Mechanical Testing of Cartilage Constructs
A key goal of functional cartilage tissue engineering is to develop constructs with mechanical properties approaching those of the native tissue. Herein we describe a number of tests to characterize the mechanical properties of tissue engineered cartilage. Specifically, methods to determine the equilibrium confined compressive (or aggregate) modulus, the equilibrium unconfined compressive (or Young’s) modulus, and the dynamic modulus of tissue engineered cartilaginous constructs are described. As these measurements are commonly used in both the articular cartilage mechanics literature and the cartilage tissue enginee...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Proteomic Analysis of Engineered Cartilage
Tissue engineering holds promise for the treatment of damaged and diseased tissues, especially for those tissues that do not undergo repair and regeneration readily in situ. Many techniques are available for cell and tissue culturing and differentiation of chondrocytes using a variety of cell types, differentiation methods, and scaffolds. In each case, it is critical to demonstrate the cellular phenotype and tissue composition, with particular attention to the extracellular matrix molecules that play a structural role and that contribute to the mechanical properties of the resulting tissue construct. Mass spectrometry prov...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Transplantation of Tissue-Engineered Cartilage in an Animal Model (Xenograft and Autograft): Construct Validation
Tissue engineering holds great promise for cartilage repair with minimal donor-site morbidity. The in vivo maturation of a tissue-engineered construct can be tested in the subcutaneous tissues of the same species for autografts or of immunocompromised animals for allografts or xenografts. This section describes detailed protocols for the surgical transplantation of a tissue-engineered construct into an animal model to assess construct validity. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Microbioreactors for Cartilage Tissue Engineering
In tissue engineering research, cell-based assays are widely utilized to fundamentally explore cellular responses to extracellular conditions. Nevertheless, the simplified cell culture models available at present have several inherent shortcomings and limitations. To tackle the issues, a wide variety of microbioreactors for cell culture have been actively proposed, especially during the past decade. Among these, micro-scale cell culture devices based on microfluidic biochip technology have particularly attracted considerable attention. In this chapter, we not only discuss the advantageous features of using micro-scale cell...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Shear and Compression Bioreactor for Cartilage Synthesis
Mechanical forces, including hydrodynamic shear, hydrostatic pressure, compression, tension, and friction, can have stimulatory effects on cartilage synthesis in tissue engineering systems. Bioreactors capable of exerting forces on cells and tissue constructs within a controlled culture environment are needed to provide appropriate mechanical stimuli. In this chapter, we describe the construction, assembly, and operation of a mechanobioreactor providing simultaneous dynamic shear and compressive loading on developing cartilage tissues to mimic the rolling and squeezing action of articular joints. The device is suitable for...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Mechanobioreactors for Cartilage Tissue Engineering
Mechanical stimulation is an effective method to increase extracellular matrix synthesis and to improve the mechanical properties of tissue-engineered cartilage constructs. In this chapter, we describe valuable methods of imposing direct mechanical stimuli (compression or shear) to tissue-engineered cartilage constructs as well as some common analytical methods used to quantify the effects of mechanical stimuli after short-term or long-term loading. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Decellularized Extracellular Matrix Scaffolds for Cartilage Regeneration
Decellularized extracellular matrix (ECM) is gaining a lot of attention as a biomaterial for tissue engineering applications. This chapter describes the processing techniques for decellularization of cell-derived ECM and protocols for the fabrication of ECM-based scaffolds in the form of hydrogels or fibrous polymer meshes by electrospinning. It describes the protocols to analyze the morphology and presence of collagen in fabricated scaffolds using scanning electron microscope and Picrosirius Red staining respectively. Methods to evaluate the metabolic activity and proliferation of cells (resazurin-based assay and DNA assa...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Hydrogels with Tunable Properties
This chapter describes the preparation of tissue engineered constructs by immobilizing chondrocytes in hydrogel with independently tunable porosity and mechanical properties. This chapter also presents the methods to characterize these tissue engineered constructs. The resulting tissue engineered constructs can be useful for the generation of cartilage tissue both in vitro and in vivo. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Stratified Scaffolds for Osteochondral Tissue Engineering
