Organ Culture Method Research Articles

Development and evaluation of a porcine in vitro colon organ culture technique.

The intestinal mucosa comprises a complex assemblage of specialized tissues that interact in numerous ways. In vitro cell culture models are generally focused on recreating a specific characteristic of this organ and do not account for the many interactions between the different tissues. In vitro organ culture (IVOC) methods offer a way to overcome these limitations, but prolonging cell viability is essential. This study aimed to determine the feasibility and optimal conditions for in vitro culture of swine colonic mucosa for use as an enteric pathogen infection model. Explants (n = 168) from commercial pigs (n = 12), aged 5 to 10wk, were used to assess the impact of various culture protocols on explant viability. Explants were cultured for up to 5d and formalin fixed at 24-h intervals. Following establishment of the culture protocol, explants (n = 208) from 13 pigs were evaluated at Day 0 and 5 of culture. Assessment of viability was based on histological changes (tissue architecture evaluated by H&E, immunostaining of cell proliferation marker Ki-67) and expression of genes encoding IL-1α, IL-8, TNF-α, IFN-γ, and e-cadherin. After 5d in culture, 20% of explants displayed over 80% of epithelial coverage, whereas 31% of explants had more than 50% of their surface covered by columnar epithelium, and 81% had crypts but with a decreased number of Ki-67-positive cells when compared to Day 0. Notably, large variability in explant quality was observed between donor pigs. Best possible explants were obtained from the distal colon of pigs, processed immediately after euthanasia, cultured at the liquid-tissue-gas interface in media supplemented with a mixture of antibiotics and antifungals and an oxygen-rich gas mix.

Culturing and Characterization of Gut Symbiont Burkholderia spp. from the Southern Chinch Bug, Blissus insularis (Hemiptera: Blissidae).

The phloem-feeding Southern chinch bug, Blissus insularis, harbors a high density of the exocellular bacterial symbiont Burkholderia in the lumen of specialized midgut crypts. Here we developed an organ culture method that initially involved incubating the B. insularis crypts in osmotically balanced insect cell culture medium. This approach enabled the crypt-inhabiting Burkholderia spp. to make a transition to an in vitro environment and to be subsequently cultured in standard bacteriological media. Examinations using ribotyping and BOX-PCR fingerprinting techniques demonstrated that most in vitro-produced bacterial cultures were identical to their crypt-inhabiting Burkholderia counterparts. Genomic and physiological analyses of gut-symbiotic Burkholderia spp. that were isolated individually from two separate B. insularis laboratory colonies revealed that the majority of individual insects harbored a single Burkholderia ribotype in their midgut crypts, resulting in a diverse Burkholderia community within each colony. The diversity was also exhibited by the phenotypic and genotypic characteristics of these Burkholderia cultures. Access to cultures of crypt-inhabiting bacteria provides an opportunity to investigate the interaction between symbiotic Burkholderia spp. and the B. insularis host. Furthermore, the culturing method provides an alternative strategy for establishing in vitro cultures of other fastidious insect-associated bacterial symbionts. An organ culture method was developed to establish in vitro cultures of a fastidious Burkholderia symbiont associated with the midgut crypts of the Southern chinch bug, Blissus insularis The identities of the resulting cultures were confirmed using the genomic and physiological features of Burkholderia cultures isolated from B. insularis crypts, showing that host insects maintained the diversity of Burkholderia spp. over multiple generations. The availability of characterized gut-symbiotic Burkholderia cultures provides a resource for genetic manipulation of these bacteria and for examination of the mechanisms underlying insect-bacterium symbiosis.

Development of a Short-Term Canine Full-Thickness Skin Organ Culture Method under Serum-Free Conditions

Full-thickness canine skin organ culture models could provide an entirely new opportunity for studying wound healing, keratinization disorders and allergic skin diseases, all of which have high prevalence and severe impact on canine quality of life. Here we present a canine organ culture method for the short-term maintenance of full-thickness, adult, canine skin in serum-free medium and investigate the possibility to induce mast cell degranulation ex vivo. General morphological features were maintained up to day 7. Epidermal thickness started to decrease from day 4 of culture. No changes were observed in epidermal pigmentation. Keratinocyte proliferation started to significantly decrease at day 7. Immunostaining for cytokeratin 10, cytokeratin 14 and loricrin was evident from day 1 to day 7. Compound 48/80 induced mast cell degranulation. This was the first attempt to establish a dog skin organ culture and document good preservation of most cutaneous structures till day 7. This method may help in dissecting canine skin biology in physiological and pathological conditions and to study drug mechanism of action in a biologically relevant environment.

Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

FTOC-Based Analysis of Negative Selection.

Potentially harmful T cell precursors are removed from the conventional T cell pool by negative selection. This process can involve the induction of apoptosis, anergy, receptor editing or deviation into a regulatory T cell lineage. As such this process is essential for the health of an organism through its contribution to central and peripheral tolerance. While a great deal is known about the process, the precise mechanisms that regulate negative selection are not clear. Furthermore, the signals that distinguish the different forms of negative selection are not fully understood. Numerous models exist with the potential to address these questions in vitro and in vivo. This chapter describes methods of fetal thymic organ culture designed to analyze the signals that determine these unique cell fates.

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Method of Studying Palatal Fusion using Static Organ Culture.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate (1). Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.

Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Twenty-Four Hour Ex Vivo Culture of Celiac Duodenal Biopsies.

Organ culture is a valuable technique in celiac disease research. It provides the opportunity to examine interactions between different cell types during the disease process without the need for invasive in vivo studies. Biopsies are maintained in an oxygen-rich environment, in contact with, but not submerged in, culture medium. A very straightforward and successful method of organ culture is described here.

Renal Stem Cells, Tissue Regeneration, and Stem Cell Therapies for Renal Diseases.

Kidney diseases are a global public health problem, with an incidence that has reached epidemic proportions and continues to climb in the USA and worldwide. This trend is projected to grow in correlation with the global rise in the aged population and the increasing prevalence of conditions that cause renal complications, such as cardiovascular disease, hypertension, and diabetes. Current treatment options for acute and chronic kidney disease include dialysis, which is also associated with substantial morbidity and mortality, and kidney transplantation, which is limited by the supply of compatible organs. Consequently, new methods to alleviate, cure, or prevent renal disease are urgently required to reduce the exponentially growing burden due to acute and chronic kidney disorders and offer alternative therapeutic options to improve patients' survival and quality of life. Several potential regenerative cell-based therapies for the treatment of renal failure are currently under development. The first one is the direct application of stem cells (SCs) to the diseased kidney, which relies on the inherent capabilities of SCs to differentiate, organize, and integrate into the existing tissues to restore function. To this aim different stem cell types have been investigated for their potential to contribute to renal regeneration. In their article “WNT/β-Catenin Signaling Is Required for Integration of CD24+ Renal Progenitor Cells into Glycerol-Damaged Adult Renal Tubules,” Z. Zhang et al. described how the endogenous canonical β-catenin/TCF pathway is reactivated during recovery from AKI and is required for the engraftment of exogenous CD24+ embryonic renal progenitor cells into damaged tubular areas upon injury. These events appear to recapitulate the WNT-dependent inductive process which drives primary nephrogenesis. Review article by F. O. Arcolino et al. entitled “Human Urine as a Noninvasive Source of Kidney Cells” summarizes the recent data regarding human urine-derived cells. Urine contains various types of kidney cells such as podocytes, proximal tubule cells, or renal stem/progenitor cells, which can be easily as well as noninvasively isolated and cultured in vitro. Although the characteristics of each cell population are not totally understood, this paper suggests the potential of urine-derived kidney cells as a tool of clinical application for the treatment of various kidney diseases. Stem cell therapy contributes for kidney regeneration not only through direct differentiation and replacement of injured cells but also by secretion of renoprotective or trophic factors. These data are summarized by K. Tsuji and S. Kitamura in their review article “Trophic Factors from Tissue Stem Cells for Renal Regeneration.” Another strategy is based on the prospective design of a therapeutic approach focused on modulation of endogenous kidney regenerative properties by conventional chemical and biological agents able to modulate the activity of resident progenitor cells. Although understanding of regeneration of the nephron may have significance to explore new therapeutic approaches, the process of regeneration of nephron has yet been poorly understood. The review article “Atlas of Cellular Dynamics during Zebrafish Adult Kidney Regeneration” by K. K. McCampbell et al. provides an essential foundation for future work aimed at elucidating the mechanisms that regulate kidney regeneration following acute kidney injury using adult zebrafish, which maintains the regenerative ability. Akito Maeshima et al. in their review “Diverse Cell Populations Involved in Regeneration of Renal Tubular Epithelium following Acute Kidney Injury” describe recent advances in understanding the regeneration mechanisms of renal tubules, particularly the characteristics of various cell populations contributing to tubular regeneration and highlight the targets for the development of regenerative medicine for treating kidney diseases in humans. Additional information on the renal regeneration capacity could be inferred from studies on kidney organ culture. The review article “Organ In Vitro Culture: What Have We Learned about Early Kidney Development?” by A. Rak-Raszewska et al. provides an introduction to the organ culture method and a summary of the progress in the field of kidney developmental biology based on it. Finally, a great advance on the comprehension of the mechanisms of renal regeneration, in particular podocyte regeneration, and on the role of putative population of renal stem/progenitor cells could be obtained from transgenic mouse models, as summarized by D. Lombardi and Laura Lasagni in their review “Transgenic Strategies to Study Podocyte Loss and Regeneration.” Finally, a number of different approaches have been applied toward tissue engineering of the kidney as a mean to replace renal function. S. Yamanaka and Takashi Yokoo in their review article “Current Bioengineering Methods for Whole Kidney Regeneration” summarized recent researches involving the use of renal stem cells and renal bioengineering to regenerate functional whole kidneys de novo. The editors hope that the original and review articles integrated in this special issue provide more insights into the advancements and challenges faced by this rapidly expanding field of regenerative medicine and will be helpful and educational for interested readers. Benedetta Bussolati Akito Maeshima Janos Peti-Peterdi Takashi Yokoo Laura Lasagni

