Gelatin Substrate Research Articles

Factors supporting long-term culture of bovine male germ cells.

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

Cellular Response of Limbal Stem Cells on PHBV/Gelatin Nanofibrous Scaffold for Ocular Epithelial Regeneration

The aim of this study was to develop a blend of nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/gelatin substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human Limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold, and human amniotic membrane) based on their phenotypic profile, viability, proliferation, and attachment ability. Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time–PCR results revealed no change in the expression profile of LSCs grown on nanofibrous substrate when compared to those grown on human amniotic membrane. In addition, electrospun nanofibrous PHBV substrate provides not only a milieu supporting LSCs expansion, but also serves as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.

Biological and Biochemical Potential of Sea Snake Venom and Characterization of Phospholipase A2 and Anticoagulation Activity.

This study is designed to isolate and purify a novel anti-clotting protein component from the venom of Enhydrina schistosa, and explore its biochemical and biological activities. The active protein was purified from the venom of E. schistosa by ion-exchange chromatography using DEAE-cellulose. The venom protein was tested by various parameters such as, proteolytic, haemolytic, phospholipase and anti-coagulant activities. 80% purity was obtained in the final stage of purification and the purity level of venom was revealed as a single protein band of about 44kDa in SDS-polyacrylamide electrophoresis under reducing conditions. The results showed that the Potent hemolytic activity was observed against cow, goat, chicken and human (A, B and O positive) erythrocytes. Furthermore, the clotting assays showed that the venom of E. schistosa significantly prolonged in activated partial thromboplastin time, thrombin time, and prothrombin time. Venomous enzymes which hydrolyzed casein and gelatin substrate were found in this venom protein. Gelatinolytic activity was optimal at pH 5-9 and (1)H NMR analysis of purified venom was the base line information for the structural determination. These results suggested that the E. schistosa venom holds good promise for the development of novel lead compounds for pharmacological applications in near future.

Improvement of the Insecticidal Capacity of Two Purpureocillium Lilacinum Strains against Tribolium Confusum.

Entomopathogenic fungi can regulate insect populations. They have extracellular enzymes that degrade cuticle components, mainly hydrocarbons, used as an energy source. The increase in insecticidal activity of fungi in a medium supplemented with cuticular hydrocarbons was assayed and the hydrolytic enzyme profiles of two strains of Purpureocillium lilacinum were evaluated. A spore suspension of P. lilacinum was inoculated in Petri plates with different values (0.99–0.97–0.95) of water activity (Aw) using the substrates gelatin, starch and tween-20. Growth rate on the different substrates and the enzymatic activity index for proteases, amylases and lipases at different incubation times, pH and Aw, was evaluated. Moreover, the insecticidal efficiency of strains grown in media supplemented with n-hexadecane and n-octacosane was analyzed. LT50 was calculated against adults of Tribolium confusum and showed that mortality increased about 15% when the strains grew in amended culture medium. High amylolytic activity was detected, but proteases were the main enzymes produced. Optimal protease production was observed in a range of acid and alkaline pH and lower Aw. The greatest growth rate was obtained in presence of gelatin. Lipase and amylase production was detected in small amounts. Fungal growth in media with hydrocarbon mixtures increased the pathogenicity of the two strains of P. lilacinum, with the strain JQ926223 being more virulent. The information obtained is important for achieving both an increase in insecticidal capacity and an understanding of physiological adaptation of the fungus.

Methylthymol blue in Fricke gels

The initial trial of methylthymol blue (MTB) as a chelator for ferric iron in Fricke gel dosimeters, used for three-dimensional (3D) dosimetry in cancer radiotherapy, is reported. MTB is a structural analogue of the conventionally used xylenol orange (XO); however, the absorbance spectrum of the ferric-MTB complex is shifted to higher wavelengths, which should allow for lower amount of light scattering during gel scanning. In this study, two gelatin substrates, two sources of XO and one source of MTB have been compared. The MTB- containing gels exhibited similar dose response and diffusion coefficient to the XO-containing gels at their wavelengths of maximum absorption (620 and 585 nm, respectively). In addition, the MTB gels gave an excellent dose response at 633 nm, which is an important wavelength that is already used with other 3D dosimeters.

