High-density culture of Vero cells in novel perfusion bioreactor system

Viruses are the obligatory intracellular parasites infecting microbes, plants, animals, and humans. They aredead outside host cell but can take-over the host’s cell machinery as soon as they are into it. Several studies oninhibitor compounds have been done for animal viruses including those that are affecting humans, but thereis inadequacy in terms of research and literature for plant viruses that are responsible for losses in crop yieldand quality loss all across the globe. This could be focal point to study plant viruses, their transmission andpathogenicity, and to establish widely used, effective, and advanced approaches for their control. The purposeof this review is to discuss various approaches to control plant viruses that have been developed and applied tocombat plant viral infections. We have divided these approaches into two categories conventional (meristemtip culture, cryotherapy, thermotherapy, and chemotherapy) and advanced (nucleic acid-based approacheslike RNA Silencing, cross-protection, transgenic plants, gene pyramiding, and protein-protein interaction).Moreover, we have discussed and compared the principles, methodologies, advantages, and disadvantages ofeach technique. The approaches have been explored to promote their application in best suited way on variousplants to control viral diseases and to improve food crops quality with increase in production.

The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor / 中华血液学杂志

Objective@#To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC).@*Methods@#The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14.@*Results@#In the two-dimensional (2D) culture, CD41+, CD41+/CD61+, CD61+ megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05).@*Conclusion@#3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

Cardiac tissue engineering: current state-of-the-art materials, cells and tissue formation / Engenharia de tecidos cardíacos: atual estado da arte a respeito de materiais, células e formação tecidual

ABSTRACT Cardiovascular diseases are the major cause of death worldwide. The heart has limited capacity of regeneration, therefore, transplantation is the only solution in some cases despite presenting many disadvantages. Tissue engineering has been considered the ideal strategy for regenerative medicine in cardiology. It is an interdisciplinary field combining many techniques that aim to maintain, regenerate or replace a tissue or organ. The main approach of cardiac tissue engineering is to create cardiac grafts, either whole heart substitutes or tissues that can be efficiently implanted in the organism, regenerating the tissue and giving rise to a fully functional heart, without causing side effects, such as immunogenicity. In this review, we systematically present and compare the techniques that have drawn the most attention in this field and that generally have focused on four important issues: the scaffold material selection, the scaffold material production, cellular selection and in vitro cell culture. Many studies used several techniques that are herein presented, including biopolymers, decellularization and bioreactors, and made significant advances, either seeking a graft or an entire bioartificial heart. However, much work remains to better understand and improve existing techniques, to develop robust, efficient and efficacious methods.

RESUMO Doenças cardiovasculares são responsáveis pelo maior número de mortes no mundo. O coração possui capacidade de regeneração limitada, e o transplante, por consequência, representa a única solução em alguns casos, apresentando várias desvantagens. A engenharia de tecidos tem sido considerada a estratégia ideal para a medicina cardíaca regenerativa. Trata-se de uma área interdisciplinar, que combina muitas técnicas as quais buscam manter, regenerar ou substituir um tecido ou órgão. A abordagem principal da engenharia de tecidos cardíacos é criar enxertos cardíacos, sejam substitutos do coração inteiro ou de tecidos que podem ser implantados de forma eficiente no organismo, regenerando o tecido e dando origem a um coração completamente funcional, sem desencadear efeitos colaterais, como imunogenicidade. Nesta revisão, apresentase e compara-se sistematicamente as técnicas que ganharam mais atenção nesta área e que geralmente focam em quatro assuntos importantes: seleção do material a ser utilizado como enxerto, produção do material, seleção das células e cultura de células in vitro. Muitos estudos, fazendo uso de várias das técnicas aqui apresentadas, incluindo biopolímeros, descelularização e biorreatores, têm apresentado avanços significativos, seja para obter um enxerto ou um coração bioartifical inteiro. No entanto, ainda resta um grande esforço para entender e melhorar as técnicas existentes, para desenvolver métodos robustos, eficientes e eficazes.