Spermatogonial stem cells: Progress and prospects.

Twenty years ago, the transplantation of spermatogonial stem cells (SSCs) from a mouse to other recipient mice was shown to be feasible, which clearly demonstrated the functional identity of SSCs. Since then, several important new findings and other technical developments have followed, which included a new hypothesis on their cell kinetics and spermatogonial hierarchy in the testis, a culture method allowing their self-renewal and proliferation, a testis tissue organ culture method, which induced their complete differentiation up to sperm, and the in vitro induction of germ cells from embryonic stem cells and induced pluripotent stem cells. These advancements reinforced or advanced our understanding of this unique cell. Nonetheless, there are many unresolved questions in the study of spermatogonial stem cells and a long road remains until these cells can be used clinically in reproductive medicine.

Establishment of ex vivo mucocele model using salivary gland organ culture

A mucocele is a common lesion originating from the minor salivary glands and frequently seen in children. For this study, we established an ex vivo mucocele model using a mouse salivary gland organ culture method. ResultsFirst, medium from the upper portion of salivary gland organ cultures was either removed or not, then culturing was continued for 10 days. After that 10-day period, 13 of 21 specimens (61.9%) in the medium removed group showed mucocele-like mucus restoration, while only 1 of 15 (6.7%) in the non-medium removed group showed restoration. Next, we examined mucocele type in the ex vivo salivary gland organ cultures and found mucous retention type mucocele formation in only the main duct of most of the cultures. Other types were observed in the main excretory and intercalated ducts, but not exclusively in the intercalated duct. We also investigated the effect of duct cutting on mucocele recrudescence. Mucoceles that developed in the cultures were cut with stainless needles under a stereomicroscope and mucus was promptly discharged. At 7 days after duct cutting, 5 of 19 specimens (26.3%) showed recrudescence mucus retention. ConclusionsThis is the first report of establishment of an ex vivo pathogenic condition for examination of salivary glands. Our model may be useful to establish mucocele treatment options including drug screening and surgical procedures.

