Substrate For Cell Growth Research Articles

Non-invasive cell counting of adherent, suspended and encapsulated mammalian cells using optical density.

In situ measurement to determine mammalian cell number in a non-invasive, non-destructive and reagent-free manner is needed to enable continuous cell manufacturing. An analytical method is presented for non-invasive cell counting by conducting multiwavelength spectral analysis of mammalian cells achieving a minimal detectable cell count of 62,500 at 295 nm. Light absorbance was insensitive to culture volume, giving an absolute cell count rather than a concentration. The activation state of cells was also considered. The study was extended to quantification within polymeric microcapsules as an advanced substrate for mammalian cell growth in bioreactor formats and resulted in an offset directly correlating with the absorbance maxima of the polymer. These studies provide feasibility for optical density as a simple end point to indirectly quantify mammalian cell number for continuous monitoring of cell cultures.

Increased Mechanical Properties of Carbon Nanofiber Mats for Possible Medical Applications

Carbon fibers belong to the materials of high interest in medical application due to their good mechanical properties and because they are chemically inert at room temperature. Carbon nanofiber mats, which can be produced by electrospinning diverse precursor polymers, followed by thermal stabilization and carbonization, are under investigation as possible substrates for cell growth, especially for possible 3D cell growth applications in tissue engineering. However, such carbon nanofiber mats may be too brittle to serve as a reliable substrate. Here we report on a simple method of creating highly robust carbon nanofiber mats by using electrospun polyacrylonitrile/ZnO nanofiber mats as substrates. We show that the ZnO-blended polyacrylonitrile (PAN) nanofiber mats have significantly increased fiber diameters, resulting in enhanced mechanical properties and thus supporting tissue engineering applications.

Scaffold-based tissue engineering approaches in treating infertility

Tissue engineering and the use of scaffolds have shown high therapeutic potentialities about male and female infertility. Nowadays, many couples are suffering from infertility problems. There are different causes for infertility including chemotherapy (for male and female), uterine injuries, and intrauterine adhesions. Extra-cellular matrix in tissue engineering provides a supportive medium for blood or lymphatic vessels making it a suitable substrate for cell implantation and growth. Dominant successes in this branch have been in use of patient-derived primary cells, these cells loaded in scaffolds and used to generate tissue for re-implantation. However, this method has limitations, because of the invasive nature of cell collection, also the cells patient-derived may be not healthy and become the source of disease. Therefore, use of stem cells, including embryonic stem (ES) cells, bone marrow mesenchymal stem cells (BM-MSCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been considered. Cell/scaffold systems have a substantial role in fertility organs or agents repair or regeneration. This review summarizes the novel scaffold-based tissue engineering approaches to treat infertility.

Cryostructurization of polymeric systems for developing macroporous cryogel as a foundational framework in bioengineering applications

Among various fabrication techniques to produce a porous scaffold, thermally-induced phase separation at controlled cryogenic condition leads to the formation of a porous polymeric cryostructure alias cryogel or cryomatrix. Cryostructurization is one of the simple and versatile methods of synthesizing a highly porous and interconnected architecture. The process of cryostructurization is present in comparable fabrication approach of fabricating advanced porous biomaterials with precise control over multiple compositions of precursor units, spatial distributions for accomplishing effective recapitulation of mechanical properties and architectural accuracy at micron-scale with bioactive functionality. The cryogenically-structured polymeric scaffolds are of noteworthy fundamental and applied interest in multi-disciplinary areas of science and showed promising matrices in various biotechnological and biomedical areas. The large interconnected pores in cryogels open up a range of applications like the three-dimensional substrate for cell growth, bioreactor for continuous production of scarce molecules, bio-processing and protein purification, an adsorbent for environmental remediation, and biosensor fabrication. Over the past two decades, significant attention to these materials with rigorous expansion in their new designs, and remarkable growth in the number of the publications and patents on cryostructured porous materials has been noticed. This article is covering the progress in cryostructurization technology in the last few decades with principles underlying the mechanism of cryogelation, process optimization, and the recent trend in cryogel for biomedical and bioengineering applications. The genesis of a highly porous interconnected architecture by the process of thermally-induced phase separation under cryo-temperature which is commonly known as cryostructurization of polymeric systems to produce porous scaffolds can be synthesized using a variety of polymer precursors for multidisciplinary applications in the fields of biomedicine, environment and bioengineering.