Isolasi dan Pengukuran Aktivitas Enzim Bromelin dari Ekstrak Kasar Bonggol Nanas (Ananas comosus) pada Variasi Suhu dan pH

This study was conducted to isolate the bromelain enzyme from pineapple weevil (Ananas comosus) and measure the protein and enzyme activity of bromelain with gelatin substrate. Research stage involves determining the protein content of the bromelain enzyme in the treatment of ammonium sulfate precipitation by 10-60% concentration and determination of bromelain enzyme activity at various pH 4.0; 5.0; 6.0; 7.0 and 8.0, with the incubation time of 10 minutes at a temperature of 650 C. Each stage performed three repetitions and analyzed using spectrometry. The results obtained showed the highest protein content in precipitation with ammonium sulfate 60% in the amount of 37.214 mg / ml and the optimum pH of bromelain enzyme activity at pH 7.0 with the value of the activity of 1,081 units / gram. Keywords: ammonium sulfate precipitation, bromelain enzyme, pineapple weevil (Ananas comosus), pH

Derivation and culture of putative parthenogenetic embryonic stem cells in new gelatin substrates modified with galactomannan

Human embryonic stem cells (ESC) lines to be used for cell therapies must be created and maintained under strict conditions, excluding the use of undefined supplements. Two key steps in the creation of a new embryonic stem cell line are adherence to the substrate and derivation towards the formation of a primary colony. The bovine parthenote embryo model was used to test different matrices of gelatin nanofibers and gelatin/galactomannan films to be used for ESC derivation and culturing. Gelatin/galactomannan films were made in two concentrations of galactomannan, 0.1 and 0.3%, in an aqueous solution of gelatin and tested for gel cytotoxicity using cumulus cells (CCs). CCs showed normal cell morphology, with no sign of lysis or degeneration in any of the matrices tested. Inner cell masses of parthenote blastocysts (n=116) were placed onto the gel matrices for culture. There were three or four repeats for each matrix. Our results showed a good rate of inner cell mass (ICM) adherence on the gelatin/galactomannan films (41%–44%) and one derivative of the gel nanofiber (17% adherence to the substrate). These results encouraged us to try new gelatin formulations to increase the rates of derivation and cell proliferation under defined culture conditions to comply with good manufacturing practice directives for the potential therapeutic use of ESCs.

Monitoring of optimized SERS active gel substrates for painting and paper substrates by unilateral NMR profilometry

In order to realize a surface enhanced Raman spectroscopy (SERS) gel protocol with portable Raman instrumentation, this contribution oversees the optimization of a removable SERS active methylcellulose gel and the applicability of an innovative gelatin substrate. Analytical evaluations by non‐invasive portable nuclear magnetic resonance (NMR) profilometry with regards to methylcellulose and gelatin film penetration and removal from an unvarnished painted surface and commercial dyed paper substrates have been carried out, respectively, following successful SERS measurements. Both gels have been specifically prepared in accordance to the substrate under exam so as to simultaneously permit sufficient surface interaction for Raman enhancements to be recorded with limited penetration into or subsequent damage on removal of the matrix under study. This work continues to bridge the gap towards non‐invasive SERS measurements and in‐situ SERS measurements. Copyright © 2014 John Wiley & Sons, Ltd.

Screening of hyaluronic acid–poly(ethylene glycol) composite hydrogels to support intervertebral disc cell biosynthesis using artificial neural network analysis

Hyaluronic acid (HA)–poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA–PEG hydrogels that may differentially affect cellular “outcomes” of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA–PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and annulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network analysis to identify relationships between HA–PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA–SH) and PEG vinylsulfone (PEG–VS) macromers, and used as substrates for NP and AF cell culture in vitro. Hydrogel mechanical properties ranged from 70 to 489kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA–PEG hydrogels formed from the lower-molecular-weight HA, with evidence of higher sulfated glycosaminoglycan production also upon lower-HA-molecular-weight composite gels. All cells formed more multi-cell clusters upon any HA–PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.

Nanofibrous gelatin substrates for long-term expansion of human pluripotent stem cells

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate, using Matrigel as control. Then, the optimal conditions were applied to long-term expansion of hPSCs with several cell lines, showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition, this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages, thereby promoting a better hPSC culture and long-term expansion.

