Two-helper-strain co-culture system: a novel method for enhancement of 2-keto-l-gulonic acid production

A novel two-helper-strain co-culture system (TSCS) was developed to enhance 2-keto-l-gulonic acid (2-KLG) productivity for vitamin C production. Bacillus megaterium and B. cereus (with a seeding culture ratio of 1:3, v/v), used as helper strains, increased the 2-KLG yield using Ketogulonigenium vulgare compared to the conventional one-helper-strain (either B. cereus or B. megaterium) co-culture system (OSCS). After 45 h cultivation, 2-KLG concentration in the TSCS (69 g l^?1) increased by 8.9 and 7 % over that of the OSCS (B. cereus: 63.4 g l^?1; B. megaterium: 64.5 g l^?1). The fermentation period of TSCS was 4 h shorter than that of OSCS (B. cereus). The increased cell numbers of K. vulgare stimulated by the two helper strains possibly explain the enhanced 2-KLG yield. The results imply that TSCS is a viable method for enhancing industrial production of 2-KLG.     

Mammalian embryo co-culture: trials and tribulations of a misunderstood method

Embryo-somatic cell co-culture was devised over 40 years ago in an attempt to improve the development and viability of mammalian preimplantation embryos generated and cultured in vitro. While initial endeavours were successful in this respect, other studies soon highlighted a number of significant long-term detrimental impacts of this approach. Surprisingly little is known about the mechanisms underlying the beneficial effects of co-culture, although the production of embryotrophic compounds, modulation of nutrient profile, protection against culture-induced stress and/or toxin clearance are all contenders. The extent to which the inadvertent exposure of embryos to serum accounts for many of these effects remains open to question. Although the popularity of somatic cell co-culture has recently declined in favour of the use of sequential media due to concerns associated with its risk of disease transmission and long-term sequelae, we argue that complete dismissal of this technique is ill advised, given that our limited understanding of basic somatic cell interactions has prevented us from fully exploiting its potential. In this respect, there is some merit in focussing future research strategies based on reconstructed maternal tract tissue. Although the use of co-culture in clinical practice is unacceptable and its implementation in domestic species for commercial purposes should be viewed with diffidence, this technique can still provide a wealth of information on the development of novel, more physiological embryo in vitro culture systems. The proviso for acquiring such information is to gain a fuller understanding of the culture requirements/biochemistry of somatic cells and their interaction with the early conceptus.     

Endosperm culture: a novel method for triploid plant production

Triploid nature of endosperm is the characteristic feature of angiosperms and is formed as a result of triple fusion. Present review discusses the morphogenic response and production of triploid plantlets by endosperm culture. Both mature and immature endosperm used for culture initiation responded differently in cultures. A key factor for the induction of cell divisions in mature endosperm cultures is the initial association of embryo but immature endosperms proliferate independent of embryo. In almost all the parasitic angiosperms, endosperm shows a tendency of direct differentiation of organs without prior callusing, whereas in autotrophic taxa the endosperm usually forms callus tissue followed by differentiation of shoot buds, roots or embryos. The endosperm tissue often shows a high degree of chromosomal variations and polyploidy. Mitotic irregularities, chromosome bridges and laggards are the other important characteristics of endosperm tissues. Triploids are usually seed sterile and is undesirable for plants where seeds are commercially useful. However, in cases where seedlessness is employed to improve the quality of fruits as in banana, apple, citrus, grapes, papaya etc. the induction of triploid plants would be of immense use. Triploid plants have more vigorous vegetative growth than their diploid counterparts. Hence, in plants where the vegetative parts are economically useful, triploids are of good use. This review focuses on the progress achieved so far in endosperm culture to obtain triploid plants.     

Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos

The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days?2?C8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P₄) were evaluated. The production of P₄ was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P₄, PGF_2??, and PGE₂ compared to fresh cell cultures. This enables the use of pools of frozen?Cthawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P₄ quantified in the medium, but had no effect on PGF_2?? and PGE₂ quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed.     

Rainbow trout leukocyte culture: a simplified method.

The addition of 10% fetal bovine serum to Leibovitz's L-15 culture medium resulted in marked growth of peripheral blood leukocytes from rainbow trout, Salmo gairdneri. Culture medium without serum or with 20% homologous serum did not induce substantial growth. In contrast to what has been reported by others, oxygenation of the culture medium was found not to be required for excellent cell growth.     

