In vitro development of one-cell embryos from outbred mice: Influence of culture medium composition

Summary One-cell embryos from outbred mice (CF1, CD-1, and Dub:ICR) were cultured in various modifications of egg culture medium (ECM). The best development was observed in medium in which inorganic salts of modified T6 medium (mT6) replaced those of ECM. In this modification (TE), 66% of one-cell CF1 embryos developed into blastocysts, comared to 46 and 43% for ECM and mT6, respectively. Moreover, the cell numbers of blastocysts developing in TE (74.9±3.3) were higher than the cell numbers of those developing in ECM (55.1±2.4). The culture requirements of embryos varied between different stocks of mice: Fewer CF1 embryos developed to the blastocyst stage than either Dub:ICR embryos (90%) or CD-1 embryos (84%). Lowering the osmolarity of the medium from 300 to 280 mOsm, increasing the concentration of KC1 from 1.42 to 25 mM, or omitting lactate from the medium during Day 1 of culture did not further improve development of embryos, in contrast to previous reports. However, the time at which embryos were transferred to outgrowth medium influenced their postblastocyst development. The best development was observed when embryos were transferred on Day 4 of culture at the late morula-early blastocyst stage. This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, Washington, DC, contract DE-AC03-76-SF01012.     

Micropropagation of Sterculia urens Roxb. — an endangered tree species

An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl^–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds.     

Platelet calcium pump activity in essential hypertensives and their first-degree relatives

Intracellular free Ca²⁺ concentration has been shown to be elevated in platelets of patients with essential hypertension. This study was designed to characterize Ca²⁺-pump activity of the platelet membranes (surface and intracellular) in these patients. A double-blind study was carried out. Untreated and treated (on R-blockers) essential hypertensives were studied in comparison with normotensive control subjects. First degree blood relatives of essential hypertensives were also studied. The Ca²⁺-activation kinetics of the enzyme showed a significant decrease in the V_max. (for the plasma- and intracellular membranes) and Km (for the intracellular membranes) in the essential hypertensive patients. Increased platelet membrane cholesterol content was observed in these patients. Lowered Ca²⁺-efflux by Ca²⁺-ATPase may lead to elevated intracellular free Ca²⁺-levels in platelets of essential hypertensives. A lowered Ca²⁺-ATPase activity may emerge as a marker for essential hypertension.     

In vitro multiplication in liquid culture of Syngonium contaminated with Bacillus spp. and Rathayibacter tritici

Contaminated Syngonium clusters were multiplied in an air lift bioreactor in liquid medium containing sucrose with the medium being circulated through a sterilizing filter. After 30 days, the culture in filtered medium produced 19.5 shoot initials per gram fresh weight of inoculum compared to 8.7 shoot initials produced in unfiltered medium. Transfer to an elongation medium with 30 mg l^-1 Rifampicin produced shoots on 67% of the clusters, while transfer to elongation medium without Rifampicin poduced shoots on 40% of the clusters. Clusters grown for three subcultures in a reactor without medium filtration had lost their multiplication ability. Clusters grown for three subcultures in a reactor with filtration, however, continued to show a two-three fold increase in fresh weight and shoot production.Abbreviations MS Murashige and Skoog     

Stimulation of the growth and solamargine production by Solanum paludosum multiple shoot cultures using a new culture medium

Organogenic callus cultures of Solanum paludosum were obtained from root, hypocotyle and cotyledon explants of plantlets cultured in sterile conditions. These callus cultures developed multiple shoots which proliferated in Murashige and Skoog basal liquid medium. These multiple shoots produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits.The optimization of the macronutrient composition of the liquid medium was performed by a method derived from the plant composition. This approach results in the establishment of an appropriate medium (SPOM medium) suitable for the improvement of both growth and solamargine production by multiple shoot cultures of S. paludosum.     

Synthesis of 6-deoxy-5-thio-d-glucose

Three routes were investigated for the conversion of d-glucose into the title compound. In the first approach, reduction of the 5,6-thürane ring of 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene α-d-glucofuranose (17) as well as that of its 3-O-allyl derivative (13) with lithium aluminium hydride was investigated; 17 afforded the corresponding 6-deoxy derivative besides di-, tri-, and poly-mers, whereas only polymers were formed from 13. In the second approach, the oxirane ring of was reduced by sodium borohydride and the resulting 6-deoxy derivative was converted into the 5-thiobenzoate; the corresponding hex-4-enofuranose was formed as a byproduct. In the third approach partial mesylation of methyl 5-thio-α-d-glucopyranoside was attempted, but the 6-mesylate 27 could be isolated only in modest yield (28%) together with rearranged 2,5-thioanhydromannofuranoside derivatives. The mechanism of this rearrangement is discussed in detail. The 6-mesylate 27 was converted via the 6-iodo derivative into the title compound.     