Stratified scaffolds are promising devices finding application in the field of osteochondral tissue engineering. In this scaffold type, different biomaterials are chosen to fulfill specific features required to mimic the complex osteochondral tissue interface, including cartilage, interlayer tissue, and subchondral bone. Here, the biomaterials and fabrication methods currently used to manufacture stratified multilayered scaffolds as well as cell seeding techniques for their characterization are presented. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Nanostructured Capsules for Cartilage Tissue Engineering
Polymeric multilayered capsules (PMCs) have found great applicability in bioencapsulation, an evolving branch of tissue engineering and regenerative medicine. Here, we describe the production of hierarchical PMCs composed by an external multilayered membrane by layer-by-layer assembly of poly(l-lysine), alginate, and chitosan. The core of the PMCs is liquified and encapsulates human adipose stem cells and surface-functionalized collagen II-TGF-β3 poly(l-lactic acid) microparticles for cartilage tissue engineering. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Scaffolds for Controlled Release of Cartilage Growth Factors
In recent years, cell-based therapies using adult stem cells have attracted considerable interest in regenerative medicine. A tissue-engineered construct for cartilage repair should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable and biocompatible poly (d,l-lactide-co-glycolide acid) (PLGA), are a unique system which combines these properties in an adaptable and simple microdevice. This device relies on nanoprecipitation of proteins encapsulated in polymer...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Bioprinted Scaffolds for Cartilage Tissue Engineering
Researchers are focusing on bioprinting technology as a viable option to overcome current difficulties in cartilage tissue engineering. Bioprinting enables a three-dimensional (3-D), free-form, computer-designed structure using biomaterials, biomolecules, and/or cells. The inner and outer shape of a scaffold can be controlled by this technology with great precision. Here, we introduce a hybrid bioprinting technology that is a co-printing process of multiple materials including high-strength synthetic polymer and cell-laden hydrogel. The synthetic polymer provides mechanical support for shape maintenance and load bearing, w...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Use of Interim Scaffolding and Neotissue Development to Produce a Scaffold-Free Living Hyaline Cartilage Graft
The fabrication of three-dimensional (3D) constructs relies heavily on the use of biomaterial-based scaffolds. These are required as mechanical supports as well as to translate two-dimensional cultures to 3D cultures for clinical applications. Regardless of the choice of scaffold, timely degradation of scaffolds is difficult to achieve and undegraded scaffold material can lead to interference in further tissue development or morphogenesis. In cartilage tissue engineering, hydrogel is the highly preferred scaffold material as it shares many similar characteristics with native cartilaginous matrix. Hence, we employed gelatin...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Gene Transfer and Gene Silencing in Stem Cells to Promote Chondrogenesis
In stem cell-based chondrogenesis for articular cartilage regeneration, TGF-β3 is dosed to the stem cells to drive differentiation into chondrocytic cells. Meanwhile, type I collagen, which is endogenously expressed in some stem cells (e.g., synovium-derived mesenchymal stem cells) and upregulated by TGF-β3, poses a threat to chondrogenesis, as type I collagen may alter the components and stiffness of articular cartilage. Therefore, a wiser strategy would be to feed the cells with TGF-β3 while at the same time silencing the expression of type I collagen. In this chapter, methods for construction of adenovira...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Differentiation of Human Induced Pluripotent Stem Cells to Chondrocytes
Human induced pluripotent stem (iPS) cells are relevant tools for modeling human skeletal development and disease, and represent a promising source of patient-specific cells for the regeneration of skeletal tissue, such as articular cartilage. Devising efficient and reproducible strategies, which closely mimic the physiological chondrogenic differentiation process, will be necessary to generate functional chondrocytes from human iPS cells. Our previous study demonstrated the generation of chondrogenically committed human iPS cells via the enrichment of a mesenchymal-like progenitor population, application of appropriate hi...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Derivation and Chondrogenic Commitment of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors
The induction of human embryonic stem cells to a mesenchymal-like progenitor population constitutes a developmentally relevant approach for efficient directed differentiation of human embryonic stem (hES) cells to the chondrogenic lineage. The initial enrichment of a hemangioblast intermediate has been shown to yield a replenishable population of highly purified progenitor cells that exhibit the typical mesenchymal stem cell (MSC) surface markers as well as the capacity for multilineage differentiation to bone, fat, and cartilage. Herein, we provide detailed methodologies for the derivation and characterization of potent m...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Mesenchymal Stem Cells Derived from Human Adipose Tissue
Human adult mesenchymal stem cells are present in fat tissue, which can be obtained using surgical procedures such as liposuction. The multilineage capacity of mesenchymal stem cells makes them very valuable for cell-based medical therapies. In this chapter, we describe how to isolate mesenchymal stem cells from human adult fat tissue, propagate the cells in culture, and cryopreserve the cells for tissue engineering applications. Flow cytometry methods are also described for identification and characterization of adipose-derived stem cells and for cell sorting. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Mesenchymal Stem Cells Derived from Human Bone Marrow
Mesenchymal stem cells are found in a number of tissues and have the potential to differentiate into a range of mesenchymal lineages. This ready availability and multipotent character means that mesenchymal stem cells have become a focus for the field of tissue engineering, particularly for the repair of bone and cartilage. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Human Fetal and Adult Chondrocytes
As the only cell type found in healthy adult cartilage, chondrocytes are the obvious and most direct starting point for cartilage tissue engineering. Human adult, juvenile, neonatal, and fetal chondrocytes have all been demonstrated to produce cartilage matrix components in vitro for production of engineered tissues. In this chapter, procedures are outlined for isolation of chondrocytes from human fetal and adult cartilage. Methods for expansion and cryopreservation of the cells and characterization of gene expression using quantitative polymerase chain reaction (Q-PCR) analysis are also described. (Source: Springer protoc...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Cartilage Tissue Engineering: What Have We Learned in Practice?
Many technologies that underpin tissue engineering as a research field were developed with the aim of producing functional human cartilage in vitro. Much of our practical experience with three-dimensional cultures, tissue bioreactors, scaffold materials, stem cells, and differentiation protocols was gained using cartilage as a model system. Despite these advances, however, generation of engineered cartilage matrix with the composition, structure, and mechanical properties of mature articular cartilage has not yet been achieved. Currently, the major obstacles to synthesis of clinically useful cartilage constructs are our in...
Source: Springer protocols feed by Biotechnology - September 14, 2015 Category: Biotechnology Source Type: news

Fluorescence Labeling of Short RNA by Oxidation at the 3′-End
In RNA nanotechnology, construction of nanoparticles involves conjugation of functionalities, cross-linking of modules, labeling of RNA subunits, and chemical modification of nucleotides. Efficiency and sensitivity are important for the RNA labeling, which also can be used as probes in microarrays, Northern blotting, and gel-shift assays. Here, we describe a method for fluorescence labeling of short RNA at the 3′-end by oxidation. The 3′-terminus of in vitro-transcribed short RNA is oxidized by sodium periodate, and fluorescein-5-thiosemicarbazide is added after removal of excess oxidant. Purified short RNA wit...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Functional Assays for Specific Targeting and Delivery of RNA Nanoparticles to Brain Tumor
Cumulative progress in nanoparticle development has opened a new era of targeted delivery of therapeutics to cancer cells and tissue. However, developing proper detection methods has lagged behind resulting in the lack of precise evaluation and monitoring of the systemically administered nanoparticles. RNA nanoparticles derived from the bacteriophage phi29 DNA packaging motor pRNA have emerged as a new generation of drugs for cancer therapy. Multifunctional RNA nanoparticles can be fabricated by bottom-up self-assembly of engineered RNA fragments harboring targeting (RNA aptamer or chemical ligand), therapeutic (siRNA, miR...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Aptamer-Mediated Nanoparticle Interactions: From Oligonucleotide–Protein Complexes to SELEX Screens
Aptamers are oligonucleotides displaying specific binding properties for a predetermined target. They can be easily immobilized on various surfaces such as nanoparticles. Functionalized particles can then be used to various aims. We took advantage of the AlphaScreen® technology for monitoring aptamer-mediated interactions. A particle bearing an aptamer contains a photosensitizer whereas another type of particle contains a chemiluminescer. Irradiation causes the formation of singlet oxygen species in the photosensitizer-containing bead that in turn activates the chemiluminescer. Luminescence emission can be observed if ...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

A High-Throughput Screening Assay for the Functional Delivery of Splice-Switching Oligonucleotides in Human Melanoma Cells