Azo Dye Mineralization by Phanerochaete Chysosporium in a Sequencing Bath Reactor

ABSTRACT The mineralization of the azo dye congo red by the fungi Phanerochaete chrysosporium was studied in two sequential batch bioreactors (R1 and R2), operated in cycles of 48 h (step I) and 24 h (step II). In step I, glucose concentration was 1 g.L-1 in both reactors and in step II, 1 g.L-1 of glucose was maintained in R1, but R2 received no addition of glucose. In step I, the average dye removal efficiencies were 76 ± 29 % (R1) and 53 ± 15% (R2), while in step II the averages recorded for dye removal for R1 and R2 were 84 ± 15 and 70 ± 28%, respectively. The rates of dye removal were 0.04 h-1 in R1 and 0.03 h-1 in R2 in step I. Higher rates were obtained in step II, 0,07 h-1 and 0,02 h-1 for R1 and R2, respectively. The highest dye removal occurred in R1 and, in R2, the residual dye was further removed. Laccase was the oxidised at higher amount, in step I was 54 μmol.min-1 for R1 and 38 μmolmin-1 for R2. The proposed treatment system was very effective in removing the azo dye, however the mineralization may not be complete and some by-products may have been formed, according to spectrofotometric analysis, were the peak corresponding to benzene, 220 nm, persisted.

Current progess and prospects of tissue-engineered trachea research / 国际外科学杂志

In recent years,Scientists paid close attention to the studies and applications of tissue-engineered trachea,which have made some advances.But how to promote tissue-engineered trachea for clinical application still needs deeper research.Here we discussed from three key aspects of tissue-engineered trachea (Scaffold,Cell,Bioreactor),which attempted to retrospect the current progress in tissue-engineered trachea and provided a guidance of further investigations.

Recent Advances for Enhancing Drug Metabolizing Functions of Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells / 한양의대학술지

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells are a promising cell source for drug screening and toxicity tests. Thus, various hepatic differentiating protocols have been developed, leading to a hepatic differentiation efficiency of approximately 90%. However, HLC drug metabolizing ability remains very low compared to human primary hepatocytes. In order to overcome this problem, several alternative methods, such as, co-culture, three-dimensional (3D) culture, bioreactor, nanochip-based, etc., have been developed, but optimization to produce fully functional HLCs is ongoing. Recently, our group reported that repeated exposure of HLCs to xenobiotics can improve the expression of hepatic metabolizing enzymes such as cytochrome P450s (CYPs) and glutathione S-transferases (GSTs). These data suggest that we should develop strategies for differentiating cells into mature HLCs by more closely mimicking in vivo fetal and postnatal liver development. Here, we review the current development of alternative methods for enhancing the drug metabolizing functions of HLCs derived from human embryonic stem cells, human-induced pluripotent stem cells, and mesenchymal stem cells as used for drug screening and toxicity tests.

Double-chamber stirred bioreactor improves the repaired effect of beta-tricalcium phosphate on goat knee cartilage defects / 中国组织工程研究

BACKGROUND:A preliminary experiment developed a double-chamber stirred bioreactor which can carry out osteogenic and cartilage induction at the same time. OBJECTIVE:To explore the effects of double-chamber stirred bioreactor on the repair of goat knee cartilage defects with tissue-engineered cartilage. METHODS:Twelve goats were selected to make bilateral femoral condyle osteochondral defects models and randomized to three groups:experimental group, implanted with the composites ofβ-tricalcium phosphate and bone marrow mesenchymal stem cells that were subjected to 2-week chondrogenic and osteogenic induction simultaneously in the double-chamber stirred bioreactor under mechanical stimulation;control group, implanted with the composites ofβ-tricalcium phosphate and bone marrow mesenchymal stem cells that were subjected to 2-week chondrogenic and osteogenic induction simultaneously in the double-chamber stirred bioreactor;blank control group, without treatment. After 12 and 24 weeks of implantation, general observation, Masson staining, II col agen immunohistochemical staining and histological scoring were performed. RESULTS AND CONCLUSION:In the experimental and control groups, new cartilage tissue and bone tissue were visible, but the experimental group showed better repair effects than the control group (P<0.05). The blank control group had no cartilage formation. These findings indicate that under the mechanical stimulation by the double-chamber stirred bioreactor in vitro, the repair effect of tissue-engineered osteochondral complex on knee joint cartilage defects can be improved.

