1.
A systems approach to plant bioprocess optimization.
Cloutier, M, Chen, J, De Dobbeleer, C, Perrier, M, Jolicoeur, M
Plant biotechnology journal. 2009;(9):939-51
Abstract
A dynamic model for plant cell metabolism was used as a basis for a rational analysis of plant production potential in in vitro cultures. The model was calibrated with data from 3-L bioreactor cultures. A dynamic sensitivity analysis framework was developed to analyse the response curves of secondary metabolite production to metabolic and medium perturbations. Simulation results suggest that a straightforward engineering of cell metabolism or medium composition might only have a limited effect on productivity. To circumvent the problem of the dynamic allocation of resources between growth and production pathways, the sensitivity analysis framework was used to assess the effect of stabilizing intracellular nutrient concentrations. Simulations showed that a stabilization of intracellular glucose and nitrogen reserves could lead to a 116% increase in the specific production of secondary metabolites compared with standard culture protocol. This culture strategy was implemented experimentally using a perfusion bioreactor. To stabilize intracellular concentrations, adaptive medium feeding was performed using model mass balances and estimations. This allowed for a completely automated culture, with controlled conditions and pre-defined decision making algorithm. The proposed culture strategy leads to a 73% increase in specific production and a 129% increase in total production, as compared with a standard batch culture protocol. The sensitivity analysis on a mathematical model of plant metabolism thus allowed producing new insights on the links between intracellular nutritional management and cell productivity. The experimental implementation was also a significant improvement on current plant bioprocess strategies.
2.
Characterization of the chondrocyte actin cytoskeleton in living three-dimensional culture: response to anabolic and catabolic stimuli.
Haudenschild, DR, Chen, J, Steklov, N, Lotz, MK, D'Lima, DD
Molecular & cellular biomechanics : MCB. 2009;(3):135-44
Abstract
The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation--precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1beta and TGF-beta. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1beta and TGF-beta differentially alter the actin cytoskeleton using time-lapse microscopy. TGF-beta increases actin extensions, and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1beta causes cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.
3.
Glycated Collagen I (GC) impairs angiogenesis in vitro: a study using an innovative chamber for cell research.
Brodsky, SV, Merks, RM, Mendelev, N, Goo, C, Chen, J
Diabetes research and clinical practice. 2007;(3):463-7
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Abstract
Studies of cell-matrix, cell-cell interaction or angiogenesis on different matrices require simultaneous comparison of read-out parameters from differently treated companion cells. The culture conditions (cell number, temperature and volume of culture medium) in different chambers are not completely equalized using conventional methods. It has been reported that cells growing in different environmental conditions may exhibit different proliferation patterns [P. Tracqui, J.W. Liu, O. Collin, J. Clement-Lacroix, E. Planus, Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays, Cell Proliferat. 38(June (3)) (2005) 119-135]. Herein we describe an innovative chamber, which could resolve this problem by significantly improving the standardization of experimental conditions. The chamber was manufactured from a standard cell culture well by its division with a septum into two sections. We utilized the chamber and recently developed topological analysis to examine the effects of glycated matrices on the capillary-like network formation by endothelial cells. Glycated Collagen I resulted in dose-dependent changes to all measured topological characteristics of the capillary-like network, such as the number of branching points, number of meshes and total capillary length. These differences were observed only in neighbored compartments coated with different matrices, but not in the compartments coated with the same matrix. The novel chamber brings an opportunity for better standardization of experimental conditions and simultaneous observation of different experimental groups, reducing the possible effect of any systematic error.