Electrochemomechanical Behavior of Polypyrrole-Coated Nanofiber Scaffolds in Cell Culture Medium.

Glucose-gelatin nanofiber scaffolds were made conductive and electroactive by chemical (conductive fiber scaffolds, CFS) and additionally electrochemical polypyrrole deposition (doped with triflouromethanesulfonate CF3SO3−, CFS-PPyTF). Both materials were investigated in their linear actuation properties in cell culture medium (CCM), as they could be potential electro-mechanically activated cell growth substrates. Independent of the deposition conditions, both materials showed relatively stable cation-driven actuation in CCM, based on the flux of mainly Na+ ions from CCM. The surprising result was attributed to re-doping by sulfate anions in CCM, as also indicated by energy-dispersive X-ray (EDX) spectroscopy results. Overall, the electrochemically coated material outperformed the one with just chemical coating in conductivity, charge density and actuation response.

Two Experimental Protocols for Accurate Measurement of Gas Component Uptake and Production Rates in Bioconversion Processes

Bioconversion processes offer many economic, environmental, and societal advantages for production of fuels and chemicals. Successful commercialization of any biotechnology usually requires accurate characterization of cell growth dynamics, substrate conversion and production excretion rates. Despite recent advancements in analytical equipment, obtaining accurate measurement of gas component uptake or production rates remains challenging due to their high sensitivity to system pressure or volume changes. Specifically, the consumption and production of various gases will result in changes in system pressure (for batch operations) or off-gas flow rate (for continuous operations). These changes would cause significant errors in the estimated gas component uptake and production rates if they were not accounted for. In this work, we propose two easy-to-implement experimental protocols and associated calculation procedures to obtain accurate measurements of gas component consumption and production rates; one is for batch operation and one is for continuous operation. For depressurized (i.e., system pressure below 1 atm) batch cultures, nitrogen (or other inert gases) is used to repressurize the system to 1 atm before taking sample; while for continuous cultures, He (or other inert gases) is used as an internal tracer to accurately measure off-gas flow rate. The effectiveness and accuracy of the two protocols and associated calculation procedures are demonstrated using several case studies with both abiotic and biotic systems.

Применение импедансной спектроскопии для оценки влияния ламининов на дифференцировку клеток кишечника in vitro

The comparative analysis of electrical parameters of a Caco-2 cell monolayer cultured on substrates coated with laminins 111,332и511 has been carried out. The cell electrical parameters reflect its state and, in particular, its degree of differentiation. Impedance spectroscopy is the most convenient method for comprehensive assessment of electrical parameters. It was shown via this method that laminin 332 was the optimal substrate for CACO-2 cell growth and differentiation in vitro. The use of laminin 511 led to significantly lower electric capacitance, and laminin 111 caused a large increase in TEER. impedance spectroscopy, laminins, barrier tissues, TEER, electric capacitance, extracellular matrix, Caco-2 The study was funded by the Russian Science Foundation (Project 16-19-10597).

Simultaneous saccharification and co-fermentation of corn stover pretreated by H2O2 oxidative degradation for ethanol production

The simultaneous saccharification and co-fermentation of Saccharomyces cerevisiae and Candida tropicalis for ethanol production using H2O2-pretreated corn stover (CS) were performed in 250-mL or 500-mL Erlenmeyer flasks at various S. cerevisiae-C. tropicalis ratios (4:1, 3:1, 2:1, 1:1, 1:2, 1:3 and 1:4), fermentation temperatures (32 °C, 35 °C and 38 °C), inoculation ratios (8%, 12% and 16%), initial pH values (4.5, 5 and 5.5) and fermentation times (96 h, 120 h and 144 h). The optimum S. cerevisiae-C. tropicalis ratio derived from mono-factor design test was 3:1. According to orthogonal design experiment results, the optimum fermentation temperature, inoculation ratio, initial pH and fermentation time with a dry solid substrate content of 25% were 32 °C 12%, 5.0 and 144 h, respectively, and the maximum ethanol concentration was 10.924 g/100 mL. The cell growth, ethanol production and total substrate consumption in the process of simultaneous saccharification and co-fermentation conformed to logistical model, Luedeking-Piret model and general substrate consumption law of mono-strain fermentation, respectively. Collectively, S. cerevisiae and C. tropicalis simultaneously produced ethanol from xylose and glucose without interference, respectively.

