50),也有各种非整倍体的细胞(2n=5—49)。     

香菇和侧耳属间原生质体的电融合研究

从培养在液体培养基中的香菇、美味侧耳和平菇的单核菌丝用酶法分离了原生质体。施加0.5MHz、500PV/cm的正弦波和μs、6000PV/cm方形脉冲的电场诱导下使其电融合。电融合后的融合子和原生质体在固体培养基上植板培养成菌落。在显微镜下检查融合子菌株菌丝的锁状联合选出从融合子长成的菌株。香菇和美味侧耳的融合菌株产生频率为61.53%,香菇和平菇的融合菌株产生频率为32.58%。根据融合菌株与亲本的拮抗作用和他们的过氧化物同工酶和酯酶同工酶的电泳酶谱与其亲本酶谱的不同,证实这些融合菌株是从融合的异核体生长成的。同时讨论了电融合方法和结果。     

Kinetic studies of phenol degradation by Rhodococcus sp. P1 II. Continuous cultivation

The degradation of phenol by Rhodococcus sp. P1 was studied in continuous culture systems. The organism could be adapted by slowly increasing concentration, step by step, up to 30.0 g · 1^-1 phenol in the influent. The degradation rate reached values of about 0.3 g · g dry mass^-1 ·h^-1. Large step increases in phenol concentration and addition of further substrates (e.g., catechol) were tolerated up to a certain concentration. With increasing dilution rate and increasing inlet phenol concentration the stability of the system decreased.     

Manganese as a controlling factor in mixed cultures of Lactobacillus plantarum and enterobacteriaceae

Addition of manganese, at levels of 50 ppm, to a liquid growth medium simulating adverse silage conditions had no effect on the growth or on the fermentation pattern of Enterobacter cloacae and Proteus vulgaris. Yet, the manganese strongly enhanced the growth of Lactobacillus plantarum. Co-cultures of L. plantarum and E. cloacae or P. vulgaris were, by addition of manganese ions, significantly altered in the favour of the former. This finding can be of use in mixed cultures where Enterobacteriaceae act as spoiler microorganisms.     

Formation of Hassall's corpuscles in vitro by the thymic epithelial cell line IT-26 R 21 of the rat

Summary In the monolayer of an established epithelial cell line from the rat thymus, IT-26R21, characteristic cell aggregates quite similar to Hassall's corpuscles were formed. These aggregates were examined by light and electron microscopy, and immunohistochemically. Their interpretation as Hassall's corpuscles is based on the following observations: (1) The aggregates are formed in the monolayer of cells that greatly resemble medullary epithelial cells of the thymus. (2) They consist of flattened epithelial cells in a concentric pattern with one or more degenerating cells in the center. (3) Loss of microvilli suggests that these cells are keratinizing. (4) The aggregates show strongly positive reactions in immunofluorescent staining with antikeratin and antiprekeratin.When Hassall's corpuscles increase in size, cellular proliferation is somewhat suppressed. Both in vivo and in vitro, they may be interpreted as an expression of a changing growth pattern in confined spaces and thus seem to have little immunological function.     

Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero

Summary Ornithine decarboxylase activity was measured during organogenesis in rat embryos grown in utero and whole rat conceptuses maintained in an in vitro culture system. Ornithine decarboxylase levels in vivo showed a distinct peak at embryonic age 10.5 d. Despite identical morphology, protein content, crown rump length and numbers of somites cultured embryos displayed a different developmental pattern and possessed less than half the ODC activity of that in vivo. The data suggest that the normal embryonic programming of ODC activity is significantly altered by the culture environment and that further biochemical comparisons of embryos growing in utero and in vitro may be required to evaluate properly the applicability of this technique to detailed studies of teratogenesis and developmental biology. This work was supported by NIH-5-507-RR5359-17 and a 1980 Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation.