Techniques for RNA extraction from cells cultured in starPEG–heparin hydrogels

Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.     

Extraction of RNA from Ca-alginate-encapsulated yeast for transcriptional profiling

We have developed a method for preparing high-quality total RNA from Ca-alginate-encapsulated Saccharomyces cerevisiae that is suitable for microarray analysis. Encapsulated cells were harvested from immobilized cell reactors and flash-frozen in liquid nitrogen. Following low-temperature mechanical disruption, cells were freed from Ca-alginate by reverse ionotropic gelation and purified by centrifugation, and then total RNA was extracted using hot acid phenol. The yield and quality of the RNA were consistently high; the RNA was free of contaminating alginate, and in microarray analysis it performed as well as RNA isolated from planktonic cells.     

An approach to modulate degradation and mesenchymal stem cell behavior in poly(ethylene glycol) networks

A simple, sequential approach for creation of hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels has been developed and characterized. The chemistry involves an initial step growth polymerization reaction between PEG-diacrylate and dithiothreitol (DTT) to form acrylate-terminated (-PEG-DTT-)n PEG chains, followed by photocross-linking to form a hydrogel network. Varying the extent of step growth polymerization prior to photocross-linking allowed for control over the equilibrium swelling ratio, degradation, and erosion of PEG hydrogels. Hydrogel degradability had a significant effect on behavior of human mesenchymal stem cells (hMSCs) encapsulated within PEG hydrogels, both in the presence and absence of an RGDSP cell adhesion ligand. In particular, enhanced network degradability resulted in enhanced hMSC viability and spreading during in vitro culture. Comparison of degradable and nondegradable hydrogels with similar physical properties (e.g., equilibrium swelling ratio) demonstrated that hMSC viability and spreading were dependent on network degradability. This study demonstrates that hydrolytically degradable PEG hydrogels can be formed via a sequential step growth polymerization and photocross-linking process and the resulting materials may serve as promising matrices for 3-dimensional stem cell culture and tissue engineering applications.     

RNA extraction from polysaccharide-based cell-laden hydrogel scaffolds

An effective method for obtaining high-quality RNA from polysaccharide-rich hydrogels would be of great interest to biomaterialists and tissue engineers. Based on the similarities between polysaccharide-based hydrogels and plant tissues, we used a plant-specific RNA extraction kit to extract RNA from mammalian cells encapsulated in hydrogel scaffolds. The results indicate that this method can be reliably used in isolating high-purity RNA from polysaccharide hydrogels.     

Synthesis and evaluation of novel biodegradable hydrogels based on poly(ethylene glycol) and sebacic acid as tissue engineering scaffolds

Novel biodegradable poly(ethylene glycol) (PEG) based hydrogels, namely, PEG sebacate diacrylate (PEGSDA) were synthesized, and their properties were evaluated. Chemical structures of these polymers were confirmed by Fourier transform infrared and proton nuclear magnetic resonance (1H NMR) spectroscopy. After photopolymerization, the dynamic shear modulus of the hydrogels was up to 0.2 MPa for 50% PEGSDA hydrogel, significantly higher than conventional hydrogels such as PEG diacrylate (PEGDA). The swelling ratios of these macromers were significantly lower than PEGDA. The in vitro degradation study demonstrated that these hydrogels were biodegradable with weight losses about 66% and 32% for 25% and 50% PEGSDA after 8 weeks of incubation in phosphate-buffered saline at 37 degrees C. In vitro biocompatibility was assessed using cultured rat bone marrow stromal cells (MSCs) in the presence of unreacted monomers or degradation products. Unlike conventional PEGDA hydrogels, PEGSDA hydrogel without RGD peptide modification induced MSC cell adhesion similar to tissue culture polystyrene. Finally, complex three-dimensional structures of PEGSDA hydrogels using solid free form technique were fabricated and their structure integrity was better maintained than PEGDA hydrogels. These hydrogels may find use as scaffolds for tissue engineering applications.     