Newly-designed collagen/polyurethane bioartificial blend as coating on bioactive glass-ceramics for bone tissue engineering applications

In the present work, a new combination of synthetic and natural biomaterials is proposed for bone tissue engineering (BTE) applications. In order to mimic the inorganic and organic phases of bone extracellular matrix (ECM), a bioactive glass-ceramic deriving from a SiO2–P2O5–CaO–MgO–Na2O–K2O parent glass, acting as a substrate in form of a slice, was surface-functionalised with a type I collagen-based coating. In particular, the collagen was blended with a water soluble polyurethane (PUR), synthesised from poly(ethylene glycol), 1,6-hexamethylene diisocyanate and N-BOC-serinol. The PUR was designed to expose amino groups on the polymeric chain, which can be exploited for the blend stabilisation through crosslinking. The newly synthesised PUR demonstrated to be non-cytotoxic, as assessed by a biological test with MG-63 osteoblast-like cells. The collagen/PUR blend showed good biocompatibility as well. The polymeric coating on the glass-ceramic samples was produced by surface-silanisation, followed by further chemical grafting of the blend, using genipin as a crosslinker. The glass-ceramic surface was characterised at each functionalisation step, demonstrating that the procedure allowed obtaining a covalent link between the blend and the substrate. Finally, biological tests performed using human periosteal derived precursor cells demonstrated that the proposed polymer-coated material was a good substrate for bone cell adhesion and growth, and a good candidate to mimic the composite nature of the bone ECM.

Graphene-based papers as substrates for cell growth: Characterisation and impact on mammalian cells

Graphene based material (GBM) substrates are promising candidates for tissue engineering and as cell scaffolds owing to their outstanding properties that favour cell adhesion and growth. In this study, we report the fabrication and use of different oxidised graphene-based ‘papers’ as substrates for cell culture, using starting materials of different thickness and lateral dimensions. Graphite oxide (GTO) consisted of thicker sheets than the thinner large graphene oxide (l-GO) and small graphene oxide (s-GO) sheets. The ‘papers’ were prepared by vacuum filtration of the material suspensions and characterised by an array of experimental techniques. The substrates were then evaluated for cellular adhesion and proliferation with two epithelial cell lines that differ in their morphologies, the human lung cell culture (A549) model and the human neural cell culture (SH-SY5Y) model using microscopic analysis. Release of lactate dehydrogenase (LDH) in cell supernatants was measured to assess any cytotoxic responses. Our results revealed that the GO papers had similar physicochemical properties but differing topographies. The papers supported cell growth and no cytotoxicity was observed. These results suggest that substrates based on graphite and graphene oxide papers are suitable biocompatible cellular supports for anchorage-dependent cell growth and can be further explored for tissue engineering, regenerative medicine, substrates for cell growth and bionic applications.

Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits

Entubulating devices to repair peripheral nerve injuries are limited in their effectiveness particularly for critical gap injuries. Current clinically used nerve guidance conduits are often simple tubes, far stiffer than that of the native tissue. This study assesses the use of poly(glycerol sebacate methacrylate) (PGSm), a photocurable formulation of the soft biodegradable material, PGS, for peripheral nerve repair. The material was synthesized, the degradation rate and mechanical properties of material were assessed and nerve guidance conduits were structured via stereolithography. In vitro cell studies confirmed PGSm as a supporting substrate for both neuronal and glial cell growth. Ex vivo studies highlight the ability of the cells from a dissociated dorsal root ganglion to grow out and align along the internal topographical grooves of printed nerve guide conduits. In vivo results in a mouse common fibular nerve injury model show regeneration of axons through the PGSm conduit into the distal stump after 21 days. After conduit repair levels of spinal cord glial activation (an indicator for neuropathic pain development) were equivalent to those seen following graft repair. In conclusion, results indicate that PGSm can be structured via additive manufacturing into functional NGCs. This study opens the route of personalized conduit manufacture for nerve injury repair. Statement of SignificanceThis study describes the use of photocurable of Poly(Glycerol Sebacate) (PGS) for light-based additive manufacturing of Nerve Guidance Conduits (NGCs). PGS is a promising flexible biomaterial for soft tissue engineering, and in particular for nerve repair. Its mechanical properties and degradation rate are within the desirable range for use in neuronal applications. The nerve regeneration supported by the PGS NGCs is similar to an autologous nerve transplant, the current gold standard. A second assessment of regeneration is the activation of glial cells within the spinal cord of the tested animals which reveals no significant increase in neuropathic pain by using the NGCs. This study highlights the successful use of a biodegradable additive manufactured NGC for peripheral nerve repair.

