Engineering superficial zone features in tissue engineered cartilage

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion‐induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Biotechnol. Bioeng. 2013; 110: 1476–1486. © 2012 Wiley Periodicals, Inc.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

A test facility for fritted spargers of production-scale-bioreactors

The production of therapeutic proteins requires qualification of equipment components and appropriate validation procedures for all operations. Since protein productions are typically performed in bioreactors using aerobic cultivation processes air sparging is an essential factor. As recorded in literature, besides ring spargers and open pipe, sinter frits are often used as sparging elements in large scale bioreactors. Due to the manufacturing process these frits have a high lot-to-lot product variability. Experience shows this is a practical problem for use in production processes of therapeutic proteins, hence frits must be tested before they can be employed. The circumstance of checking quality and performance of frits as sparging elements was investigated and various possibilities have been compared. Criteria have been developed in order to evaluate the sparging performance under conditions comparable to those in production bioreactors. The oxygen mass transfer coefficient (k _L a) was chosen as the evaluation criterion. It is well known as an essential performance measure for fermenters in the monoclonal antibody production. Therefore a test rig was constructed able to automatically test frit-spargers with respect to their k _L a-values at various gas throughputs. Performance differences in the percent range could be detected.     

我国巴马小型猪SLA-2基因克隆及分子特征

为研究我国巴马小型猪SLA-Ⅰ分子特征,设计引物克隆了SLA-Ⅰ类分子SLA-2基因(SLA-2bm),并通过分子生物学软件分析其分子特征。经克隆及序列测定分析,SLA-2bm为1119bp,其中3~1097为ORF区,共编码364个氨基酸,分别在第125、188、227和283位置出现半胱氨酸残基,含有两对链内二硫键。氨基酸同源性分析显示SLA-2bm与其它SLA-2、SLA-3和SLA-1序列的同源率分别为88.4%~96.4%、88.3%~90.5%和87.7%~92.7%。系统进化树显示SLA-2bm与其它SLA-2等位基因在遗传关系上相对独立,进化程度较低;各功能区分析,SLA-2bm与人HLA-A2和小鼠H-2K分子结构相似,且保留了人HLA-A2基因的部分功能位点。结果表明,SLA-2bm属于一个新的等位基因,巴马小型猪是保留原始基因特征的品种。     

植物基因组中微型反向重复转座元件研究进展

微型反向重复转座元件(miniature inverted repeat transposable element,MITE)是一类特殊的转座元件,在结构上与有缺失的DNA转座子相似,但具有反转录转座子高拷贝数的特点.MITE时常与基因相伴,对基因调控可能起重要作用,因此,MITE正逐渐成为基因和基因组进化及生物多样性研究的一种重要工具.本文综述了植物基因组MITE的结构、分类、活性及其应用研究进展.     

Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19K and Aven in the production of a monoclonal antibody with Chinese hamster ovary cells

Mammalian cells are used for the production of numerous biologics including monoclonal antibodies. Unfortunately, mammalian cells can lose viability at later stages in the cell culture process. In this study, the effects of expressing the anti-apoptosis genes, E1B-19K and Aven, separately and in combination on cell growth, survival, and monoclonal antibody (MAb) production were investigated for a commercial Chinese Hamster Ovary (CHO) mammalian cell line. CHO cells were observed to undergo apoptosis following a model insult, glucose deprivation, and at later stages of batch cell culture. The CHO cell line was then genetically modified to express the anti-apoptotic proteins E1B-19K and/or Aven using an ecdysone-inducible expression system. Stable transfected pools induced to express Aven or E1B-19K alone were found to survive 1-2 days longer than the parent cell line following glucose deprivation while the expression of both genes in concert increased cell survival by 3 days. In spinner flask batch studies, a clonal isolate engineered to express both anti-apoptosis genes exhibited a longer operating lifetime and higher final MAb titer as a result of higher viable cell densities and viabilities. Interestingly, survival was increased in the absence of an inducer, most likely as a result of leaky expression of the anti-apoptosis genes confirmed in subsequent PCR studies. In fed-batch bioreactors, the expression of both anti-apoptosis genes resulted in higher growth rates and cell densities in the exponential phase and significantly higher viable cell densities, viabilities, and extended survival during the post-exponential phase. As a result, the integral of viable cells (IVC) was between 40 and 100% higher for cell lines engineered to express both Aven and E1B-19K in concert, and the operational lifetime of the fed-batch bioreactors was increased from 2 to 5 days. The maximum titers of MAb were also increased by 40-55% for bioreactors containing cells expressing Aven and E1B-19K. These increases in volumetric productivity arose primarily from enhancements in viable cell density over the course of the fed-batch culture period since the specific productivities for the cells expressing anti-apoptosis genes were comparable or slightly lower than the parental hosts. These results demonstrate that expression of anti-apoptosis genes can enhance culture performance and increase MAb titers for mammalian CHO cell cultures especially under conditions such as extended fed-batch bioreactor operation.     

Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment

The present study was carried out to determine (1) the optimal duty cycle of ultrasound for activation of pig oocytes and cloned embryos derived from miniature pig fetal fibroblasts and (2) whether cloned embryos can develop to term following activation by ultrasound stimulation. When oocytes were exposed to ultrasound with 20% or 30% duty cycle, the blastocyst formation rates were significantly (P < 0.05) higher than that of oocytes exposed to ultrasound with 10% duty cycle. In contrast, the blastocyst formation rate of cloned embryos decreased as the duty cycle of ultrasound increased; the value of embryos exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that of embryos exposed to ultrasound with 50% duty cycle. When cloned embryos exposed to ultrasound with 10% duty cycle were transferred into the oviducts of two recipient gilts to assess their development in vivo, the pregnancy of one of the gilts was maintained to term and two piglets were delivered via Cesarean section. The results of the present study showed that (1) although the duty cycle of ultrasound affects in vitro development after activation of both pig oocytes and miniature pig cloned embryos, the optimal duty cycle is different between them and (2) miniature pig cloned embryos have the ability to develop into piglets after activation by ultrasound stimulation.     

Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral-mediated gene transfer

BACKGROUND: Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig. METHODS: Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR). RESULTS: The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues. CONCLUSIONS: Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs.     

Microalgae wastewater treatment: Biological and technological approaches

Current global environmental issues raise unavoidable challenges for our use of natural resources. Supplying the human population with clean water is becoming a global problem. Numerous organic and inorganic impurities in municipal, industrial, and agricultural waters, ranging from microplastics to high nutrient loads and heavy metals, endanger our nutrition and health. The development of efficient wastewater treatment technologies and circular economic approaches is thus becoming increasingly important. The biomass production of microalgae using industrial wastewater offers the possibility of recycling industrial residues to create new sources of raw materials for energy and material use. This review discusses algae‐based wastewater treatment technologies with a special focus on industrial wastewater sources, the potential of non‐conventional extremophilic (thermophilic, acidophilic, and psychrophilic) microalgae, and industrial algae‐wastewater treatment concepts that have already been put into practice.