High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations.

Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 107 cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 107 cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures.

We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors.

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.

Orientation of cutinase adsorbed onto PMMA nanoparticles probed by tryptophan fluorescence.

The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d=106.0+/-0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3-0.4, 2-3, and 6-7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp-Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.

Thermodynamics and mechanism of cutinase stabilization by trehalose.

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.

Aggregation as the basis for complex behaviour of cutinase in different denaturants.

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.

Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Adsorption of human IgG on to poly(N-isopropylacrylamide)-based polymer particles.

Thermosensitive poly(N-isopropylacrylamide)-based polymer particles were synthesised, and screened for the adsorption of human immunoglobulin G (hIgG). At pH 9 the adsorption on microgel particles was strongly affected by temperature, approximately 40 mg hIgG/g support (90% of initial hIgG) being adsorbed at 40 degrees C but only 10% of initial hIgG at 25 degrees C. At pH 5 the maximum adsorbed amount (20 mg hIgG/g support) was similar for both temperatures. The adsorption of hIgG on to charged poly(methyl methacrylate)/poly(N-isopropylacrylamide) core-shell latexes was negligible (5-10 mg hIgG/g support) at the same temperature and pH conditions. The lower adsorption of hIgG onto the core-shell particles is explained by steric interactions due to the small size of the shell.

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS.

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.

Crystal structures of the free and sterol-bound forms of beta-cinnamomin.

The crystal structure of the elicitin beta-cinnamomin (beta-CIN) was determined in complex with ergosterol at 1.1 A resolution. beta-CIN/ergosterol complex crystallized in the monoclinic space group P2(1), with unit cell parameters of a = 31.0, b = 62.8, c = 50.0 A and beta = 93.4 degrees and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 A structure of beta-CIN (P4(3)2(1)2 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of beta-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to beta-CIN provided an explanation for the apparent inability of beta-CIN to bind this ligand, as observed experimentally.