Scaffold Architecture Controls Insulinoma Clustering, Viability, and Insulin Production

Recently, in vitro diagnostic tools have shifted focus toward personalized medicine by incorporating patient cells into traditional test beds. These cell-based platforms commonly utilize two-dimensional substrates that lack the ability to support three-dimensional cell structures seen in vivo. As monolayer cell cultures have previously been shown to function differently than cells in vivo, the results of such in vitro tests may not accurately reflect cell response in vivo. It is therefore of interest to determine the relationships between substrate architecture, cell structure, and cell function in 3D cell-based platforms. To investigate the effect of substrate architecture on insulinoma organization and function, insulinomas were seeded onto 2D gelatin substrates and 3D fibrous gelatin scaffolds with three distinct fiber diameters and fiber densities. Cell viability and clustering was assessed at culture days 3, 5, and 7 with baseline insulin secretion and glucose-stimulated insulin production measured at day 7. Small, closely spaced gelatin fibers promoted the formation of large, rounded insulinoma clusters, whereas monolayer organization and large fibers prevented cell clustering and reduced glucose-stimulated insulin production. Taken together, these data show that scaffold properties can be used to control the organization and function of insulin-producing cells and may be useful as a 3D test bed for diabetes drug development.

Characterization and cytological effects of a novel glycated gelatine substrate

Hyperglycemia in diabetes results in the glycation of long-lived proteins. Protein glycation leads to the formation of advanced glycation end products (AGEs), which are implicated in delayed wound healing and other diabetes-associated pathologies, one of which is periodontal disease. Research into the mechanisms by which glycated long-lived proteins such as collagen exert their effects can allow for the understanding of diabetic pathologies and the development of appropriate treatments. However, the high cost of purified protein can be a limitation for many laboratories around the world. The objective of this study was to develop a low-cost in vitro model of glycated gelatine as an alternative to the glycated collagen model. We investigated the glycation of gelatine type A, a denatured form of collagen, which is low-cost and abundantly available. In this study, gelatine was incubated for 7 days with ribose or methylglyoxal (MG). Cross-linking, autofluorescence and UV–Vis spectrophotometry assays were performed and indicated a dose-dependent linear increase in cross-linking and autofluorescence of gelatine by ribose and MG. MG produced more cross-linking compared to ribose at the same concentrations. The UV–Vis spectra of the glycated gelatines confirmed the presence of AGE fluorophores. Because diabetes is a risk factor for periodontal disease, the effect of the glycated substrates on the basic behaviour of human periodontal ligament (HPDL) cells was evaluated. Glycation dose dependently reduced HPDL attachment and cell spreading, indicating that the novel glycated gelatine substrate affects cell behaviour. These results show that gelatine glycated with ribose or MG can be used as low-cost in vitro models to study the effects of protein glycation on cell behaviour in diabetes and ageing.

Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine.

Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.

Purification and characterization of five snake venom metalloproteinases from Egyptian Echis pyramidum pyramidum venom.

New five P-III snake venom metalloproteinases (SVMPs): EpyB2 (62 kDa), EpyB3 (62+23 kDa), EpyB4 (60 kDa), EpyB5 (67 kDa) and EpyB6 (66 kDa) of the most dangerous viper, Echis pyramidum pyramidum (Epy), were purified and characterized in a set of biochemical assays. The SVMPs were purified by applying a protocol of two successive chromatographic steps. Three purified SVMPs "EpyB2, EpyB4, and EpyB5" have hemorrhagic activity with MHDs, 7 μg, 7.6 μg and 15 μg, respectively; furthermore, they have high preference towards fibronectin, collagen, gelatin, fibrin and hemoglobin substrates compared with non-hemorrhagic SVMPs (EpyB3 and EpyB6). All the purified SVMPs showed remarkable thermal and pH stability, inhibited by metalloproteinase inhibitors and Zn(2+), Mn(2+), Ni(2+), Co(2+), Cu(2+), and Hg(2+). The purified SVMPs act as α-fibrinogenases, prothrombin activators and procoagulants. In conclusion, Epy venom has multiple SVMPs that are responsible for hemorrhagic events and thus represent a significant health hazard for victims of envenomation, however, they may be useful for treating diseases involving abnormal blood clot formation.

Substrate gelatin zymogrophy analysis of matrix metalloproteinase-2 and -9 (gelatinase A and B) in sera from patients with benign and malignant prostate disease

Substrate gelatin zymogrophy analysis of matrix metalloproteinase-2 and -9 (gelatinase A and B) in sera from patients with benign and malignant prostate disease

Fabrication of Poly(N-isopropylacrylamide) Films Containing Submicrometer Grooves for Constructing Aligned Cell Sheets

Transplantation of cell sheets including an intact extracellular matrix is one tissue-engineering strategy for tissue regeneration. Temperature-responsive substrates based on poly(N-isopropylacrylamide) (PNIPAAm) have been used to harvest intact cell sheets by temperature change. In this work, we immobilized PNIPAAm on plastic substrates by a UV-activated azide-based cross-linking mechanism. We demonstrated that the UV-cross-linked PNIPAAm films could respond to temperature changes and be used for cell-sheet fabrication. Next, grooved PNIPAAm substrates were fabricated by imprinting from grooved poly(dimethylsiloxane) (PDMS) molds (800 nm in groove width and 500 nm in depth). C2C12 cells formed aligned cell sheets on the grooved PNIPAAm surface. The aligned cell sheet could be transferred to a gelatin substrate without losing cell alignment. We expect that this simple time-saving technique for the fabrication of grooved PNIPAAm substrates will benefit from the application of cellular alignment in tissue-engineering products.

Hypothetical cathepsin-like protein from Nematostella vectensis and its silicatein-like cathepsin mutant for biosilica production

Silicatein has high sequence identity and similarity with that of cathepsin L. In silicatein, serine replaces the active-site cysteine that is found in cathepsin L. Here, we obtained hypothetical cathepsin-like protein (CAT) from Nematostella vectensis which is 55% identical and 75% similar to mature silicatein alpha (SIL) of Suberites domuncula. When this protein was expressed in Escherichia coli, it displayed protease activity with both N-carbobenzoxy-l-phenylalanyl-l-arginine-4-methylcoumaryl-7-amide (Z-FR-AMC) and gelatin substrates, as well as silica-condensing activity using the tetraethoxy silane (TEOS) substrate. To increase its silica-forming activity and stability, some residues including the active site cysteine, were mutated into conserved silicatein residues, resulting in a mutant with 65% identity and 79% similarity to SIL. The mutant silicatein-like cathepsin (SLC) had increased expression levels in E. coli, and silica-forming activity comparable to that of SIL. In addition, SLC exhibited decreased protease activity as compared to that of CAT. Both CAT and SLC produced silica particles of sizes smaller than 50nm, which increased to 200–300nm in the presence of a structure-directing agent, such as Triton X-100. In conclusion, CAT was evolved to function as a biosilica-forming protein, and SLC was engineered by mutating CAT residues into conserved SIL residues to produce various silica-based materials.

Abstract 230: Evaluation of Hyperglycemia-Driven Alterations on Vascular Endothelial Function