Cytotoxicity analysis of indoor air pollution from biomass combustion in human keratinocytes on a multilayered dynamic cell culture platform

Skin tissue is the first barrier against ambient harmful matter and has direct contact with indoor air pollutants. Nevertheless, a comprehensive understanding of cytotoxicity of indoor air pollution on skin cells is insufficiently clear. Herein, for the first time a multilayered dynamic cell culture platform was established to study the cytotoxicity of indoor air pollutant from biomass combustion in human skin keratinocytes. The platform consisted of seven repetitive polydimethylsiloxane modules carrying six pieces of polycarbonate membrane between them as substrate for cell growth to realize the simultaneous dynamic culture of 12 layers of keratinocytes. After exposure to biomass combustion soluble constituents (BCSCs), cell viability under microfluidic platform conditions declined more significantly, and apoptosis rates increased more obviously compared with well plate conditions. Transmission electron microscope showed that keratinocyte microstructures displayed obvious signs of cellular damage. Our study confirmed that the nuclear factor of kappa B (NF-κB) signaling pathway was activated, which significantly increased the Bax/Bcl-2 ratio and tumor necrosis factor-alpha and interleukin 6 expression, indicating that NF-κB signaling pathway was the major factor in BCSCs-induced cytotoxicity. These findings offer an insight into the mechanism of BCSCs-induced cytotoxicity in keratinocytes and provide a theoretical basis for future studies on skin cells.

Multi-objective versus single-objective optimization of batch bioethanol production based on a time-dependent fermentation model

Traditional process simulators, such as Aspen Plus, are inadequate for optimizing multiple-objective systems in fermentation-based processes. This work uses a novel integrated platform of the robust genetic algorithm optimization in MATLAB linked with an Aspen Plus unsteady-state batch fermentation simulation to optimize the batch ethanolic fermentation process with respect to initial substrate concentration, fermentation time, and in situ product removal. A time-dependent fermentation model that utilizes both glucose and xylose, the major sugars present in lignocellulosic hydrolysate, with Monod cell growth kinetics, substrate and product inhibitions, is used as a model system. The optimized design variables from the multi-objective optimization (MOO) and single-objective optimization (SOO) suggest the typical concentrations of sugars from lignocellulosic hydrolysate must be concentrated to optimize the performance of the batch fermentation process. Furthermore, time-dependent information from an unsteady-state simulation was used to design an integrated batch fermentation with in situ product recovery, allowing higher initial sugars concentrations to be used in the fermentation process (about 50%, for the best optimal solution in the MOO). This resulted in 15% ethanol productivity, 143% total ethanol produced, and 67% fraction of sugar converted improvements relative to the batch fermentation without product recovery. Unlike the single optimal solution from the SOO, MOO presents many equally optimal solutions that can be used as a decision-support tool to guide the choice of design variables for optimum process performance. This study creates a platform that can be used to optimize integrated biorefinery and refinery processes.

