The methylene blue colorimetric microassay for determining cell line response to growth factors

The validity of the methylene blue colorimetric microassay for determining the response of monolayers of human ovarian tumour cell lines to different growth factors was investigated. Linearity of the relationship between cell density and optical density was confirmed for each cell line (r=0.989–0.999,p<0.001), and when initial cell density was optimised to give exponential growth over the assay period, differences in response to medium supplements were obvious. The response of target cells to growth factors, obtained using the methylene blue assay, were compared with, and found to parallel, previously documented responses obtained non-colorimetrically. Thus Mink lung epithelial cells (MLEC) were inhibited by TG (Holleyet al., 1983), EGF had an inhibitory effect on A431 cells (Gill & Lazar, 1981; Barnes, 1982), and the mesothelial cell line showed a proliferative response to EGF and hydrocortisone (Connell and Rheinwald, 1983).The methylene blue colorimetric microssay was found to be a simple, reliable, sensitive method with low variability, for determining the response of cultured cells to growth factors.     

Enhancement of antibody production by growth factor addition in perfusion and hollow-fiber culture systems

The effects of the high-molecular-weight growth factors, transferrin and bovine serum albumin (BSA), on antibody production were analyzed quantitatively in continuous hollow-fiber cultivation over a period of 60 days. Transferrin enhanced cell growth but had no significant effect on the specific antibody production rate, whereas BSA significantly enhanced antibody production. The antibody production rate was increased 4- and 14-fold respectively by feeding BSA at 2 and 5 g L(-1) into the EC side of the system (the side connected to the cell-containing outer part of the hollow-fiber unit) compared with the production achieved without BSA. Addition of 5 g L(1) BSA into the IC side of the system (the side connected to the inner part of the hollow-fiber unit) resulted in a 2.5-fold increase in the antibody production rate. The effect of BSA was also analyzed using the perfusion culture system with a separation unit. When fresh medium containing either 2 or 5 g L(-1) BSA was fed into the reactor, both the specific growth rate and specific death rate increased, while the specific antibody production rate was increased 2- and 25-fold, respectively, by feeding BSA at these two concentrations compared with no addition. Comparing the two systems, the increase in the antibody production rate achieved with the hollow-fiber system was threefold greater than that in the perfusion culture system with the same concentration of BSA feeding. (c) 1995 John Wiley & Sons, Inc.     

Selective effect of the herbicide DCMU on unicellular algae — a potential tool to maintain monoalgal mass culture ofNannochloropsis

The selective effect of DCMU on photosynthetic activity and growth rate was examined in several marine unicellular algae:Nannochloropsis sp. (Eustigmatohyceae),Dunaliella salina (Chlorophyceae)Isochrysis galbana (Prymnesiophyceae) andChaetoceros sp. (Bacillariophyceae). DCMU at 10^–7 M caused an immediate decrease in the photosynthetic rate ofDunaliella andIsochrysis (about 50% inhibition), whereas 10^–6 M imposed 80% inhibition in the photosynthetic rate ofChaetoceros. InNannochloropsis the rate was affected only when DCMU concentration exceeded 10^–6M. Cellular growth rate of all studied algae was affected by DCMU in a similar manner to photosynthesis. The differential effect of DCMU was further examined in mixed cultures in whichNannochloropsis was cultivated together with an additional species simulating a contamination situation of aNannochloropsis culture. When DCMU was added at concentrations higher than 10^–7 M, the growth of the competing algae significantly decreased, whileNannochloropsis maintained a relatively high growth rate. Consequently, after a growth period of 4 to 7 days a clear domination ofNannochloropsis was observed. These results demonstrate that DCMU and probably other herbicides of similar characteristics can be used effectively as a selective tool to suppress contaminating unicellular algae in open ponds in order to maintain a monoculture ofNannochloropsis.     

新疆紫草细胞生产阶段培养中细胞生长及产物合成的研究I：悬浮培养

新疆紫草细胞生产阶段培养时生物产量随接种量的增加而增大,但生物量的倍增数并不增大。色素产量只在一定范围内与接种量成正相关关系,超过一定范围反而下降。当选定适当接种量时,加大培养基的浓度可提高色素产率。扩大培养规模,色素产量下降。     

茄子子叶和下胚轴的组织培养和植株再生

以4种茄子品种——六叶茄、七叶茄、九叶茄和长茄的子叶和下胚轴为外植体,探讨了不同生长调节物质对外植体分化的影响和不同品种、不同外植体以及外植体不同苗龄的分化能力差异。观察到外植体分化呈极性,并建立了4个品种茄子下胚轴的高效再生体系。     

Callus culture ofPterocladia capillacea (Rhodophyta) and analysis of cell wall polysaccharides

Calluses induced fromPterocladia capillacea have been kept in culture for more than three years. They exhibit a fast growth rate, owing to the release of single cells, which in turn develop into new callus. The effect of various media and culture conditions upon growth was investigated. In order to confirm the identity of the callus cells, a 0,45 mg incoculum was grown that yielded 15 g dried callus within six weeks. Polysaccharides from this material (5.5 g) were analysed by¹³C NMR spectroscopy. This produced a spectrum typical of agar and very similar to the one obtained for agar extracted fromP. capillacea plants. However, the callus agar displayed no gel-forming properties, even after alkali modification.author for correspondence     

Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola

When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l^–1 to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g^–1 dry wt, 9 mg g^–1 dry wt and 57 mg g^–1 dry wt, respectively. The final biomass (1.3 mg dry wt ml^–1) was not affected.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

Rate of Helicobacter pylori infection in children and clonality of Taiwan strains

The infection rate of Helicobacter pylori in children from < 1 to 17 years old was investigated. Three techniques, namely culture, CLO test, and PCR, were employed to check the presence or absence of the organism in the antrum of the stomach. Several PCR positives without viable cultures were observed in babies of less than one year old. On the other hand, only two viable cultures were obtained from toddlers of less than two years old. The percentage of positive cultures steadily increased from 8% (3 of 42 cases) in the 0-4 years old age group to 32% (32 of 99 cases) in the 13-17 years old age group. A steady increase also was observed in the result of the CLO test. In PCR, the percentage of positives was greatly higher than that seen with the culture or CLO test. The rate of PCR positives also showed an increase with age but of a much slower rate. The overall infection rate in 295 children was 22% (64 of 295 cases) positive with culture and 76% (225 of 295 cases) with PCR, in contrast to 85% (40 of 49 cases) and 92% (43 of 47 cases), respectively, in adults. The urease activity of the H. pylori derived from children was much lower than that derived from adults (P < 0.001). Taken together, these results suggest that a child might be repeatedly infected and some infecting strains eventually might obtain a steady infection, perhaps by a strain of higher virulence such as higher urease activity. The base variations in the nucleotide sequences did not correlate to the varied urease activities or to the age of the child. The sequences, however, indicated that there were two types of strains. The strains in Taiwan appeared to be derived from the French type strain and not the English type strain. The amino acid sequences of the ureA and the phylogenetic relationship of the 29 strains indicated that the strains in Taiwan are rapidly evolving into a unique clone.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.