Trout hepatocyte culture: Isolation and primary culture

Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×10⁶ liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep ₄₅₀ activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep ₄₅₀ activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture. This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support Grant 5507RR05700010.     

Anther and microspore culture ofLupinus albus in liquid culture medium

Embryo-like structures (ELS) with clearly defined cotyledons and radicles have been regenerated fromLupinus albus L. microspores. ELS induction from microspores in liquid medium was successful, with a maximum of over 3,500 ELS per anther being produced from microspores predominantly at the early binucleate stage of development. Cytological analysis revealed that first pollen grain mitosis occurred in closed buds with maximum ELS production being obtained from buds at the point of first petal emergence. Generally there was a lack of synchronisation of microspores within anthers from the less mature bud germination from ELS has not been observed; recurrent somatic embryogenesis occurred following internal cleavage within the ELS or on the surface of the ELS. Methods of increasing the level of mature ELS capable of germination are under investigation.Abbreviations BA 6-benzyladenine - 24-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 2iP N-isopentyl-adenine - GA₃ gibberellic acid - S&H Schenk & Hildebrandt medium (1972) - M&S Murashige & Skoog medium (1962) - N&N Nitsch & Nitsch medium (1969) - B5 Gamborg's B5 medium (1968) - NNB5 N&N salts plus B5 vitamins - ELS Embryo Like Structures - DAPI 4, 6-Diamidino-2-Phenylindole     

Relationship between culture redox potential and culture fluorescence inCorynebacterium glutamicum

Summary Transient experiments were performed during a batch fermentation ofCorynebacterium glutamicum ATCC 14296 in which the flow of air to the reactor was cut off. The fluorescence of the culture was observed to vary linearly with the culture redox potential during the transient. This observation was made below the sensitivity limit of a dissolved oxygen probe. It appears that redox potential in conjunction with fluorescence measurements are of utility in studying extra- and intracellular reduction states, particularly in the microaerobic regime.     

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Hydrobiologia - Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20 000 tons...     

Microcarrier culture of fish cells and viruses in cell culture bioreactor.

This article deals with the culture of grass carp (Ctenopharyngodon idellus) lip and embryo cells on Cytodex 3 and GT-2 microcarriers in a 1.5-L cell culture bioreactor to propagate grass carp hemorrhage virus. The cells and viruses were successfully cultivated at 26 degrees C, pH 7.0, and dissolved oxygen 40% of air saturation. The cell density achieved was as high as 7.4 x 10(6) cells/mL, and the virus titre reached 6.75 log LD50/0.5 mL from an initial 3.00 log LD50/0.5 mL. The results present broad prospects for fish virus vaccine production.     

Biotechnology and fish culture

Biotechnology can currently be considered of importance in aquaculture. The increase in the production of aquatic organisms over the last two decades through the use of biotechnology indicates that in a few generations biotechnology may overtake conventional techniques, at least for the commercially more valuable species. In the last few years, genetics has contributed greatly to fish culture through the application of the more recent techniques developed in biotechnology and in genetic engineering. At present, the most commonly used methods in fish biotechnology are chromosome manipulation and hormonal treatments, which can be used to produce triploid, tetraploid, haploid, gynogenetic and androgenetic fish. These result in the production of individuals and lineages of sterile, monosex or highly endogamic fish. The use of such strategies in fish culture has as a practical objective the control of precocious sexual maturation in certain species; other uses are the production of larger specimens by control of the reproductive process and the attainment of monosex lines containing only those individuals of greater commercial value. The use of new technologies, such as those involved in gene transfer in many species, can result in modified individuals of great interest to aquaculturists and play important roles in specific programmes of fish production in the near future.     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

Fed-batch culture automated by uses of continuously measured cell concentration and culture volume

An automated control method of fed-batch culture in which the nutrient feed rate was determined from continuously measured cell concentration and culture broth volume was developed. Theoretical background was elucidated, from which it was found that the method is unique in that it controls specific substrate consumption rate of the microorganism. The method was experimentally applied to the fed-batch cultures of recombinant Escherichia coli HB101. It was observed that the specific substrate feed rate affects not only the specific growth rate but also the growth yield. If some conditions are satisfied, this type of automated fedbatch culture can be applied widely to any microbial systems and seems especially useful when the culture medium is composed of natural complex nutrient(s) because their concentrations are very difficult to detect and control.     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

Bringing culture back in: the class origins and ethnoracial destinations of culture and achievement

ABSTRACT

Notably absent from assimilation scholarship are analyses that seriously engage, rather than dismiss, cultural explanations for differences in group outcomes. That dismissal has left the assimilation scholars ill-equipped to thoroughly respond to popular and quasi-academic explanations that rely on an all-encompassing view of culture to explain immigrant group differences in socioeconomic outcomes. Jennifer Lee and Min Zhou’s The Asian American Achievement Paradox embraces the cultural explanation, but traces the root of an ethnoracial culture to its class origins. In so doing, Lee and Zhou force scholars to contend seriously with ‘culture’ as part of a larger explanation for group outcomes.     

A comparison of barley isolated microspore and anther culture and the influence of cell culture density

Comparisons were made between the efficiency of barley plant regeneration from anther culture (AC) and isolated microspore culture (IMC) for the European winter cultivar `Igri' and the spring F<sub>1</sub> Australian breeder's hybrid Amagi Nijo×WI2585. In both cases, IMC produced a higher number of green regenerant plantlets per anther than AC. For `Igri' there was a 100- to 200-fold improvement and for Amagi Nijo×WI2585 there was a five- to ninefold improvement of IMC over AC. To improve the consistency and reliability of the IMC method, we investigated several parameters, including maltose concentration, subculture protocol, microspore plating density and colony plating density. Subculturing during the liquid culture phase produced no significant improvement in the number of microspores developing into colonies. The optimal concentration of maltose in the liquid induction medium was found to be 90 g l^–1. Both microspore plating density and colony plating density were found to influence plant regeneration. Microspores produced the highest numbers of colonies when plated at densities greater than 5×10⁴ ml^–1, and colonies produced optimal numbers of green plantlets when plated at 12.5–25 colonies/cm². Received: 23 March 1997 / Revision received: 29 May 1997 / Accepted: 25 June 1997