Since the conception of RNA nanotechnology (Cell, 94:147, 1998), there has been tremendous interest in its application for the functional delivery of RNA into cells. Splice-switching oligonucleotides (SSOs) are an emerging antisense drug class with the ability to therapeutically modify gene expression. A wide variety of chemical modifications have been devised to try to increase the activity and stability of SSOs. Also, as with most nucleic acid therapeutics, delivery into the cell is the major hurdle for in vivo and clinical applications. As a result, various RNA nanoparticles are being constructed for targeted delivery o...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Methods for Assembling B-Cell Lymphoma Specific and Internalizing Aptamer–siRNA Nanoparticles Via the Sticky Bridge
Structured functional RNA entities, including aptamers and siRNAs, have amazing versatility in structure and function. These molecules can serve as powerful, attractive building blocks for the bottom-up assembly of complex nanostructures. Here, we describe novel cell-type specific and internalizing B-cell activating factor receptor (BAFF-R) aptamer–siRNA delivery systems for B-cell lymphoma therapy, in which both the aptamer and the Dicer substrate siRNA (DsiRNA) portions are conjugated through a “sticky bridge.” The BAFF-R is overexpressed on the surface of B-cell malignancies, allowing binding and inter...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Design, Assembly, and Evaluation of RNA–Protein Nanostructures
The use of RNA–protein interaction motifs (RNP motifs) to design and build nanoscale objects has the potential to expand the field of RNA nanotechnology. In principle, RNP motifs can be integrated easily into RNA nano objects, providing an alternative technique to increase the functional and structural complexities of the RNA. Investigating the design principles of RNP nanostructures will enable the construction of highly sophisticated biomacromolecular complexes such as ribosomes from scratch. As an initial step towards this goal, we designed and constructed triangular-like nanostructures by employing box C/D kink-t...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Mapping RNA Interactions to Proteins in Virions Using CLIP-Seq
RNA nanotechnology often involves protein–RNA complexes that require significant understanding of how the proteins and RNAs contact each other. The CLIP-Seq (cross-linking immunoprecipitation, and DNA sequencing) protocol can be used to probe the RNA molecules that interact with proteins. We have optimized the procedures for RNA fragmentation, immunoprecipitation, and library construction in CLIP-Seq to map the interactions between the RNA and the capsid of a simple positive-strand RNA virus. The results show that distinct portions of the viral RNA contact the capsid. The protocol should be applicable to other RNA vi...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Multiple Approaches for the Investigation of Bacterial Small Regulatory RNAs Self-assembly
We present here the various approaches that can be used for the detection and analysis of bacterial small noncoding RNA self-assemblies. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Overview of Methods in RNA Nanotechnology: Synthesis, Purification, and Characterization of RNA Nanoparticles
RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149–155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833–842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applicati...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Mapping Protein–RNA Interactions by RCAP, RNA-Cross-Linking and Peptide Fingerprinting
RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein–RNA contacts within virions. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Measuring the Elasticity of Ribonucleotide(s)-Containing DNA Molecules Using AFM
Ribonucleotides, ribonucleoside monophosphates (rNMPs), have been revealed as possibly the most noncanonical nucleotides in genomic DNA. rNMPs, either not removed from Okazaki fragments during DNA replication or incorporated and scattered throughout the genome, pose a perturbation to the structure and a threat to the stability of DNA. The instability of DNA is mainly due to the extra 2′-hydroxyl (OH) group of rNMPs which give rise to local structural effects, which may disturb various molecular interactions in cells. As a result of these structural perturbations by rNMPs, the elastic properties of DNA are also affect...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Silver Nanoclusters for RNA Nanotechnology: Steps Towards Visualization and Tracking of RNA Nanoparticle Assemblies
The growing interest in designing functionalized, RNA-based nanoparticles (NPs) for applications such as cancer therapeutics requires simple, efficient assembly assays. Common methods for tracking RNA assemblies such as native polyacrylamide gels and atomic force microscopy are often time-intensive and, therefore, undesirable. Here we describe a technique for rapid analysis of RNA NP assembly stages using the formation of fluorescent silver nanoclusters (Ag NCs). This method exploits the single-stranded specificity and sequence dependence of Ag NC formation to produce unique optical readouts for each stage of RNA NP assemb...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Large Scale Purification of RNA Nanoparticles by Preparative Ultracentrifugation
Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradie...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

HPLC Purification of RNA Aptamers up to 59 Nucleotides with Single-Nucleotide Resolution
An RNA sample is usually heterogeneous. RNA heterogeneity refers to difference in length or size (i.e., number of nucleotides [nt]), sequence, or alternative but coexisting conformations. Separation and purification of RNA is generally required for investigating the structure and function of RNA, such as RNA catalysis and RNA structure determination by nuclear magnetic resonance or crystallography. Separation and purification of RNA is also required for using RNAs as functional probes and therapeutics as well as building blocks for RNA nanoparticles. Previously established protocols are limited in separating RNAs longer th...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Using RNA Nanoparticles with Thermostable Motifs and Fluorogenic Modules for Real-Time Detection of RNA Folding and Turnover In Prokaryotic and Eukaryotic Cells
RNA nanotechnology is an emerging field at the interface of biochemistry and nanomaterials that shows immense promise for applications in nanomedicines, therapeutics and nanotechnology. Noncoding RNAs, such as siRNA, miRNA, ribozymes, and riboswitches, play important roles in the regulation of cellular processes. They carry out highly specific functions on a compact and efficient footprint. The properties of specificity and small size make them excellent modules in the construction of multifaceted RNA nanoparticles for targeted delivery and therapy. Biological activity of RNA molecules, however, relies on their proper fold...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Methods and Assays for Specific Targeting and Delivery of RNA Nanoparticles to Cancer Metastases
In recent years, RNA nanotechnology has become increasingly attractive due to its potential for applications in nanomedicine. RNA nanotechnology refers to the design and synthesis of nanoparticles composed mainly of RNA via bottom-up self-assembly. RNA nanoparticle is a suitable candidate for targeted delivery of therapeutics to cancer cells due to its multivalency, which allows the combination of therapeutic, targeting, and detection moieties all into one nanoparticle. To date, a system capable of exclusively targeting metastatic cancers that have spread to distant organs or lymph nodes is in demand. In this chapter, we r...
Source: Springer protocols feed by Biotechnology - April 25, 2015 Category: Biotechnology Source Type: news

Freeze-Drying of Mammalian Sperm
Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) at 4 °C for several years and at ambient temper...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Freeze-Drying of Decellularized Heart Valve Tissues
Decellularized xeno-antigen-depleted porcine pulmonary heart valves tissues may be used as matrix implants for patients with malfunctioning heart valves. Decellularized tissues are biological scaffolds composed of extracellular matrix components. Biological scaffolds closely resemble properties of native tissue, but lack immunogenic factors of cellular components. Decellularized heart valve scaffolds need to be stored to be readily available whenever needed. Scaffolds can be stored at reduced supra-zero temperatures, cryopreserved or freeze-dried. The advantage of freeze-drying is that it allows long-term storage at room t...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Cord Blood Clinical Processing, Cryopreservation, and Storage
Allogeneic umbilical cord blood (UCB) hematopoietic stem cell transplantation has become a crucial advancement in the treatment for a variety of diseases including hematopoietic and non-hematopoietic malignancies, BM failure syndromes, hemoglobinopathies, and metabolic and immunodeficiency disorders. It has been well documented that the success of UCB engraftment is tied to UCB banking processes, and now there are established guidelines for standardization of collection, banking, processing, and cryopreservation for unrelated UCB units with purpose of achieving consistent production of high quality placental and UCB units ...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Directional Freezing for Large Volume Cryopreservation
Cryopreservation is currently the method of choice when it comes to long-term preservation of viable biological samples. The process, and consequently the volume of the sample, however, is limited by the ability to achieve homogenous and efficient heat removal. When this cannot be properly managed, ice crystals will grow uncontrollably resulting in extensive damage to the cryopreserved cells or tissues. Directional freezing is a technique that can be used to precisely control heat dissipation and ice crystal growth and morphology even when freezing large volumes. The technique has been used over the years to cryopreserve s...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Vitrification of Heart Valve Tissues
Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80 % cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, has several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preserv...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Cryopreservation of Plant Cell Lines