A novel strategy and kinetics analysis of half-fractional high cell density fed-batch cultivation of Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii is an important microorganism for aroma production in traditional fermented foods. Using Z. rouxii as the original strain, the batch was split after glucose depletion in the culture medium. Half of the volume of the culture medium was released, and fresh culture medium was fed in. The exponential culture kinetics and the formula for the half‐fractional fed‐batch cultivations were determined to achieve a new strategy for high cell density culturing of Z. rouxii. Based on a full cultivation, three half‐fractional fed‐batch cultivations were performed after every 10 hr of culture. The specific growth rates of Z. rouxii at the different stages were in the order μX0>μX1>μX2>μX3 (0.525 to 0.229 hr−1). The glucose substrate consumption rates gradually decreased following the order μS0>μS1>μS2>μS3 (−1.165 to −0.722, g/g). The equation models for cell growth and glucose substrate consumption showed typical exponential behavior. The total cell yield was 1.78‐fold higher than the yield from a full cultivation, and this continuous subculture strategy also indicated a higher efficiency than traditional full cultivation. A new strategy for highly efficient culturing of Z. rouxii was achieved in a pilot scale. A foundation with data support for the production and application of Z. rouxii was developed.

Biomimetic porous high-density polyethylene/polyethylene-grafted-maleic anhydride scaffold with improved in vitro cytocompatibility

A major challenge for tissue engineering is to design and to develop a porous biocompatible scaffold, which can mimic the properties of natural tissue. As a first step towards this endeavour, we here demonstrate a distinct methodology in biomimetically synthesized porous high-density polyethylene scaffolds. Co-extrusion approach was adopted, whereby high-density polyethylene was melt mixed with polyethylene oxide to form an immiscible binary blend. Selective dissolution of polyethylene oxide from the biphasic system revealed droplet-matrix-type morphology. An attempt to stabilize such morphology against thermal and shear effects was made by the addition of polyethylene- grafted-maleic anhydride as a compatibilizer. A maximum ultimate tensile strength of 7 MPa and elastic modulus of 370 MPa were displayed by the high-density polyethylene/polyethylene oxide binary blend with 5% maleated polyethylene during uniaxial tensile loading. The cell culture experiments with murine myoblast C2C12 cell line indicated that compared to neat high-density polyethylene and high-density polyethylene/polyethylene oxide, the high-density polyethylene/polyethylene oxide with 5% polyethylene- grafted-maleic anhydride scaffold significantly increased muscle cell attachment and proliferation with distinct elongated threadlike appearance and highly stained nuclei, in vitro. This has been partly attributed to the change in surface wettability property with a reduced contact angle (∼72°) for 5% PE- g-MA blends. These findings suggest that the high-density polyethylene/polyethylene oxide with 5% polyethylene- grafted-maleic anhydride can be treated as a cell growth substrate in bioengineering applications.

Enhanced biosynthesis of 3,4-dihydroxybutyric acid by engineered Escherichia coli in a dual-substrate system

3,4-Dihydroxybutyric acid (3,4-DHBA), a versatile platform four carbon (C4) chemical, can be used as a precursor in the production of many commercially important chemicals. Here, a dual-substrate biosynthesis system was developed for 3,4-DHBA production via a synthetic pathway established in an engineered Escherichia coli, and using xylose as a synthetic substrate and glucose as a cell growth substrate. The deletion of genes xylA, yjhH and yagE and others encoding for alcohol dehydrogenases in E. coli is essential for the production of 3,4-DHBA. Blocking competing pathway by removing the gene yiaE encoding for a 2-keto-3-deoxy-D-xylonate reductase also facilitated carbon flow towards the synthesis of 3,4-DHBA. Furthermore, regulation the availability of NAD+ resulted in further improved 3,4-DHBA production. The combinational optimization of the biosynthesis system led to a production of 0.38g/L 3,4-DHBA. This study provides an alternative 3,4-DHBA biosynthesis approach with the possibility of utilizing hydrolysates of lignocellulosic biomass as substrates.

Monitoring cell substrate interactions in exopolysaccharide-based films reinforced with chitin whiskers and starch nanoparticles used as cell substrates

ABSTRACTExopolysaccharides (EPS) extracted from Nostoc commune cyanobacteria were used to prepare low-adhesion cell substrates for tumor-selective cell growth. The substrates were reinforced with chitin whiskers and starch nanoparticles and characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and tensile tests. Two anchorage independent cells, green fluorescent protein-modified HeLa and THP-1 cells were seeded on the samples to assess their interaction with the composites. The results confirmed the cell–surface interaction of EPS-based composites. The mechanical properties of these films can be tailored by controlling the type and concentration of the reinforcement, which can be used in the design of different types of biomaterials.