Effects of oscillatory flow on shear stress distributions in perfusion bioreactor for bone tissue engineering / 医用生物力学

Objective To study the effects of oscillatory flow, as well as pore size and porosity of the 3D scaffold on distributions of flow rate and shear stress in perfusion bioreactor, and to propose optimization methods for preparing the 3D decellularized bone scaffold and perfusion bioreactor based on theoretical results. Methods Based on the previously established 3D decelluarized scaffold and perfusion bioreactor for bone tissue engineering in the laboratory, the decelluarized scaffold was simplified as an isotropic porous media. The velocity and shear stress distributions in the bioreactor were further simulated theoretically. Results Under the oscillatory flow, the Darcy shear stress and velocity in the 3D porous scaffold presented a consistently regular pattern. Compared with the unidirectional flow, the difference of velocity and Darcy shear stress decreased at different radius, which could contribute to the homogeneous 3D culture of seed cells in bone tissue engineering. Increasing the inlet perfusion velocity could improve the average Darcy shear stress. Increasing the pore diameter or porosity of the scaffold had no obvious effects on the peak value of velocity, but sharply reduced the average Darcy shear stress. Increasing inlet oscillation frequency could decrease the peak value of velocity and obviously decrease the difference of velocity at different radius. Conclusions Appropriate oscillatory flow was beneficial for generating required shear stress for stem cells in bone tissue engineering. The research findings in this study are expected to provide theoretical guidance to optimize the 3D culture method of seed cells for bone tissue engineering.

Cartilage tissue engineering by electrospun PCL scaffolds seeded with rabbit chondrocytes under flow perfusion culture in vitro / 中华骨科杂志

Objective To investigate the chondrocyte proliferation and extracellular matrix biosynthesis of electrospun PCL scaffolds seeded with rabbit chondrocytes under flow perfusion culture in vitro.Methods Nonwoven PCL microfiber mats were fabricated,and contra-aperture cylindrical glass equipment as a perfusion bioreactor was designed and manufactured on our own.The experiment included peffusion culture group and static culture group.Primary chondrocytes were isolated from the knee joints of two-month-old New Zealand white rabbits and seeded into scaffolds.The scaffold-cell complexes were harvested at 3,7,and 14 days of culture for scanning electron micrograph (SEM) analysis,biochemical assay,real-time PCR and histology analysis.Results Electrospun PCL scaffolds were composed of microfibers with a diameter of 1.67±0.76 μm and pores with a diameter of 17.65±7.11 μm.SEM showed a better cell proliferation with typical morphology of chondrocytes under perfusion culture.At 7 days of culture,DNA content in perfusion culture group was higher than in static culture group.At 3,7 and 14 days of culture,compared with the static culture group,glycosaminoglycan (GAG) content and GAG/DNA ratio in perfusion culture group were higher,and the differences were statistically significant.At 14 days of culture,real-time PCR showed aggrecan and collagen type Ⅱ gene expression and collagen type Ⅱ to collagen type Ⅰ ratio were higher in perfusion culture group than in static culture group; HE and safranin O staining showed a significant cell proliferation,infiltration,as well as extracellular matrix biosynthesis in perfusion culture group.Conclusion Under flow perfusion culture,the electrospun PCL scaffolds seeded with rabbit chondrocytes can enhance chondrocyte proliferation and extracellular matrix biosynthesis,which is a promising method for cartilage tissue engineering.

Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05), indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

The optimum flow shear stress and the mass transport benefiting for construction of the tissue-engineered bone combining computational fluid dynamics / 中华骨科杂志