Isolation of Adipose Tissue Stem Cells with Organ Culture Method

Background Isolation of adipose tissue mesenchymal stem cells (MSCs) with current enzymatic methods has some limitations. For example, it is costly and time-consuming and results in a heterogeneous cell population that making compromise proliferation and differentiation of MSCs. Also, it is accompanied with the increased risk of culture contaminations because of several handling steps.In this article we present a non-enzymatic method for isolation of MSCs. Methods Small pieces of rat adipose tissue and also human liposuction sample were placed in the culture flask, covered with fetal bovine serum (FBS) and maintained in incubator for 24 h. Then, the FBS was changed with DMEM medium containing 20 FBS. When the fibroblast-like cells were appeared around the tissue they were expanded through 3 passages and used for Immunophenotype and differentiation assays. Results Flow cytometric analysis showed that the cells isolated with organ culture method expressed CD44 and CD105 but did not express CD34 and CD45 markers. The isolated cells also differentiated into adipocyte and osteoblast.Therefore, consistent with classically isolated MSCs, the cells isolated with our method express the stem cell surface markers and have pluripotent property. Conclusion The presented method is an easy and cheap procedure and can be used for harvesting MSCs from very small fat samples of human or animals. Key Words: Adipocyte, Human, Osteoblast, Stem cell.

Methylglyoxal causes endothelial dysfunction: the role of endothelial nitric oxide synthase and AMP-activated protein kinase α

Methylglyoxal is a major precursor in the formation of advanced glycation end products and is associated with the pathogenesis of diabetes-related vascular complications. The aim of this study was to evaluate whether methylglyoxal induces endothelial dysfunction and to determine the contributors involved in this process. Rat thoracic aortic rings were treated for 24 h with 100 μM methylglyoxal by using an organ culture method. A cumulative dose-response curve to acetylcholine was obtained to determine endothelium-dependent relaxation. The protein levels of endothelial nitric oxide synthase (eNOS) and its phosphorylated form at the serine 1177 site [p-eNOS (Ser1177)], heat shock protein 90 (Hsp90), AMP-activated protein kinase α (AMPKα) and its phosphorylated form at the threonine 172 site [p-AMPKα (Thr172)] were evaluated. Superoxide production was determined by lucigenin-chemiluminescence. Treatment with 100 μM methylglyoxal for 24 h decreased acetylcholine-induced vascular relaxation. The levels of eNOS and p-eNOS (Ser1177) were reduced while no effect on Hsp90 was observed. Levels of p-AMPKα (Thr172) were significantly decreased without any change in total AMPKα protein levels. Superoxide level was not affected by methylglyoxal treatment. In rat aortic rings, methylglyoxal determines a reduction in endothelium-dependent relaxation. This effect seems to be mediated via a reduction in p-eNOS (Ser1177) and p-AMPKα (Thr172).

In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method

The in vitro propagation of mouse spermatogonial stem cells (SSCs) became possible in 2003; these cultured SSCs were named germ-line stem (GS) cells. To date, however, it has not been possible to induce spermatogenesis from GS cells in vitro. Recently, we succeeded in producing functional sperm from primitive spermatogonia in explanted neonatal mouse testis tissues. Here we describe a protocol that can support spermatogenesis from GS cells up to sperm formation in vitro using an organ culture method. GS cells transplanted in the extracted testis form colonies in the tissue fragments and differentiate into sperm under the described in vitro organ culture conditions. It takes about 6 weeks to obtain sperm from GS cells. The sperm are viable, resulting in healthy offspring through micro-insemination. Thus, this protocol should be a valuable tool for the study of mammalian spermatogenesis.

Anin vitroMethod for Producing Sperm for ICSI

It is a challenge to induce spermatogenesis in vitro, because it involves a complicated process of sequential cell proliferation and differentiation, from spermatogonial stem cells to sperm formation. Recently, we have succeeded, using a classical organ culture method, to produce sperm from spermatogonial stem cells, using several modifications. The produced spermatids and sperm were fertile, giving rise to healthy progeny. This new in vitro system for spermatogenesis could be useful for addressing the issue of sperm quality that is becoming more important in this ICSI era. We also hope that an in vitro system of human spermatogenesis will be developed in the near future.

A role for hedgehog signaling in the differentiation of the insertion site of the patellar tendon in the mouse.