Type 2 diabetes mellitus (T2DM) afflicts ~25 million people in the United States and hyperglycemia is a major causative factor contributing to vascular dysfunction among this population. This study evaluated the hypothesis that hyperglycemia-induced changes in endothelial glycoproteome leads to alterations of important homeostatic signaling pathways that result in vascular dysfunction. Utilizing targeted glycoprotein enrichment coupled with tandem mass spectrometry for protein identification and quantification, the cell surface glycoproteins were assessed from rat microvascular endothelial cells (RMVECs) cultured in ‘normal’ glucose (5 mM) or ‘high’ glucose (25 mM). Altogether, 273 N-glycosylation modified proteins were identified; 65 proteins uniquely N-glycosylated and 22 proteins significantly increased in N-glycosylation (p&lt;0.05) from ‘high’ glucose RMVECs. Additionally, 499 O-glycoproteins were identified; 78 proteins uniquely O-glycosylated and 65 proteins significantly increased in O-glycoslyation (p&lt;0.05) from ‘high’ glucose RMVECs. The type-1A angiotensin II receptor (AT1; p=0.029), along with numerous cell adhesion molecules, had increased N-glycosylation in ‘high’ glucose RMVECs. Numerous ion channels important for vascular function and the 5-HT2A serotonin receptor (p=5.3e-28), recently shown to contribute to vascular dysfunction in T2DM, had elevated O-glycosylation in ‘high’ glucose RMVECs. In order to evaluate these targets, functional assays were developed to measure endothelial and vascular function. Immobilization assays in a parallel plate flow chamber (PPFC) indicated that ‘high’ glucose significantly increased RMVEC adhesion to gelatin substrate under laminar flow (p&lt;0.05). In addition, incubation of RMVECs under ‘high’ glucose conditions increased the adherence of healthy platelets to RMVECs, suggesting the importance of adhesion protein glycosylation in endothelial dysfunction. Finally, studies in human subcutaneous arterioles demonstrated that acute exposure to ‘high’ glucose impaired acetylcholine-induced vasodilation. Utilizing these assays, differentially hyperglycemia-regulated targets will be functionally evaluated for potential therapeutic intervention.

Degradation of feather waste by Aspergillus niger keratinases: Comparison of submerged and solid-state fermentation

The isolation of native and/or the production of genetically modified enzyme-producing microorganisms may have substantial impacts on industrial processes. In this work twenty-eight Aspergillus niger mutants were screened for peptidases and keratinases production on a basal medium containing chicken feathers (1%). Four strains were selected after preliminary assays: 3T5B8, 9D40, 9D80, and 11D40. The keratinase production was higher when the A. niger strains were cultivated in a solid-state condition rather than a submerged condition: the keratinolytic activity of 3T5B8 strain was 7 times greater when cultivated by solid-state fermentation (SSF). A. niger 3T5B8 had the highest keratinase activity (172.7 U/ml) after seven days at pH 5.0 from solid-state fermentation, whereas the lowest activity was given by A. niger 9D40 after four days (21.3 U/ml) from submerged fermentation. Zymography of culture supernatant showed multiple bands migrating at 40–130, 14–130, displaying activity towards keratin and gelatin substrates, respectively. This is the first study to report production of high molecular mass peptidases using a feather-degrading Aspergillus. Peptidases from strains 3T5B8, 9D40, 9D80, and 11D40 were inhibited by PMSF, except the approximately 40-kDa peptidase, which was inhibited by phenanthroline, indicating the presence of serine and metallopeptidases. The results therefore suggest that the isolates are promising keratinase producers for biotechnological purposes.

Norepinephrine Modulates the Motility of Resting and Activated Microglia via Different Adrenergic Receptors

Microglia, the resident immune cells of the central nervous system (CNS), monitor the brain for disturbances of tissue homeostasis by constantly moving their fine processes. Microglia respond to tissue damage through activation of ATP/ADP receptors followed by directional process extension to the damaged area. A common feature of several neurodegenerative diseases is the loss of norepinephrine, which might contribute to the associated neuroinflammation. We carried out a high resolution analysis of the effects of norepinephrine (NE) on microglial process dynamics in acute brain slices from mice that exhibit microglia-specific enhanced green fluorescent protein expression. Bath application of NE to the slices resulted in significant process retraction in microglia. Analysis of adrenergic receptor expression with quantitative PCR indicated that resting microglia primarily express β2 receptors but switch expression to α2A receptors under proinflammatory conditions modeled by LPS treatment. Despite the differential receptor expression, NE caused process retraction in both resting and LPS-activated microglia cultured in the gelatinous substrate Matrigel in vitro. The use of subtype-selective receptor agonists and antagonists confirmed the involvement of β2 receptors in mediating microglial process dynamics in resting cells and α2A receptors in activated cells. Co-application of NE with ATP to resting microglia blocked the ATP-induced process extension and migration in isolated microglia, and β2 receptor antagonists prolonged ATP effects in brain slice tissues, suggesting the presence of cross-talk between adrenergic and purinergic signaling in microglia. These data show that the neurotransmitter NE can modulate microglial motility, which could affect microglial functions in pathogenic situations of either elevated or reduced NE levels.