Serum-free non-toxic freezing solution for cryopreservation of human adipose tissue-derived mesenchymal stem cells

Objective

To develop a cost-effective, non-toxic and xeno-free freezing solution for the preservation of adipose tissue-derived stem cells (hADSC) with a long shelf-life.

Results

The potential of various hydrocolloids and organic osmolytes as cryoprotectants and individual components of phosphate buffered saline (PBS) as carrier media were evaluated to formulate a freezing solution for the cryopreservation of hADSCs. Among the hydrocolloids, the highest viability, 55 %, was achieved with post-thawed (after 48 h storage at ?80 °C) hADSCs cryopreserved in 10 % (v/v) polyvinylpyrrolidone (PVP) using PBS as carrier media. 0.9 % NaCl was a superior carrier medium resulting an enhanced cell viability (70 %) when used in 10 % PVP than other components of PBS. A higher cell viability (81 %) was achieved when 10 % PVP/0.9 % NaCl was supplemented with 60 mM ectoin. The cryopreserved cells retained normal cytoskeletal distribution pattern and adipogenic and osteogenic differentiation ability during 14 and 21 days of incubation.

Conclusion

A serum-free and non-toxic 10 % PVP/0.9 % NaCl/60 mM ectoin freezing solution was developed for cryopreservation of hADSC for application in tissue engineering and regenerative medicine.

     

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.     

Design and Synthesis of LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers

Three LNA-based mercaptoacetamido-linked nonionic nucleoside dimers T^L-S-T, T-S-T^L , and T^L-S-T^L have been synthesized by HOBT and HBTU catalyzed condensation of silyl-protected 2-S-(thymidin-5?′-yl)mercaptoacetic acid or 2-S-(2?′-O,4?′-C-methylenethymidin-5?′-yl)mercaptoacetic acid with 3?′-amino-3?′-deoxy-5?′-O-DMT-2?′-O,4?′-C-methylenethymidine or with 3?′-amino-3?′-deoxy-5?′-O-DMT-β-thymidine followed by desilylation of the protected dimers. The 3?′-O-phosphoramidite derivative of one of the nucleoside dimers was successfully prepared by condensation with [P(-Cl)(-OCH₂CH₂CN)-N(iPr)₂}] in DCM in the presence of N,N-diisopropylethylamine (DIPEA), which is a building block for the preparation of mercaptoacetamido-linked oligonucleotides of therapeutic applications.     

Early response of endothelial cells to flow is mediated by VE-cadherin

Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.     

Inhibition of Infectious Bursal Disease Virus by Vector Delivered SiRNA in Cell Culture

Infectious Bursal Disease (IBD) is major threat to poultry industry. It causes severe immunosuppression and mortality in chicken generally at 3 to 6 weeks of age. RNA intereference (RNAi) emerges as a potent gene regulatory tool in last few years. The present study was conducted to evaluate the efficiency of RNAi to inhibit the IBD virus (IDBV) replication in-vitro. VP2 gene of virus encodes protein involved in capsid formation, cell entry and induction of protective immune responses against it. Thus, VP2 gene of IBDV is the candidate target for the molecular techniques applied for IBDV detection and inhibition assay. In this study, IBDV was isolated from field cases and confirmed by RT-PCR. The virus was then adapted on chicken embryo fibroblast cells (CEF) in which it showed severe cytopathic effects (CPE). The short hairpin RNA (shRNAs) constructs homologous to the VP2 gene were designed and one, having maximum score and fulfilling maximum Reynolds criteria, was selected for evaluation of effective inhibition. Selected shRNA construct (i.e., VP2-shRNA) was observed to be the most effective for inhibiting VP2 gene expression. Real time PCR analysis was performed to measure the relative expression of VP2 gene in different experimental groups. The VP2 gene was less expressed in virus infected cells co-transfected with VP2-shRNA as compared to mock transfected cells and IBDV+ cells (control) at dose 1.6 µg. The result showed ～95% efficient down regulation of VP2 gene mRNA in VP2-shRNA treated cells. These findings suggested that designed shRNA construct achieved high level of inhibition of VP2 gene expression in-vitro.     

Interaction of metal ions with D-glucobenzothiazoline: isolation and characterization of the resultant products

Six different metal-ion complexes of D-glucobenzothiazoline were synthesized and characterized by analytical and spectral techniques. Formation of different types of species (ML and ML(2)) were observed with Cu(2+), Ag(+), Cd(2+), Hg(2+), and Zn(2+) ions. Existence of an anomeric mixture in the case of the Cu(2+) complex is identified from the EPR spectra, and the results were further supported by the simulated spectra. The structures were proposed based on different studies.     

Quick PCR based diagnosis of typhoid using specific genetic markers

A quick multiplex PCR based detection method was developed for early diagnosis of typhoid using specific genetic markers of S. typhi. Primers of tyv gene, flag gene, viaB gene and ratA gene confirmed the specificity and sensitivity of the PCR. The serum samples of the suspected typhoid patients were taken directly for PCR without culturing and isolating genomic DNA. Overall diagnosis required 2 h which is the least time ever reported for a PCR based methods. The sensitivity of the method is up to 5 fg genomic DNA. The genetic markers are specific and the four pairs of primers give selective amplification and differentiate S. typhi from closely related S. typhimurium.     

Functional genomics as a tool in virus research

Genomics is the study of an organism’s entire genome. It started out as a great scientific endeavor in the 1990s which aimed to sequence the complete genomes of certain biological species. However viruses are not new to this field as complete viral genomes have routinely been sequenced since the past thirty years. The ‘genomic era’ has been said to have revolutionized biology. This knowledge of full genomes has created the field of functional genomics in today’s post-genomic era, which, is in most part concerned with the studies on the expression of the organism’s genome under different conditions. This article is an attempt to introduce its readers to the application of functional genomics to address and answer several complex biological issues in virus research.