Objective To explore the optimum flow shear stress and mass transport for the construction of tissue-engineered bone.Methods The β-tricalcium phosphate (β-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (HBMMSCs) were cultured in perfusion bioreactor.When the same flow rate was applied,the flow shear stress was separately 1×,2× and 3×.When the same flow shear stress was applied,the flow rates were separately 3 ml/min,6 ml/min and 9 ml/min.Cell proliferation was measured by MTT method.The construction of tissue-engineered bone was evaluated by measuring alkaline phosphatase (AKP) activity,secretion of osteopontin (OP) and osteocalcin (OC),and the mineralization of extracellular matrix (ECM).The flow shear stress and the mass transport were obtained using computational fluid dynamics.Results When the flow rate was same,the most cell proliferation was found in 2× group.The AKP activity and secretion of OC was higher in 2× and 3× groups than in those in 1× group.After 28days,the highest amount of mineralization of ECM was found in 3× group.When the flow shear stress was same,the AKP activity was highest in 6 ml/min group.After 28 days,secretion of OC and formation of mineralized ECM was highest in 3 ml/min group.When the flow rate was same,the flow shear stress was separately 0.004-0.007 Pa,0.009-0.013 Pa and 0.013-0.018 Pa.When the flow shear stress was same,the flow rate was separately 0.267-0.384 mm/s,0.521-0.765 mm/s and 0.765-1.177 mm/s.Conclusion When the tissue-engineered bone was constructed,0.013-0.018 Pa flow shear stress and 0.267-0.384 mm/s mass transport velocity could improve the construction of the tissue-engineered bone in vitro.

Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation.

Use of Candida rugosa lipase immobilized on sepabeads for the amyl caprylate synthesis: batch and fluidized bed reactor study

Lipase from Candida rugosa was covalently immobilized on Sepabeads EC-EP for application for amyl caprylate synthesis in an organic solvent system. Several solvents were tested in terms of biocatalyst stability and the best result was obtained with isooctane. The lipase-catalyzed esterification in the selected system was performed in batch and fluidized bed reactor systems. The influence of several important reaction parameters including temperature, initial water content, enzyme loading, acid/alcohol molar ratio, and time of addition of molecular sieves is carefully analyzed by means of an experimental design. Almost complete conversion (> 99 percent) of the substrate to ester could be performed in a batch reactor system, using lipase loading as low as 37 mg g-1 dry support and in a relatively short time (24 hrs) at 37°C, when high initial substrate molar ratio of 2.2 is used. Kinetics in a fluidized bed reactor system seems to still have a slightly better profile than in the batch system (90.2 percent yields after 14 hrs). The fluidized bed reactor operated for up 70 hrs almost with no loss in productivity, implying that the proposed process and the immobilized system could provide a promising approach for the amyl caprylate synthesis at the industrial scale.

Overview of parameters influencing biomass and bioreactor performance used for extracellular ligninase production from Phanerochaete chrysosporium

The production of extracellular enzymes is gaining momentum as commercial interests seek alternative ways to improve the productivity in the biotechnology and pharmaceutical industries. Early research studies looked at improving batch bioreactor operational challenges; however, the use of continuous cultures was indicated to be favourable. This led to a new approach developed to produce extracellular enzymes continuously using fixed-film bioreactors from biofilms immobilised on polymeric and inorganic membranes. In this review, the performance of P. chrysosporium biomass, evaluated in terms of ligninase production using different bioreactor operation conditions, is highlighted. Furthermore, the limitations related to the implementation of optimised batch culture conditions to continuous fixed-film bioreactors are discussed. DO transportation, trace element toxicity and lipid peroxidation effects on P. chrysosporium biomass in fixed-film bioreactors operated for elongated periods, are also discussed.

Aproximación al cultivo de condrocitos en la Universidad Nacional de Colombia. Reporte técnico / Approach to chondrocytes culture in National University of Colombia. A technical report

La pérdida de un tejido o de su función, debido a defectos congénitos, enfermedad o trauma, es uno de los problemas más difíciles, frecuentes y costosos que enfrenta la medicina humana. La degeneración del cartílago articular se conoce como osteoartrosis (OA), la cual se manifiesta mediante daño enzimático y mecánico de la matriz extracelular, que genera dolor y conduce a pérdida del movimiento e inestabilidad articular. El cartílago articular tiene una baja capacidad de reparación, por su avascularidad. El presente trabajo pretende realizar una aproximación a la ingeniería tisular del cartílago con el propósito de establecer la fundamentación teórica en el tema para comprender el comportamiento in vitro del tejido que permita a futuro construir un equivalente de cartílago mediante los procedimientos actuales de ingeniería tisular, que ofrezca respuesta a la problemática social y de salud pública. Las conclusiones del presente trabajo posibilitan futuras investigaciones en el campo de la regeneración de cartílago articular y su aplicación clínica e igualmente brindan las bases para plantear investigaciones in vivo o in vitro que profundicen en el tema

