Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

Relationship between production of carrot somatic embryos and dissolved oxygen concentration in liquid culture

To evaluate the relationship between somatic embryogenesis and dissolved oxygen concentration, somatic embryo cultures of carrot (Daucus carota L.) were cultured under various dissolved oxygen concentration levels (bubble free aeration with 4%, 7%, 20%, 30%, and 40% oxygen in flasks). The system used allows dissolved oxygen concentration control without bubble aeration or mixing speed modification. The total number of somatic embryos was not affected by the dissolved oxygen (DO) concentration tested. Even if globular-stage embryos were induced at a low level of oxygen aeration, heart-stage embryo formation was still repressed. Oxygen enrichment (20%, 30% and 40% oxygen) enhanced torpedo and cotyledonary-stage embryo production. The oxygen-enriched aeration was effective in promoting the growth of the late developmental stages. Sugar consumption did not increase when the oxygen concentration was enriched above the ambient level. The number of heart-stage embryos increased as oxygen concentration increased up to the 7% level, while above the 20% level no change in production was observed. The production of cotyledonary-stage embryos was directly related to oxygen concentration. These results support that oxygen-enriched aeration provides oxygen to the low oxygen areas in somatic embryo. After the heat-stage embryos, which were grown at the 7% level were transferred to a flask with ambient, they developed an elongated root part and eventually grew to normal plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of single heart cells from chick embryos for the Na+ current measurements

To record the fast Na⁺ current, spheroidal heart cells enzymatically-dispersed from 3 18-day-old chick embryos were used for voltage clamping. The peak of currents in response to voltage steps of 200 ms long from holding potentials of -90 -105 mV were measured. The current-voltage curves for the peak inward current showed U-shaped relations; the averaged peak current of about -1400 pA was observed at about -30 mV and the current reversed sign at +40 + 50 mV. Both the peak current and the reversal potential values showed marked [Na]_o- dependence, i.e. reduced by 36% and by 20 mV, respectively, for a halved [Na]_o. Tetrodotoxin (TTX) partially (10^-6 M) or completely (10^-5 M) suppressed the current. The steady-state inactivation of the current (h) was characterized by the half inactivation voltage of around -80 mV and the slope factor of -4 -8 mV. The half activation voltage and the slope factor for the steady-state activation (m) were -55 mV and 4-6 mV, respectively. The electrophysiological and pharmacological properties were similar between young (3-day-old) and old (15-18-day-old) embryonic heart cells, excepting the much smaller current and the slower onset of TTX action in young embryonic hearts.     

Physico-chemical properties of the encapsulation matrix and germination of carrot somatic embryos

Carrot somatic embryos were encapsulated in alginate gel beads. To improve the quality of a "synthetic seed" coating, the rheology and dehydration properties of different matrices were tested. By increasing alginate and CaCl(2) concentrations, additional mineral elements were shown to increase resistance to rupture, and to depress the germination of somatic embryos. A polysaccharide addition was found to slow the alginate matrix dehydration; alginate-gellan gum and alginate-kaolin matrices could preserve the viability of somatic embryos at low relative humidities (30% to 35% germinations at 50% relative humidity) to a greater extent than other matrices. (c) 1995 John Wiley & Sons, Inc.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.     

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However,in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.     

Isolation and culture of neural crest cells from embryonic murine neural tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from protocols optimized for the culture of rat NC. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC.     

Primary cell culture from embryos of the Japanese scallop Mizuchopecten yessoensis (Bivalvia)

Primary cell cultures obtained from embryos of Mizuchopecten yessoensis (Bivalvia) survived for four months. Although the number of cells progressively decreased during the cultivation, mitotic cells were observed both at the first stages and at the end. A possibility of growing marine invertebrates cells in long term primary culture is discussed.     

Stage-specific nitrogen metabolism in developing carrot somatic embryos

The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and ¹⁴C-labeling. ¹⁵N NMR spectroscopy was used to examine the uptake and incorporation of ¹⁵N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α-amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non-embryogenic cells and germinating embryos were glutamine and α-amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y-aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon-14 labeling indicated that labeled glutamine pools in non-embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non-embryogenic cells and germinating stage embryos. Furthermore, during early to mid-embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.     

Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro culture of blood cells from the colonial protochordateBotryllus schlosseri

Summary Primary cultures of circulatory blood cells from the colonial tunicateBotryllus schlosseri were cultivated in 96-well plates for up to 3 mo. in a medium based on Dulbecco’s modified Eagle’s medium, supplemented with salts to the botryllid ascidian hemolymph osmolarity, HEPES buffer,l-glutamin, fetal bovine serum, and antibiotics. Intercellular bridges between granular pigment cells were established within 24 h. The viability of these cells decreased slowly, and most died within 1 mo. without any sign of cell proliferation. Other cell types remained in an arrested state and were subjected to a weekly medium exchange. Spontaneous cell proliferation was randomly recorded in 6 to 10% of the wells from 2 wk to 1 mo. This proliferation was followed by the formation of masses of cell clumps, from which uniform hemocytes (5 μm, lymphocytelike cells) migrated peripherally. Stress conditions, which included longer intervals between medium exchanges and partial medium replacement, increased the probability of cell proliferation. From each proliferating primary culture, we successfully performed up to 10 plating cycles over a period of 15 wk, during which the cells differentiate in size but are uniformly structured. This produced the firstBotryllus lymphocytelike cell line. From this stage, cell numbers remained constant for up to 6 mo. without increase in cell number. Several mitogenic factors were employed on primary cultures.Botryllus and sea cucumber hemolymphs and mixed interleukins were found to augment significantly proliferation of at least one specific cell size, whereas cells were not markedly responsive to lectins (Concavalin A, wheat germ agglutinin,Ulex europaeus agglutinin), insulin, and retinoic acid. The results are discussed with respect to future efforts in the development of tunicate blood cell cultures.     

