Preparation of intact bovine tail intervertebral discs for organ culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

Modulation of arginine decarboxylase activity in cucumber (Cucumis sativus) cotyledons in short-term organ culture

Among the various amines administered to excisedCucumis sativus cotyledons in short-term organ culture, agmatine (AGM) inhibited arginine decarboxylase (ADC) activity to around 50%, and putrescine was the most potent entity in this regard. Homoarginine (HARG) dramatically stimulated (3- to 4-fold) the enzyme activity. Both AGM inhibition and HARG stimulation of ADC were transient, the maximum response being elicited at 12 h of culture. Mixing experiments ruled out involvement of a macromolecular effector in the observed modulation of ADC. HARG-stimulated ADC activity was completely abolished by cycloheximide, whereas AGM-mediated inhibition was unaffected. Half-life of the enzyme did not alter on treatment with either HARG or AGM. The observed alterations in ADC activity are accompanied by change in Km of the enzyme. HARG-stimulated ADC activity is additive to that induced by benzyladenine (BA) whereas in presence of KCl, HARG failed to enhance ADC activity, thus demonstrating the overriding influence of K+ on amine metabolism.

Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni.

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.

Seed production and culture techniques of Genetically Improved Farmed Tilapia (GIFT) in brackishwater environment

Effects of different levels of salinity on survival, growth and gonadal development of Genetically Improved Farmed Tilapia (GIFT) were studied under laboratory conditions in glass aquarium, for a period of ten weeks. The initial individual size of the GIFT was 20.23±4.45 and the salinity levels tested were 0, 5, 10, 15 and 20 ppt. The highest survival of 87.5% was found in 0 ppt and the lowest 60.5% in 20 ppt. Though the survival decreased progressively with increased salinity, there were no significant differences (P>0.05) among 0, 5, and 10 ppt. Similar to what has been observed in survival, the specific growth rate (SGR %/day) also decreased as of 1.30, 1.24, 1.08, 0.90 and 0.71, respectively, with the increased salinity of 0, 5, 10, 15 and 20 ppt. The gonadal development was highest in 0 ppt with a GSI value of 3.75 and lowest of 2.01 in 20 ppt. In the second experiment, gonadal development and seed production performance of GIFT in brackishwater condition were investigated for a period of three months. Each of the three fine meshed hapas of 20 square meters made from nylon net was placed in a freshwater (0 ppt) and in a brackish water (10-15 ppt) pond of the Brackishwater Station (BS). GIFT of 65 g average weight from a single cohort were stocked into three hapas at a rate of 2 per m. The male vs female ratio was 1:3. The development of gonad was faster with the higher gonadosomatic index (GSI %) of 3.85 % in freshwater condition than that of 2.73 % in brackish water. Within three months of the study period, a total of 70,510 and 44,250 GIFT fry were produced respectively, in freshwater and brackishwater conditions. Finally under third experiment, a participatory on-farm trial was carried out to evaluate the production performance of GIFT in monoculture and in polyculture with silver barb in coastal freshwater pond conditions. Nine ponds were selected for three treatment combinations of GIFT monoculture (T1), GIFT and silver barb polyculture (T2), and silver barb monoculture (T3). The ponds have been stocked in April, 05 at a density of 25,000 fry per ha. Fishes were fed with rice bran at the rate of 6% bw per day. In one month culture period, GIFT attained an average weight of 16.27 g in monoculture and 17.23 g in polyculture, against an average stocking weight of 0.37 g. Silver barb reached an average weight of 16.62 g in polyculture with GIFT and 10.01 g in monoculture, against an average stocking weight of 3.79 g.

Seed production and culture techniques of climbing perch, Anabas testudineus

Six treatments each with 12 replications designed to optimize the dose of inducing agent PG to achieve fertilization and hatching success of climbing perch, Anabas testudineus were tested. The females were given single injection of 7-12 mg PG/kg body weight and the males were given 4 mg PG/kg body weight. Fertilization and hatching rate varied from 67±4.55% to 66±3.0% and 59±4.88% to 57±6.21% for the doses of 10, 11 and 12 mg PG/kg of body weight, respectively. The hormone dose had significant (P<0.05) effect on fertilization and hatching. Six mini shallow cisterns (570 cm x 105 cm) were used to investigate the efficacy of zooplankton and Artemia nauplii as feed for spawn rearing. Three-day old spawns were stocked in six mini shallow cisterns at a stocking density of 100 individuals/L of water. Two treatments each with three replications were used to develop culture technique of the climbing perch. In case of treatment-1, the spawns were fed with Artemia nauplii three times daily, while in treatment-2, zooplankton were used as feed in the same manner as in treatment-1. After 14 days of rearing, mean final weight of the fry of treatments-1 and 2 were 95.55±6.71 and 57.69±5.40 mg, respectively. In treatment-1, spawn fed with Artemia nauplii showed significantly (P<0.05) higher mean weight than the spawn fed with zooplankton (treatment 2).

Generation of Variability for Salt Tolerance in Rice Using Tissue Culture Techniques

The study deals with the generation of variability for salt tolerance in rice using tissue culture techniques. Rice is the staple food of more than half of the world’s population. The management of drought, salinity and acidity in soils are all energy intensive agricultural practices. The Genetic variability is the basis of crop improvement. Somaclonal and androclonal variation can be effectively used for this purpose. In the present study, eight isozymes were studied and esterase and isocitric dehydrogenase was found to have varietal specific, developmental stage specific and stress specific banding pattern in rice. Under salt stress thickness of bands and enzyme activity showed changes. Pokkali, a moderately salt tolerant variety, had a specific band 7, which was present only in this variety and showed slight changes under stress. This band was faint in tillering and flowering stage .Based on the results obtained in the present study it is suggested that esterase could possibly be used as an isozyme marker for salt tolerance in rice. Varietal differences and stage specific variations could be detected using esterase and isocitric dehydrogenase . Moreover somaclonal and androclonal variation could be effectively detected using isozyme markers.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Homing of mesenchymal stem cells in induced degenerative intervertebral discs in a whole organ culture system

Homing of human bone marrow-derived mesenchymal stem cells (BMSCs) was studied using ex vivo cultured bovine caudal intervertebral discs (IVDs).