The loss of a tissue or its function due to congenital defects, disease or trauma, is one of the major, frequent and expensive problems challenging the human medicine. The articular cartilage degeneration is known as osteoarthritis (OA), which is characterized by enzyme and mechanical damage of extracellular matrix, generating pain and lead to the loss of movement and articular instability. The articular cartilage has low repair ability due to its avascularization. The aim of present paper is to make an approach to cartilage tissue engineering to establish the theoretical basis on this subject to understand the tissue in vitro behavior allowing in the future, to construct a cartilage equivalent by means of current procedures of tissue engineering, offering a response to social problems and of the public health. Conclusions from present paper make easy futures researches in the field of articular cartilage regeneration and its clinical application and also, providing the bases to planning in vivo and in vitro researches to deepen in this subject

Modification of bioreactor for tissue-engineered heart valve and its application / 第二军医大学学报

Objective: To modify the pulsatile bioreactor we constructed previously for simulating the high-flow, high-pressure hemodynamics of heart valve in vivo, and to experimentally cultivate the tissue-engineered heart valves (TEHV) in the modified bioreactor. Methods: T-PLS system (NewheartBio Co., Ltd Korea) was used to generate pulsatile f1ow in the modified bioreactor and we designed a new air-exchange pathway to avoid contamination. The TEHV were made by seeding human bone mesenchymal stem cells (BMSCs) on de cellularized porcine heart valve. After cultured under static condition for 4 d, the TEHVs were moved to the modified bioreactor and exposed to low-flow (0-600 ml/min) or high-flow(0-4 800 ml/min) pulsatile hydrodynamics for 7d, then the cells on TEHVs were evaluated. Results: After modification, the flow range expanded from (0-1 200) ml/min to (0-6000) ml/min and the pressure range expanded from (0 40) mmHg to (0-180) mmHg. In culture experiments, 26.3% of the seeded cells remained under low-flow environment and cells were completely lost under the high-flow dynamics. Conclusion: The modified bioreactor can basically simulate the dynamics of heart valve in vivo and can be used in TEHV cultivation research. However, the current TEHV can not tolerate the high-flow pulsatile hydrodynamics.

Repair of cartilage defects using chondrocyte and osteoblast composites in vitro cultured in bioreactor / 中华创伤杂志

Objective To assess the feasibility of chondrocyte and osteoblast composites in vitro cultured in bioreactor in repairing cartilage defects.Methods Marrow mesenchymal stem cells were isolated and cultured in vitro,and then were induced to chondrocytes and osteoblasts by growth factor.Chondrocytes and osteoblasts were cocultured in bioreactor for 21 days to form the composites.The adhesion,extension and proliferation of chondrocytes and osteoblasts were observed under scanning electron microscope.The cartilage defects on canine model were repaired with the chondrocyte and osteoblast composites.Results The induced chondrocytes and osteoblasts had fine adhesion,extension and proliferation in the β-TCP scaffold.The repaired tissues in experimental group maintained their thickness to the full depth of the original tissues.A statistical difference was observed between negative control group and experimental group(q=12.337 0,P ＜ 0.01)and between blank control group and experimental group (q=31.539 3,P ＜0.01).Conclusion Perfusion bioreactor makes chondrocyte and osteoblast survive and proliferate in a three-dimensional scaffold and increases the composition rate of the chondrocyte and osteoblast.