Development of a biostable replacement for PEGDA hydrogels

The exceptional tunability of poly(ethylene glycol) (PEG) hydrogel chemical, mechanical, and biological properties enables their successful use in a wide range of biomedical applications. Although PEG diacrylate (PEGDA) hydrogels are often used as nondegradable controls in short-term in vitro studies, it is widely acknowledged that the hydrolytically labile esters formed upon acrylation of the PEG diol make them susceptible to slow degradation in vivo. A PEG hydrogel system that maintains the desirable properties of PEGDA while improving biostability would be valuable in preventing degradation-related failure of gel-based devices in long-term in vivo applications. To this end, PEG diacrylamide (PEGDAA) hydrogels were synthesized and characterized in quantitative comparison to traditional PEGDA hydrogels. It was found that PEGDAA hydrogel modulus and swelling can be tuned over a similar range and to comparable degrees as PEGDA hydrogels with changes in macromer molecular weight and concentration. Additionally, PEGDAA cytocompatibility, low cell adhesion, and capacity for incorporation of bioactivity were analogous to that of PEGDA. In vitro hydrolytic degradation studies showed that the amide-based PEGDAA had significantly increased biostability relative to PEGDA. Overall, these findings indicate that PEGDAA hydrogels are a suitable replacement for PEGDA hydrogels with enhanced hydrolytic resistance. In addition, these studies provide a quantitative measure of the hydrolytic degradation rate of PEGDA hydrogels which was previously lacking in the literature.     

Hepatocyte viability and protein expression within hydrogel microstructures

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.     

Tailoring the degradation of hydrogels formed from multivinyl poly(ethylene glycol) and poly(vinyl alcohol) macromers for cartilage tissue engineering

Tuning the degradation profiles of polymer cell carriers to match cell and tissue growth is an important design parameter for (cartilage) tissue engineering. In this study, degradable hydrogels were fabricated from divinyl, tetrafunctional poly(ethylene glycol) (PEG) and multivinyl, multifunctional poly(vinyl alcohol) (PVA) macromers to form homopolymer and copolymer gels. These gels were characterized by their volumetric swelling ratio and mass loss profiles as a function of degradation time. By variation of the macromer chemistry and functionality, the degradation time changed from less than 1 day for homopolymer PVA gels to 34 days for pure PEG gels. Furthermore, the degrading medium influenced mass loss, and a marked decrease in degradation time, from 34 to 12 days, was observed with the PEG gels when a chondrocyte-specific medium containing fetal bovine serum was employed. Interestingly, when copolymer gels of PEG and PVA were formed, PVA was released throughout the degradation (as determined by gel permeation chromatography) suggesting that covalent cross-linking of the PVA in the network was facilitated by copolymerizing with the PEG macromer. To assess these novel gels for cartilage tissue engineering applications, chondrocytes were photoencapsulated in the copolymer networks and cultured in vitro for up to 6 weeks. DNA, glycosaminoglycan (GAG), and total collagen contents increased with culture time, and the resulting neocartilaginous tissue at 6 weeks was homogeneously distributed as seen histologically. Biochemical analysis revealed that the constructs were comprised of 0.66 +/- 0.04 microg of DNA/mg wet weight (ww), 1.0 +/- 0.05% GAG/ww, and 0.29 +/- 0.07% total collagen/ww at 6 weeks. Furthermore, the compressive modulus increased during culture from 7 to 97 kPa as the neocartilaginous tissue evolved and the gel degraded. In summary, fabricating hydrogels through the copolymerization of PEG and PVA macromers is an effective tool for encapsulating chondrocytes, controlling gel degradation profiles, and generating cartilaginous tissue.     

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.     