Modeling of the Degradation of Poly(ethylene glycol)-co-(lactic acid)-dimethacrylate Hydrogels

Because of their similarity with extracellular matrix, hydrogels are ideal substrates for cell growth. Hydrogels made of synthetic polymers are excellent alternatives to natural ones and offer the key advantage of precisely controllable degradation times. In this work, hydrogels have been prepared from modified poly(ethylene glycol) macromonomers, functionalized on both ends first with a few lactic acid units, and then with methacrylate groups. A library of hydrogels has been prepared using free-radical polymerization of the macromonomers, by changing both the macromonomer concentration and their type, i.e., the number of lactic acid repeating units. The degradation kinetics of these hydrogels, caused by the hydrolysis of the lactic acid units, have been carefully monitored in terms of swelling ratio, mass loss, and Young’s modulus. A complete mathematical model, accounting for hydrogel degradation, swelling, and reverse gelation, has been developed and used to predict all the measured quantities until co...

LC-hrms metabolomics profiling in breast cancer: Searching for biomarkers in human plasma samples.

e23053 Background: Breast cancer (BC) is most common cancer in women and development of new technologies for understanding molecular changes involved in BC progression is essential. Metabolic changes precede phenotypic changes, due to cellular regulation affects use of small-molecule substrates for cell division, growth or environmental changes, such as hypoxia. Metabolomics (profiling of metabolites in biofluid, cell and tissue) is routinely applied as a tool for biomarker discovery. Liquid chromatography−mass spectrometry(LC−MS)is the dominating platform. Methods: With the aim to find metabolomic profiles for diagnostic and prognostic of newly diagnosticated breast cancer (BC) we have investigated plasma samples in neoadjuvant setting (134 BC patients with median age of 51 years, stages T1-4,N1-2,M0 and 135 healthy controls) using high resolution mass spectrometry couple to liquid chromatography in reverse phase and HILIC modes which provided resolved chromatography of highly polar as well as hydrophobic analytes, enabling the analysis of a wide range of chemicals, necessary for untargeted metabolomics. Chromatograms were processed with software (Markerview) to generate a table listing retention times with associated ion masses and intensities. To identify potential biomarkers, we used in combination 2 independent variable selection techniques: principal component analysis and Student t-test. Results: We observed a significant difference in metabolic profile between the 2 groups. 15 molecular features [oleamide, KDNalpha2-3Galbeta1-4Glcbeta-Cer(d18:1/22:0)] were found significantly down/up regulated in BC patients compared with healthy subjects, and they were selected as diagnostic biomarkers. Additionally, ROC curve analysis was used to assess their clinical usefulness, obtaining an AUC of 0.963 (95% CI 0.919-0.989) when using a multivariate model of 3 features. As an outcome, we showed that selected biomarkers are useful as diagnostic biomarkers. Conclusions: The present study demonstrates potential of metabolomics in identifying novel biomarkers for BC. Further studies may reveal the potential of metabolites as diagnostics biomarkers for BC and their role in pathogenesis.

Fabrication of high-transmission microporous membranes by proton beam writing-based molding technique

Porous membranes are widely used as filters in a broad range of micro and nanofluidic applications, e.g. organelle sorters, permeable cell growth substrates, and plasma filtration. Conventional silicon fabrication approaches are not suitable for microporous membranes due to the low mechanical stability of thin film substrates. Other techniques like ion track etching are limited to the production of randomly distributed and randomly orientated pores with non-uniform pore sizes.In this project, we developed a procedure for fabricating high-transmission microporous membranes by proton beam writing (PBW) with a combination of spin-casting and soft lithography. In this approach, focused 2MeV protons were used to lithographically write patterns consisting of hexagonal arrays of high-density pillars of few µm size in a SU-8 layer coated on a silicon wafer. After development, the pillars were conformably coated with a thin film of poly-para-xylylene (Parylene)-C release agent and spin-coated with polydimethylsiloxane (PDMS). To facilitate demolding, a special technique based on the use of a laser-cut sealing tape ring was developed. This method facilitated the successful delamination of 20-μm thick PDMS membrane with high-density micropores from the mold without rupture or damage.