Tendons are typically composed of two histologically different regions: the midsubstance and insertion site. We previously showed that Gli1, a downstream effector of the hedgehog (Hh) signaling pathway, is expressed only in the insertion site of the mouse patellar tendon during its differentiation. To test for a functional role of Hh signaling, we targeted the Smoothened (Smo) gene in vivo using a Cre/Lox system. Constitutive activation of the Hh pathway in the mid-substance caused molecular markers of the insertion site, e.g. type II collagen, to be ectopically expressed or up-regulated in the midsubstance. This was confirmed using a novel organ culture method in vitro. Conversely, when Smo was excised in the scleraxis-positive cell population, the development of the fibrocartilaginous insertion site was affected. Whole transcriptome analysis revealed that the expression of genes involved in chondrogenesis and mineralization was down-regulated in the insertion site, and expression of insertion site markers was decreased. Biomechanical testing of murine adult patellar tendon, which developed in the absence of Hh signaling, showed impairment of tendon structural properties (lower linear stiffness and greater displacement) and material properties (greater strain), although the linear modulus of the mutant group was not significantly lower than controls. These studies provide new insights into the role of Hh signaling during tendon development.

PLANT TISSUE CULTURE IN BIOTECHNOLOGY: RECENT ADVANCES IN TRANSFORMATION THROUGH SOMATIC EMBRYOGENESIS

118 The development of cell, tissue and organ culture methods was rapidly accelerated in the second half of the previous century after establishing robust cell culture techniques and media formulation for in vitro growth of plant material [1–8]. A stunning number of articles have been published on in vitro induction and maintenance of non-differentiated cells and the regeneration the plants from them whether through organogenesis or somatic embryogenesis. Fine protocols were established for the culture of enzymatically isolated single cells and protoplasts [9], which were able to regenerate into plants [10–12]. In vitro culture methods became an essential part of many micropropagation protocols. Culture of plant cells and organs in bioreactors were used for the production of different secondary metabolites and pharmaceuticals. In vitro techniques were used for production of mutants, haploids, virus-free material, and also for maintenance and preservation of rare genotypes and specific cell cultures [13–15]. The first positive results on Agro bac te rium-mediated transformation in plants were reported in 1983 [16–18]. A simple method for transferring genes into plants through the inoculation of leaf discs with Agrobacterium tumefaciens followed by in vitro culture and regeneration of whole plants was reported by Horsch et al. [19]. Different methods for DNA delivery into plant cell, including electroporation [20], PEG treatment [21, 22], microinjection [23], sonication [24], biolistics or particle bombardment [25], silicon carbide Whiskers treatment [26, 27], were used for specific transformation purposes and different types of cells and genotypes. However, Agrobac terium-mediated transformation became a preferred method. Agrobacterium tumefaciens is a natural vector system for transgenes delivery into a wide range of plants species, providing an efficient and «clean» insertion of DNA into the plant genome and deserved to be called the «tzar of genetic engineering» [28]. In the past 30 years the discovery and application of new transformation technologies essentially sped up the improvement of major cultivated crops. The first, really commercially grown plants hit the market in the mid — 1990s. Practically all transformation systems were based on in vitro culture methods. It was impossible to transform whole plant organism at once. All techniques were based on transforming single cells of callus, leaves, pollen, roots or other organs and than regeneration of plants through somatic embryogenesis or organogenesis. Routine and highly efficient transformation methods for many important crop and particular genotypes were implemented in many biotechnology companies. UDK 575327/275.854

Bioactive Derivatives of Curcumin Attenuate Cataract Formation In Vitro

In this study, curcumin derivatives salicylidenecurcumin (CD1) and benzalidenecurcumin (CD2)] were prepared, and their biological activity was compared in in vitro selenite-induced cataract model. The antioxidant activity was studied using DPPH radical scavenging assay. Knoevenagel condensates of curcumin exhibited higher DPPH radical scavenging activity compared with curcumin. The anticataractogenic potential of curcumin derivatives was analyzed using lens organ culture method. The activity of antioxidant enzymes and calcium homeostasis was reversed to near normal levels following treatment in organ cultured rat lenses. These results indicated that curcumin and its derivatives--CD1 and CD2--are beneficial against selenite-induced cataract in vitro. Of these, CD1 is having higher bioactive potential compared with curcumin and CD2.