A method for obtaining a long-lived intestinal tissue culture

The interest in the studies of the intestines using the method of tissue organ culture has considerably grown in recent years. It can be explained by the great possibilities of obtaining unique data about the state of intestines in normal and pathological condition, e.g. malabsorption syndrome. The paper deals with the method modified by the authors to obtain long-living (24 hours) intestinal tissue organ culture. The investigations used bioptic sections which were obtained by jejunoscopy with spot biopsy of children suffering from intestinal malabsorption. The viability of the explants was proved by histological and histochemical tests. The promise held by the methods is emphasized.     

Growth of a facultative anaerobe under oxygen-limiting conditions in pure culture and in co-culture with a sulfate-reducing bacterium

Abstract The occurrence and properties were studied of glucose-metabolizing bacteria present in the anaerobic sediment 5–10 cm below the surface of an estuarine tidal mud-flat. Of all these bacteria (10⁴– 10⁵ per g wet sediment) 80–90% were facultatively anaerobic species. Chemostat enrichments on glucose under aerobic, oxygen-limited and alternately aerobic-anaerobic conditions also yielded cultures dominated by facultative anaerobes. One of the dominant species, tentatively identified as a Vibrio sp., was studied in more detail under oxygen-limiting conditions. Fermentative and respiratory metabolisms were found to operate simultaneously, and the ratio between the two was regulated by the extent of oxygen limitation. A small fraction of the acetate formed under such growth conditions was shown to be subsequently respired. A co-culture was established of the Vibrio sp. and a sulfate-reducing bacterium ( Desulfovibrio HL21 ) in an aerated chemostat. The importance of these observations is discussed in relation to the role of facultative anaerobes in anaerobic habitats.     

Optimal culture conditions for producing conjugated linoleic acid in skim-milk by co-culture of different Lactobacillus strains

The ability of different Lactobacillus strains to produce conjugated linoleic acid (CLA) was determined. Three species—Lactobacillus plantarum (Lp), Lactobacillus acidophilus (La) and Streptococcus thermophilus (St)—were co-cultured in a medium containing skim-milk supplemented with hydrolyzed safflower oil. This study was aimed at future applications in dairy products. The optimal operation parameters were established by response surface methodology. More CLA was produced by co-culture than by single strain culture. The CLA produced by co-culture of La and Lp (La–Lp) was more than that produced by La and St (La–St). Maximum CLA production of 316.52 μg/mL was obtained with La–Lp co-culture using a substrate concentration of 5.0 %, inoculum size of 5.0 %, an initial medium pH of 6.4 and a temperature of 36.4 °C for 48 h. To our knowledge, this is the first report in the literature of the use of co-cultures of La–St and La–Lp to produce CLA.     

一种新的稀释定量方法在标本检测中的应用

我们使用注射器装入小金属棒在磁力搅拌器上作连续定量稀释方法与传播的吸管或移液器稀释方法,用于含菌量大的粪便标本及含菌量较小的烧伤病人创面和血液标本进行标本细菌的定量分析,结果证实两种方法结果较准确,重复性好,且无统计学差异。     

Image processing: a non-destructive method for measuring growth in cell and tissue culture

The use of machine vision in evaluating callus growth directly in Petri dishes is reported for carrot cell suspension growing in the presence of increasing concentrations of various herbicides. Machine vision was much more accurate and sensitive than visual counting of the cell aggregates. Image processing also gives more detailed information than does counting because of its ability to distinguish between intermediate levels of callus growth.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - MS Murashige and Skoog - GR Gelrite     

The advantages of co-culture over mono cell culture in simulating in vivo environment

Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.     

Pineal gland cells in culture : Morphology, biochemistry, differentiation, and co-culture with sympathetic neurons

We present here the initial report of a method for reproducibly obtaining primary cell cultures from pineal glands of 2-day-old rats. During culture, the putative pinealocytes became associated with each other in “nests”. Cells in these nests displayed vesicle-crowned rodlets and cilia, which are fine structural features in vivo of pinealocytes from neonatal rats. Treatment of the cultured cells with either norepinephrine or dibutyryl-cyclic AMP (db-cAMP) resulted in an increase in the activity of serotonin N-acetyltransferase, a marker activity for pineal function. This stimulation could be blocked by either cycloheximide or actinomycin D, and norepinephrine stimulation was also blocked by -propranolol. Further, the pineal cell cultures were able to support the growth of dispersed cells of rat superior cervical ganglia and to allow neurite outgrowth in these co-cultures, though the presence of nerve growth factor (NGF) in the medium of these cultures could not be detected.     

