In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

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

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Intestinal Tritrichomonas foetus infection in cats in Switzerland detected by in vitro cultivation and PCR

Tritrichomonas foetus, a parasite well known for its significance as venereally transmitted pathogen in cattle, has recently been identified as a cause of chronic large-bowel diarrhea in domestic cats in the US, UK, and, more recently, also in Norway. In a period of 3 months (October to December 2007), 45 cats of Switzerland suffering from chronic diarrhea were investigated for intestinal infections, including a search for trichomonads. A commercially available in vitro culture system was used to screen for infection, complemented with a PCR and subsequent amplicon sequencing to support speciation. The PCR is based upon amplification of a sequence derived from the internal transcribed spacer region 1 (ITS1) on the ribosomal RNA gene (rRNA) using primers designed to detect a broad range of genera and species belonging to the family of Trichomonadidae. The method was furthermore adapted to the uracil DNA glycosylase (UDG) system in order to prevent carry-over contamination and it included a recombinant internal control to track for inhibitory reactions. Eleven out of the 45 cats were culture-positive, as revealed by microscopic identification of trichomonadid organisms. One of the isolates was subjected to scanning electron microscopy and findings revealed the presence of three flagella, thus placing the isolate into the gender Tritrichomonas sp. PCR and subsequent amplicon sequencing were carried out with ten of the 11 isolates. A total homology with published T. foetus sequences was confirmed in all of the cases. T. foetus therefore appears to range among those organisms that can cause chronic diarrhea in cats in Switzerland.

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 mum. Round-shaped primmorphs of less than 100 gm were formed 3 days after transferring the dissociated cells into seawater containing Ca2+ and Mg2+. The effect of various cell dissociation conditions, inoculum. cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum. cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled. (C) 2002 Elsevier Science B.V. All rights reserved.

Orchid in vitro growth as affected by nitrogen levels in the culture medium

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito de poliaminas exógenas no crescimento inical in vitro e nos teores de fenóis, poliaminas e atividade da peroxidase em Aloe vera (L.) Burm.

Lateral shoots of the Aloe vera (L.) Burm. cultivated in vitro, without addition vegetal regulators, for 90 days, were inoculated in MS culture-medium, containing or not spermine and/or spermidine. After 30 days of cultivation, the plants were submitted to biochemical analysis together with micropropagated plants - that were under in vitro cultivation for 90 days - (denominated as characterization), and matrix plants (in vivo). The levels of free polyamines, total phenols, total flavonoids, and the activity of peroxidase were evaluated in the biochemical analyses. The exogenous application of spermidine have promoted large number of shoots. Spermidine and spermine have promoted, when associated, an increase in the number of shoots as well as an increase of the contents of putrescine and and flavonoids. The putrescine has presented the most significant alterations, enabling to be utilized as marker of morphogenesis in the micropropagated Aloe vera. Tissues under active growth have presented high activity of peroxidase as well as those with greater rate of oxidation. In these tissues, there were noticed also higher contents of total flavonoids, indicating the need of antioxidative compounds. The action of polyamines jointly tseemed to be benefic for the shooting of micropropagated Aloe vera.

Micropropagação e conservação in vitro de Croton antisyphiliticus Mart

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Paclobutrazol no crescimento e desenvolvimento de Zygopetalum crinitum Lodd. e Cattleya schilleriana Rchb.f. (Orchidaceae) durante as fases in vitro e de aclimatização

Pós-graduação em Agronomia (Produção Vegetal) - FCAV

In vitro propagation of Sacha inchi

The aim of this study was to perform an in vitro evaluation of the auxin: cytokinine ratio in different segments of the epicotyl and hypocotyl of Sacha inchi (Plukenetia Volubilis Linneo) seeds germinated in vitro. The segments apical (A), median (B) and basal (C) were introduced into semi-solid MS culture medium (2.0g L-1 Phytagel), supplemented with MS vitamins, sucrose (30.0g L-1) and submitted to three doses of auxin indolebutyric acid - IBA (0; 0.1; 0.5mg L-1), associated with four doses of the cytokinine benzylaminopurine - BAP (0; 0.1; 0.5; 1.0mg L-1), totaling 36 treatments. After nine weeks of in vitro cultivation, the apical segment ( A) presented shoot formation by direct organogenesis at the concentrations of 0.5 and 1.0 of BAP associated with 0.0 and 0.1 of IBA. It is feasible to use in vitro cultivation with the apical region of seeds germinated in vitro used as explants.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

Propagação in vitro de barueiro (Dipteryx alata Vog.)

Savannah is the second biome in biodiversity in Brazil, presenting great vegetation endemism. Dipteryx alata Vog. (Fabaceae), native from this biome, is an economically important species, with an incipient market due to the lack of commercial plantations. This highlights the need to develop and provide the basis for the domestication of this species. Thus, this study determined the best conditions for in vitro establishment, multiplication, elongation and rooting of stem tips of D. alata plantlets grown vitro. Two culture media (MS and WPM) were evaluated in different salt concentrations (25, 50, 75 and 100%) for plantlet establishment. Four concentrations of 6– Benzylaminopurine (BAP) (0, 1, 2, 3 and 4 mg L-1) amended with 0.25 mg L-1 naphthalene-acetic acid (NAA) were studied for multiplication. Simultaneous elongation and rooting were studied with four concentrations of NAA (0, 1, 2, 3 and 4 mg L-1) together with 0.5 mg L-1 IBA. The variables analyzed were: shoot length (CPA), root length (CP), fresh matter (MF), dry matter (MSC), stem diameter (DC) and number of leaves (NF), 120 days after inoculation, with the exception of number of shoots, which was evaluated in the multiplication stage only. The medium MS at the original salt concentration (100%) was effective for the in vitro establishment of E. alata, resulting in greater root length (27.65 cm) and number of leaves per plantlet (26.0). The concentration of 4 mg L-1 BAP was the best one for multiplication; however, greater concentrations can boost multiplication. The effect of NAA and IBA were noticeable on in vitro elongation and rooting, with best CPA (3.14 cm) and CR (15.84 cm). Therefore, it is possible to state that the medium MS increases the success probability of in vitro establishment of stem tips of Dipteryx alata. NAA concentrations below 3 mg L-1 were favorable for in vitro development of the species, with essential characteristics for acclimatization success|.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

An in-vitro 3-D cell culture model for studying pathomechanisms in AMD

Purpose To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Methods Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing within a 24 G needle. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model of choroidal neovascularization.