Purification,immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells

A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium. An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall. EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells. Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident. The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells. In seedlings, EP4 proteins were mainly found in roots. EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence. The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.DLO Centre for Plant Breeding and Reproduction Research (CPRO-DLO)     

Persistence of the developmental block of in vitro fertilized domestic cat embryos to temporal variations in culture conditions

A series of studies examined the influence and temporal interaction of energy substrate, media complexity, and tissue co-culture on the development of in vitro fertilized cat embryos and the persistence of the morula-to-blastocyst developmental block. In Study I, oocytes were fertilized and cultured for 144 hr in a simple culture medium (modified Krebs Ringer bicarbonate; mKrb), containing either glucose or glutamine, or cultured in mKrb w/ glutamine for the initial 72 hr with transfer to mKrb w/ glucose for the final 72 hr. Fertilization rate, percent development to morulae, and cell number per embryo were similar (P > 0.05) between treatments and blastocyst formation was universally low (<10%). In Study II, oocytes were fertilized and cultured in either mKrb (w/ glucose or glutamine) or in a complex medium, Ham's F10 (w/ 10% fetal bovine serum [FBS]). After 72 hr of initial culture, embryos in mKrb were transferred into Ham's F10. Fertilization rate was lower (P < 0.01) in Ham's F10 but embryo development to the morulae stage and cell number per embryo were comparable (P > 0.05) for all treatments. A higher percentage of blastocysts and morulae becoming blastocysts were observed after initial culture in mKrb w/ glutamine than after initial culture in mKrb w/ glucose. In Study III, oocytes were fertilized and cultured initially in mKrb (w/ glutamine), and then switched to either Ham's F10 or cat oviductal cell monolayers (in Ham's F10). Additional embryos were cultured exclusively in Ham's F10 or on cat oviductal cell monolayers. Fertilization rates were lower (P < 0.05) on oviductal cells but cell number per embryo was similar (P > 0.05) in all treatments. Blastocyst formation was lower (P < 0.05) on oviductal cells than in mKrb-Ham's F10 treatment and was <20% in all treatments. In summary, while in vitro fertilization-derived cat embryos develop to morulae under a variety of culture conditions, the morula-to-blastocyst developmental block was minimally responsive to alterations in energy substrate and medium complexity or fluctuations in their temporal availability. In addition, oviductal cell culture, alone or in combination with other culture variations, was ineffective in overcoming the developmental block. © 1996 Wiley-Liss, Inc.     

Isolation and characterization of mouse neural precursor cells in primary culture

Summary Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-d-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system. This study was entrusted to the Institute of Physical and Chemical Research (RIKEN) by the Science and Technology Agency (STA) and was financially supported by the Special Coordination Funds for Promoting Science and Technology. R. Shiurba is a recipient of a STA fellowship award from the Japanese Union of Scientists and Engineers.     

A medium and simplified procedure for growing single cells from Solanum species

A nutrient medium that allows rapid growth of calli from individual cells of several Solanum species has been developed. The medium is based upon that of Kao and Michayluk (Planta, 126 (1975) 105–110). Modifications that improve plating efficiencies at low density include omission of pyruvate, malate, citrate and fumarate and increasing the phosphate level from 1.25 to 5 mM. The inclusion of 0.1–0.2% bovine serum albumin was essential for growth at low density. At a plating density of 80 protoplasts/ml, plating efficiencies of 1.5—2.0% for Solanum tuberosum L. and S. cardiophyllum Lindl. are often obtained. Single cells of these species were mechanically isolated after 48 h of culture at 800 or 8000 protoplasts/ml and plated singly on fresh medium. The single cells divided and formed rapidly-growing celli with plating efficiencies of 37–75%. Plants have been regenerated from these calli.     

Isolation and long-term culture of rat,rabbit, and human nasal turbinate epithelial cells

Summary Nasal turbinate epithelial cells were isolated from rats, rabbits, and humans using either a surgical or an in situ enzyme incubation technique. The culture conditions that permit optimal cell attachment and selective growth of the nasal epithelial cells were determined. These conditions will permit the long-term culture of these cells where typically 20 to 30 population doublings were observed. Differences between rat and human nasal epithelial cells were seen in substrate requirements, colony-forming efficiency, and response to fetal bovine serum and bovine serum albumin. These methodology and results will permit mechanistic studies of normal and abnormal cellular function and comparative response studies between nasal epithelial cells from rats and humans. This work was supported under U.S. Environmental Protection Agency contract 68-02-4032.     

Isolation and culture of rat microvascular endothelial cells

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.     

大鼠应激耐受多系分化细胞的分离、扩增改良技术

目的 探讨体外从大鼠骨髓间充质干细胞（BMSCs）中分离应激耐受多系分化细胞（Muse cells）及其扩增培养的技术方法.方法 分离、扩增、鉴定BMSCs,应用长时间应激方法酶消化分离Muse细胞,悬浮扩增培养4d后Western blot鉴定特异性抗原.结果 第3代生长良好的BMSCs中表达CD29、CD90而不表达CD45的占细胞总数的97.89%.长时间酶消化结合悬浮培养后,以酶消化8h组细胞表达CD105显著高于其余各组（P〈0.05）,符合Muse细胞特征.结论 Muse细胞可由BMSCs经消化分离扩增获得,可望为其进一步研究应用奠定基础.