Modular co-culture engineering,a new approach for metabolic engineering

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.     

Genome-scale metabolic modeling of a clostridial co-culture for consolidated bioprocessing

An alternative consolidated bioprocessing approach is the use of a co-culture containing cellulolytic and solventogenic clostridia. It has been demonstrated that the rate of cellulose utilization in the co-culture of Clostridium acetobutylicum and Clostridium cellulolyticum is improved compared to the mono-culture of C. cellulolyticum, suggesting the presence of syntrophy between these two species. However, the metabolic interactions in the co-culture are not well understood. To understand the metabolic interactions in the co-culture, we developed a genome-scale metabolic model of C. cellulolyticum comprising of 431 genes, 621 reactions, and 603 metabolites. The C. cellulolyticum model can successfully predict the chemostat growth and byproduct secretion with cellulose as the substrate. However, a growth arrest phenomenon, which occurs in batch cultures of C. cellulolyticum at cellulose concentrations higher than 6.7 g/L, cannot be predicted by dynamic flux balance analysis due to the lack of understanding of the underlying mechanism. These genome-scale metabolic models of the pure cultures have also been integrated using a community modeling framework to develop a dynamic model of metabolic interactions in the co-culture. Co-culture simulations suggest that cellobiose inhibition cannot be the main factor that is responsible for improved cellulose utilization relative to mono-culture of C. cellulolyticum.     

Fed-batch culture with a modified DO-stat method

To support a high growth rate of microorganism in fed-batch culture with high cell density, a modified DO-stat method was developed. In this method, an exponential substrate feed was coupled with the usual DO-stat method, i.e., a fixed amount of substrate per DO signal was exponentially fed to the culture based on the estimation of the substrate consumption rate and thereafter the feed was stopped in order to prevent the oversupply of substrate until an abrupt increase in the concentration of dissolved oxygen (DO) in the broth appeared. After that, the feed was started again and this cyclic operation was repeated throughout the cultivation. This method was applied to the fed-batch cultivation of ethanol utilizing yeast, Candida brassicae. At high cell densities (> 10 g/l), this modified method was more effective than the usual one in keeping a higher growth rate.     

Toxoplasma gondii maintenance in tissue culture: a new efficient method for culturing RH tachyzoites

We describe here a new tissue culture method for prolonged laboratory maintenance of tachyzoites of the highly virulent RH strain of Toxoplasma gondii. Using a rapidly proliferating murine tumor cell line (YAC-1), the method described is easy to perform and is as or more efficient (both in terms of yield and cost) than other traditional methods for maintenance of the parasite. Furthermore, upon prolonged maintenance (greater than 160 days) in YAC-1 tissue culture, the pathogenicity of the parasite, as well as its capacity to elicit an immune response, are comparable to that of organisms maintained in mice. We conclude therefore, that the method described herein is a suitable alternative to the traditional method of maintenance of virulent RH strain T. gondii tachyzoites.     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture

The expanded application of commercial bovine IVM, IVF, and IVC systems is dependent on the ability to produce embryos in culture that are capable of producing normal pregnancies. Because serum containing culture systems can induce neonatal and fetal problems there exists a definite need for a serum-free culture system that produces viable blastocysts. This study demonstrated that the physiological sequential media system G1.2/G2.2 could produce bovine blastocysts at rates equivalent to co-culture. Additionally, these blastocysts had equivalent or increased cell numbers and inner cell mass development. Blastocysts produced in the G1.2/G2.2 culture system produced pregnancies following both fresh transfer and cryopreservation at equivalent rates to co-culture. Finally, this study demonstrated that the media system G1.2/G2.2 could be used in a commercial OPU transfer program without any loss in the numbers of blastocysts produced or the numbers of pregnancies resulting following transfer from either fresh or cryopreserved blastocysts.     

Submerged organ culture: An improved method

Summary Explants of whole ovaries and oviducts from postnatal rats were completely submerged during cultivation as organ cultures in chemically defined medium. The oxygen concentration in the culture chamber was raised to over 90%, and the stainless steel platform, used for cultures at the surface of the medium, was abandoned and excluded from further use. Thus, all of the periphery of an explant had equal access to nutrients and oxygen. Throughout the ovarian explants the tissues appeared uniformly viable, and mitotic figures were distributed evenly. These observations are in contrast to those on organs cultivated at the surface of the medium where a lack of structural uniformity had been detected. The method has general application to a variety of organs.