Evaluation of Physical and Mechanical Properties of Porous Poly (Ethylene Glycol)-co-(L-Lactic Acid) Hydrogels during Degradation

Porous hydrogels of poly(ethylene glycol) (PEG) have been shown to facilitate vascularized tissue formation. However, PEG hydrogels exhibit limited degradation under physiological conditions which hinders their ultimate applicability for tissue engineering therapies. Introduction of poly(_L-lactic acid) (PLLA) chains into the PEG backbone results in copolymers that exhibit degradation via hydrolysis that can be controlled, in part, by the copolymer conditions. In this study, porous, PEG-PLLA hydrogels were generated by solvent casting/particulate leaching and photopolymerization. The influence of polymer conditions on hydrogel architecture, degradation and mechanical properties was investigated. Autofluorescence exhibited by the hydrogels allowed for three-dimensional, non-destructive monitoring of hydrogel structure under fully swelled conditions. The initial pore size depended on particulate size but not polymer concentration, while degradation time was dependent on polymer concentration. Compressive modulus was a function of polymer concentration and decreased as the hydrogels degraded. Interestingly, pore size did not vary during degradation contrary to what has been observed in other polymer systems. These results provide a technique for generating porous, degradable PEG-PLLA hydrogels and insight into how the degradation, structure, and mechanical properties depend on synthesis conditions.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.     

Extraction of high-quality RNA from pancreatic tissues for gene expression studies

Extracting RNA from pancreatic tissue is notoriously challenging because of the organ's high RNase content. Standard methods using TriPure or TRIzol classically yield RNA of sufficient quality for routine gene expression analysis but not for microarray or deep sequencing analysis. Here we developed a simple method to extract high-quality RNA from mouse pancreas. Our method uses an RNase inhibitor and combines different protocols using guanidium thiocyanate–phenol extraction. It enables reproducible isolation of RNA with an RNA integrity number around 9.     

Hydrogel design for supporting neurite outgrowth and promoting gene delivery to maximize neurite extension

Hydrogels capable of gene delivery provide a combinatorial approach for nerve regeneration, with the hydrogel supporting neurite outgrowth and gene delivery inducing the expression of inductive factors. This report investigates the design of hydrogels that balance the requirements for supporting neurite growth with those requirements for promoting gene delivery. Enzymatically-degradable PEG hydrogels encapsulating dorsal root ganglia explants, fibroblasts, and lipoplexes encoding nerve growth factor were gelled within channels that can physically guide neurite outgrowth. Transfection of fibroblasts increased with increasing concentration of Arg-Gly-Asp (RGD) cell adhesion sites and decreasing PEG content. The neurite length increased with increasing RGD concentration within 10% PEG hydrogels, yet was maximal within 7.5% PEG hydrogels at intermediate RGD levels. Delivering lipoplexes within the gel produced longer neurites than culture in NGF-supplemented media or co-culture with cells exposed to DNA prior to encapsulation. Hydrogels designed to support neurite outgrowth and deliver gene therapy vectors locally may ultimately be employed to address multiple barriers that limit regeneration.     

Combined influence of substrate stiffness and surface topography on the antiadhesive properties of Acr-sP(EO-stat-PO) hydrogels

Biomaterials that prevent nonspecific protein adsorption and cell adhesion are of high relevance for diverse applications in tissue engineering and diagnostics. One of the most widely applied materials for this purpose is Poly(ethylene glycol) (PEG). We have investigated how micrometer line topography and substrate elasticity act upon the antiadhesive properties of PEG-based hydrogels. In our studies we apply bulk hydrogel cross-linked from star-shaped poly(ethylene oxide-stat-propylene oxide) macromonomers. Substrate surfaces were topographically patterned via replica molding. Additionally, the mechanical properties were altered by variations in the cross-linking density. Surface patterns with dimensions in the range of the cells' own size, namely 10 μm wide grooves, induced significant cell adhesion and spreading on the Acr-sP(EO-stat-PO) hydrogels. In contrast, there was only little adhesion to smaller and larger pattern sizes and no adhesion at all on the smooth substrates, regardless the rigidity of the gel. The effect of varied substrate stiffness on cell behavior was only manifest in combination with topography. Softer substrates with line patterns lead to significantly higher cell adhesion and spreading than stiff substrates. We conclude that the physical and mechanical surface characteristics can eliminate the nonadhesive properties of PEG-based hydrogels to a large extent. This has to be taken into account when designing surfaces for biomedical application such as scaffolds for tissue engineering which rely on the inertness of PEG.