Continuous,high capacity reconstitution of nutrient media from concentrated intermediates

We designed an Integrated Media Preparation System (IMPS) for continuous, on-line preparation of cell culture media and delivery to intermediate storage vessels or directly to a bioreactor. Key components of the IMPS include: a high precision, continuous fluid mixing device; formulation-specific liquid medium concentrates; validated process controls and membrane filtration; and automated dispensing into large volume flexible plastic containers. The IMPS system is designed to produce sterile, single-strength liquid medium from common raw materials at a delivery rate of 1000–3000 liters per hour and will manufacture homogenous batches from several thousand liters to over 60,000 liters. Fortified nutrient media prepared from multi-component 50X concentrates have been demonstrated to accelerate bioreactor seed chains, increase product yield, and reduce the overall manufacturing cost of nutrient medium. A productivity matrix will analyze the fully-loaded costs and contrast alternative methods for media preparation against projected biological yield.Abbreviations IMPS Integrated Media Preparation System - 50X Nutrient fluid components formulated at fifty-fold final use concentration - 1X Nutrient fluid formulated at final, single-strength use concentration - cGMP Current Good Manufacturing Practices - SCADA Supervisory Control and Data Acquisition - PLC Process Logic Controller - LTI Life Technologies, Inc. - WFI Water for Injection - CIP Clean in place - SIP Sterilize in place - HPLC High performance liquid chromatography - DMEM Dulbecco's Modified Eagle's Medium     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster

We have established a primary culture system for Drosophila eye imaginal discs. With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells. Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons. Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23°C. These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium (“4+5”); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS). Eye disc fragments survived in this medium for at least 20 days. Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20-hydroxyecdysone (20-HE) (1 μg/ml), whereas neurite outgrowth was seen in the absence of 20-HE. Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro. Eye discs from 5-h pupae (P+5) or older commenced ommachrome synthesis in vitro, in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies. Average neurite length was not affected by age among pupae younger than P+5; but neurite outgrowth from P+24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro. Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro. Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro. Morphogenetic changes were manifested in cultures. Viability tests with calcein AM and ethidium bromide revealed few dead cells in living cultures. © 1995 John Wiley & Sons, Inc.     

肾综合征出血热人体组织中病毒包涵体的性质及意义的进一步研究

应用免疫组化、原位分子杂交、电镜及免疫电镜等方法进一步对肾综合征出血热人体尸检组织中病毒包涵体（ＩＢ）的抗原、核酸性质和超微结构特点作了进一步观察。结果,在３９例中的２０例尸检病例组织中显示出病毒核蛋白抗原和血凝素抗抗原阳性的ＩＢ,其中包括１６例陕西尸检病例组织中的６例休克期和１例多尿期病例,１７例上海病例中的３例休克期和９例少尿期病例及３例江西病例中的１例休克期病例。ＩＢ主要分布在呼吸道和肺泡、肾远曲小管和集合管、胃肠道、腺垂体、扁桃体、胰腺、前列腺等组织的粘膜上皮和腺上皮细胞及肝细胞和睾丸生精上皮细胞胞浆中,阳性细胞形态基本正常。应用原位分子杂交,可在该组细胞小同时检测到病毒ＲＮＡ,多为胞浆内弥漫阳性,仅少数组织中显示出病毒ＲＮＡ阳性ＩＢ结构。电镜观察阳性组织细胞中出现由大量微丝微管及颗粒样结构组成的ＩＢ结构,其小的病毒颗粒状结构、内质网及纤维丝状结构呈病毒抗原阳性,上述结构位于高尔基体区。结果说明该病毒有感染上皮细胞的特性,对其宿主细胞的致细胞病变作用是极其温和的,且多表现在亚细胞水平。ＩＢ可能是病毒过量表达抗原的堆积或病毒复制部位,而微丝微管结构可能参与病毒的感染过程。