Plant cell cultures may consist of dedifferentiated cells as well as of cells showing embryogenic potential. They can be used for very different purposes in research and biotechnology as well as for plant propagation. For such cell cultures, cryopreservation is the only means for long-term preservation. Most of the different cryopreservation approaches, which are generally used for plant tissues, have also been applied to plant cell cultures; they include slow freezing, vitrification, and encapsulation/dehydration approaches. The controlled-rate slow freezing approach which is described here, however, remains to be the gol...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Writing Standard Operating Procedures (SOPs) for Cryostorage Protocols: Using Shoot Meristem Cryopreservation as an Example
Standard operating procedures are a systematic way of making sure that biopreservation processes, tasks, protocols, and operations are correctly and consistently performed. They are the basic documents of biorepository quality management systems and are used in quality assurance, control, and improvement. Methodologies for constructing workflows and writing standard operating procedures and work instructions are described using a plant cryopreservation protocol as an example. This chapter is pertinent to other biopreservation sectors because how methods are written, interpreted, and implemented can affect the quality of st...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Freeze-Drying of Proteins
Freeze-drying has become one of the most important processes for the preservation of biological products. This chapter provides protocols for freeze-drying of proteins and discusses the importance of formulation, cycle development, and validation. Specific formulations for stabilization of proteins are presented as well as advice on common problems with freeze-drying of proteins. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Cryopreservation of Red Blood Cells
Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we desc...
Source: Springer protocols feed by Biotechnology - December 2, 2014 Category: Biotechnology Source Type: news

Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Engineering, Bio-arming Technology
The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of proteins/peptides library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-ac...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Yeast Metabolomics: Sample Preparation for a GC/MS-Based Analysis
Metabolome sample preparation is one of the key factors in metabolomics analyses. The quality of the metabolome data will depend on the suitability of the experimental procedures to the cellular system (e.g., yeast cells) and the analytical performance. Here, we summarize a protocol for metabolome analysis of yeast cells using gas chromatography–mass spectrometry (GC–MS). First, the main phases of a metabolomics analysis are identified: sample preparation, metabolite extraction, and analysis. We also provide an overview on different methods used to quench samples and extract intracellular metabolites from yeast...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Genetic Engineering of Industrial Saccharomyces cerevisiae Strains Using a Selection/Counter-selection Approach
Gene modification of laboratory yeast strains is currently a very straightforward task thanks to the availability of the entire yeast genome sequence and the high frequency with which yeast can incorporate exogenous DNA into its genome. Unfortunately, laboratory strains do not perform well in industrial settings, indicating the need for strategies to modify industrial strains to enable strain development for industrial applications. Here we describe approaches we have used to genetically modify industrial strains used in winemaking. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

13C-Based Metabolic Flux Analysis in Yeast: The Pichia pastoris Case
Metabolic flux analysis based on tracing patterns of stable isotopes, particularly 13C, comprises a set of methodologies to experimentally quantify intracellular biochemical reaction rates, i.e., to measure carbon flux distributions through a metabolic network. This allows quantifying the response of a metabolic network to an environmental or genetic perturbation (i.e., the metabolic phenotype). Here, we describe a protocol based on growing yeast on a 13C-labelled substrate and subsequent NMR detection of 13C-patterns in proteinogenic amino acids. To calculate metabolic fluxes, we describe two complementary mathematical ap...
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news

Pathway Activity Profiling (PAPi): A Tool for Metabolic Pathway Analysis
Pathway Activity Profiling (PAPi) is a method developed to correlate levels of metabolites to the activity of metabolic pathways operating within biological systems. Based solely on a metabolomics data set and the Kyoto Encyclopedia of Genes and Genomes, PAPi predicts and compares the activity of metabolic pathways across experimental conditions, which considerably improves the hypothesis generation process for achieving the biological interpretation of biological studies. In this chapter, we describe how to apply PAPi to a metabolomics data set using the R-software. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - July 1, 2014 Category: Biotechnology Source Type: news