Utilization of the biorreactor of imersion by bubbles at the micropropagation of Ananas comosus L. Merril

The research for new techniques of in vitro cultivation is being object of many studies around the world, in order to optimize and decrease production costs of seedlings with agronomical interest. The main goal of this work was to compare different systems of in vitro cultivations using Ananas comosus L. Merril. So, the in vitro growth of the plantlets was promoted in two different bioreactors: Bioreactor of Immersion by Bubbles (B.I.B.®) and the Reactor of Temporary Immersion (R.I.T.A.®) with immersion cycle every 2 hours for 15 minutes and the traditional system in flasks with 200 mL. All cultivation systems used the MS liquid nutritive solution, supplemented with BAP (1 mgL-1), ANA (0.25 mgL-1), sucrose (30 gL-1) and Tween 20® (0.5 µL). The pH was adjusted to 5.8 and sterilized at 120°C for 15 minutes. The cultures were kept into a growth room during 30 days, with controlled temperature of 25±2°C, under white cold light (46.8 µmol.m-2.s-1), with photoperiod of 16 hours. The experimental design used was randomized, with three treatments, three repetitions and ten plants each stage. Among the evaluated systems, the BIB® presented the best results for the tested variables, mainly the total number of shoots, being 2.3 e 3.1 times superior when compared with the system R.I.T.A.® and the traditional consecutively. So the system of immersion by bubbles turns into an effective equipment to produce seedlings of pineapple in large scale.

A busca por novas técnicas de cultivo in vitro vem sendo amplamente estudadas, visando otimizar e baixar o custo de produção das mudas que tenham interesse agronômico. O objetivo deste trabalho foi comparar diferentes sistemas de cultivo in vitro de Ananas comosus L. Merril. Para tanto, o crescimento in vitro de plântulas foi promovido em sistemas de biorreatores (B.I.B.® e R.I.T.A.®) com ciclo de imersão a cada 2 horas por 15 minutos e o sistema tradicional em frascos de 200 mL. Em todos os sistemas de cultivo, foram utilizadas solução nutritiva líquida MS, suplementado com 1 mg L-1 de BAP, 0,25 mg L-1 de ANA, 30 g L-1 de sacarose e 0,5 µL de Tween® 20, pH ajustado para 5,8 e autoclavagem a 120°C por 15 minutos. As culturas foram mantidas em sala de crescimento durante 30 dias, temperatura controlada de 25±2°C, sob luz branca fria (46,8 µmol.m-2.s-1), com 16 horas de fotoperíodo. O delineamento foi inteiramente casualizado, com três tratamentos, três repetições e dez plantas por estágios. Para os sistemas avaliados, o biorreator de imersão por bolhas apresentou os melhores resultados dentre as variáveis analisadas, com destaque ao número total de brotações, sendo 2,3 e 3,1 vezes superiores quando comparado com o sistema R.I.T.A.® e sistema tradicional respectivamente. Portanto, o sistema de imersão por bolhas torna-se um equipamento eficaz na produção de mudas de abacaxizeiro em larga escala.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Proteins in a DNA world: expression systems for their study / Proteínas en el mundo del DNA: Sistemas de expresión para su estudio

Every day, new proteins are discovered and the need to understand its function arises. Human proteins have a special interest, because to know its role in the cell may lead to the design of a cure for a disease. In order to obtain such information, we need enough protein with a high degree of purity, and in the case of the human proteins, it is almost impossible to achieve this by working on human tissues. For that reason, the use of expression systems is needed. Bacteria, yeast, animals and plants have been genetically modified to produce proteins from different species. Even "cell-free" systems have been developed for that purpose. Here, we briefly review the options with their advantages and drawback, and the purification systems and analysis that can be done to gain understanding on the function and structure of the protein of interest.

Cada día, nuevas proteínas son descubiertas y surge la necesidad de caracterizarlas, siendo las de origen humano las que presentan un mayor interés. Conocer su función nos ayudará a entender padecimientos y diseñar una posible cura. Sin embargo, obtener suficiente cantidad de proteínas humanas en cantidad para llevar a cabo los análisis pertinentes, presenta una gran dificultad. Por tal razón, es necesario el uso de sistemas de expresión de proteínas heterólogas. Bacterias, levaduras, animales y plantas han sido modificados genéticamente para expresar proteínas de otras especies, e incluso sistemas in vitro han sido desarrollados para producir proteínas. En este artículo se revisan brevemente las opciones con sus ventajas y desventajas, así como las estrategias de purificación y los análisis que se pueden llevar a cabo para avanzar en el conocimiento de la función y estructura de la proteína de interés.