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Optimization of a chondrogenic medium through the use of factorial design of experiments.

ABSTRACT: The standard culture system for in vitro cartilage research is based on cells in a three-dimensional micromass culture and a defined medium containing the chondrogenic key growth factor, transforming growth factor (TGF)-?1. The aim of this study was to optimize the medium for chondrocyte micromass culture. Human chondrocytes were cultured in different media formulations, designed with a factorial design of experiments (DoE) approach and based on the standard medium for redifferentiation. The significant factors for the redifferentiation of the chondrocytes were determined and optimized in a two-step process through the use of response surface methodology. TGF-?1, dexamethasone, and glucose were significant factors for differentiating the chondrocytes. Compared to the standard medium, TGF-?1 was increased 30%, dexamethasone reduced 50%, and glucose increased 22%. The potency of the optimized medium was validated in a comparative study against the standard medium. The optimized medium resulted in micromass cultures with increased expression of genes important for the articular chondrocyte phenotype and in cultures with increased glycosaminoglycan/DNA content. Optimizing the standard medium with the efficient DoE method, a new medium that gave better redifferentiation for articular chondrocytes was determined.

SUBMITTER: Enochson L

PROVIDER: S-EPMC3559199 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Optimization of a chondrogenic medium through the use of factorial design of experiments.

Enochson Lars L Brittberg Mats M Lindahl Anders A

BioResearch open access 20121201 6

The standard culture system for in vitro cartilage research is based on cells in a three-dimensional micromass culture and a defined medium containing the chondrogenic key growth factor, transforming growth factor (TGF)-β1. The aim of this study was to optimize the medium for chondrocyte micromass culture. Human chondrocytes were cultured in different media formulations, designed with a factorial design of experiments (DoE) approach and based on the standard medium for redifferentiation. The sig ...[more]

PMID: 23514743

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Project description:BackgroundPretreatments are one of the main bottlenecks for the lignocellulose conversion process and the search for cheaper and effective pretreatment methodologies for each biomass is a complex but fundamental task. Here, we used a 2?5-1 fractional factorial design (FFD) to optimize five pretreatment variables: milling time, temperature, double treatment, chemical concentration, and pretreatment time in acid-alkali (EA) and acid-organosolv (EO) pretreatments, applied to elephant grass leaves.ResultsFFD allowed optimization of the pretreatment conditions using a reduced number of experiments and allowed the identification of secondary interactions between the factors. FFD showed that the temperature can be kept at its lower level and that the first acid step can be eliminated in both pretreatments, without significant losses to enzymatic hydrolysis. EA resulted in the highest release of reducing sugars (maximum of 205 mg/g substrate in comparison to 152 mg/g in EO and 40 mg/g in the untreated sample), using the following conditions in the alkali step: [NaOH]?=?4.5% w/v; 85 °C and 100 min after ball milling the sample. The factors statistically significant (P?<?0.05) in EA pretreatment were NaOH concentration, which contributes to improved hydrolysis by lignin and silica removal, and the milling time, which has a mechanical effect. For EO samples, the statistically significant factors to improved hydrolysis were ethanol and catalyst concentrations, which are both correlated to higher cellulose amounts in the pretreated substrates. The catalyst is also correlated to lignin removal. The detailed characterization of the main hemicellulosic sugars in the solids after pretreatments revealed their distinct recalcitrance: glucose was typically more recalcitrant than xylose and arabinose, which could be almost completely removed under specific pretreatments. In EA samples, the removal of hemicellulose derivatives was very dependent on the acid step, especially arabinose removal.ConclusionThe results presented herewith contribute to the development of more efficient and viable pretreatments to produce cellulosic ethanol from grass biomasses, saving time, costs and energy. They also facilitate the design of enzymatic cocktails and a more appropriate use of the sugars contained in the pretreatment liquors, by establishing the key recalcitrant polymers in the solids resulting from each processing step.

Project description:Realization of graphene-based sensors and electronic devices remains challenging, in part due to integration challenges with current fabrication and manufacturing processes. Thus, scalable methods for in situ fabrication of high-quality graphene-like materials are essential. Low-cost CO2 laser engravers can be used for site-selective conversion of polyimide under ambient conditions to create 3-D, rotationally disordered, few-layer, porous, graphene-like electrodes. However, the influences of non-linear parameter terms and interactions between key parameters on the graphitization process present challenges for rapid, resource-efficient optimization. An iterative optimization strategy was developed to identify promising regions in parameter space for two key parameters, laser power and scan speed, with the goal of optimizing electrode performance while maximizing scan speed and hence fabrication throughput. The strategy employed iterations of Design of Experiments Response Surface (DoE-RS) methods combined with choices of readily measurable parameters to minimize measurement resources and time. The initial DoE-RS experiment set employed visual response parameters, while subsequent iterations used sheet resistance as the optimization parameter. The final model clearly demonstrates that laser graphitization through raster scanning is a highly non-linear process requiring polynomial terms in scan speed and laser power up to fifth order. Two regions of interest in parameter space were identified using this strategy: Region 1 represents the global minimum for sheet resistance for this laser (∼16 Ω/sq), found at a low scan speed (70 mm/s) and a low average power (2.1 W) . Region 2 is a local minimum for sheet resistance (36 Ω/sq), found at higher values for scan speed (340 mm/s) and average power (3.4 W), allowing ∼5-fold reduction in write time. Importantly, these minima do not correspond to constant ratios of average laser power to scan speed. This highlights the benefits of DoE-RS methods in rapid identification of optimum parameter combinations that would be difficult to discover using traditional one-factor-at-a-time optimization. Verification data from Raman spectroscopy showed sharp 2D peaks with mean full-width-at-half-maximum intensity values <80 cm-1 for both regions, consistent with high-quality 3D graphene-like carbon. Graphene-based electrodes fabricated using the parameters from the respective regions yielded similar performance when employed as capacitive humidity sensors with hygroscopic dielectric layers. Devices fabricated using Region 1 parameters (16 Ω/sq) yielded capacitance responses of 0.78 ± 0.04 pF at 0% relative humidity (RH), increasing to 31 ± 7 pF at 85.1% RH. Region 2 devices (36 Ω/sq) showed comparable responses (0.88 ± 0.04 pF at 0% RH, 28 ± 5 pF at 85.1% RH).

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Optimization of a chondrogenic medium through the use of factorial design of experiments.

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Optimization of a chondrogenic medium through the use of factorial design of experiments.

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