A high-rate perfusion bioreactor for plant cells

A perfusion bioreactor allowing continuous extraction of secondary metabolites was designed and challenged for Eschscholtzia californica plant cell suspensions. Four sedimentation columns mounted inside a 2.5-L bioreactor separated single cells and cell aggregates from the culture medium. Cells were elicited with chitin at day 4 and the liquid medium free of cells and debris was then continuously pumped to the extraction columns containing fluidized XAD-7 resins, and then recirculated back to the cell suspension. A medium upward velocity corresponding to cell sedimentation velocity maintained a stable cell/medium separation front in the columns for sedimented cell volume (SCV) of 90% (70% packed cell volume, PCV). Two perfusion bioreactor cultures of 10 and 14 days were performed. A maximum dilution rate of 20.4/day was reached from day 4 to day 6, and was then reduced to 5/day at day 9 for 55% SCV. Control cultures were performed without and with free extraction resins into the cell suspension. Perfusion cultures showed similar specific growth rates of 0.24 +/- 0.04/day before and after elicitation. However, production level in the perfusion cultures was similar to that from the culture without resins with a maximum of 2.06 micromole/gDW total alkaloids, with 1.54 micromole/gDW in the resins. Cultures with free resins resulted in 30.94 micromole/gDW with 28.4 +/- 8.8 micromole/gDW in the resins. Difference in the cells nutritional state from elicitation was identified as a major cause in the production reduction. However, pathway to chelilutine was favored in the continuous extraction culture.     

Effects of pipette geometry on the time course of solution change in patch clamp experiments.

The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.     

Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.     

The effects of plant traits on species' responses to present and historical patch configurations and patch age

Patch configuration is viewed as an important factor affecting the distributions of plant species. Although a number of studies have explored the relationship between plant life‐history traits and species’ distributions in fragmented landscapes, the effect of individual traits on the dependence of species on historical versus current landscape configurations remains unclear. We identified the extent to which present (2000s) and historical (1843, 1954, 1980s) patch configurations (area and connectivity) and patch age affected the distributions of 99 species inhabiting dry calcareous grasslands. We used traits related to dispersal, survival, growth and habitat preferences to explain the dependence of 60 of these species on present and historical configurations, and the age of grassland patches. We found that most of the species had an affinity to currently or historically large, older and more isolated patches. This suggests that many dry grassland species are not in equilibrium with the current landscape, as their distributions still reflect past landscape structures. Rapidly growing species with higher seed bank longevity and nutrient requirements primarily occur in young, large grassland patches whereas species with the opposite traits occur in older, historically large and currently more isolated smaller patches. We hypothesise that patch quality is the reason why different species occupy patches of different age. Species occupying young, large patches commonly disperse by endozoochory. By contrast, no dispersal trait is associated with species occupying old, usually isolated patches. Our results suggest that species occupying old patches are exposed to higher potential risk of extinction, as their distributions are probably limited by the low number and connectivity of available suitable patches and poor dispersal ability. We thus suggest that the dynamics of these species can effectively be supported by improving the quality of young grassland patches.     

Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts

Summary Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2±0.2 pA (mean±se,n=21) at±40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg²⁺ in 0.15m KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with aP _k/P _Cl=32 (n=6). Treatment with alkaline pH (8.3) increased the current and anion permeability (P _K/P _Cl=16,n=6). The membrane becomes completely cation selective when low concentrations (12 m) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 m), propranolol (70 m) and quinine (0.6mm) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.     

Chromatin techniques for plant cells

A large number of recent studies have demonstrated that many important aspects of plant development are regulated by heritable changes in gene expression that do not involve changes in DNA sequence. Rather, these regulatory mechanisms involve modifications of chromatin structure that affect the accessibility of target genes to regulatory factors that can control their expression. The central component of chromatin is the nucleosome, containing the highly conserved histone proteins that are known to be subject to a wide range of post-translational modifications, which act as recognition codes for the binding of chromatin-associated factors. In addition to these histone modifications, DNA methylation can also have a dramatic influence on gene expression. To accommodate the burgeoning interest of the plant science community in the epigenetic control of plant development, a series of methods used routinely in our laboratories have been compiled that can facilitate the characterization of putative chromatin-binding factors at the biochemical, molecular and cellular levels.     

Voltage-independent barium-permeable channel activated inLymnaea neurons by internal perfusion or patch excision

Summary Isolated nerve cells fromLymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba²⁺-permeable channel with a slope conductance of 27 pS at negative potentials (50mm Ba²⁺). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca²⁺ below 1mm or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca²⁺ or Ba²⁺. The channel can be activated in on-cell patches by either internal perfusion with high Ca²⁺ or the long-term internal perfusion of low Ca²⁺ solutions not containing ATP. These channels may carry the inward Ca²⁺ current which causes a regenerative increase in intracellular Ca⁺ when snail neurons are perfused with high Ca²⁺ solutions. High internal Ca²⁺, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (–50 mV) whole-cell Ba²⁺ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca²⁺ solution containing ATP and Mg²⁺. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.     

Serum-free solutions for cryopreservation of cells

With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37° C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these formulations.     

Experimental analysis of patch dynamics in tallgrass prairie plant communities

Abstract. Previous research has indicated that patch structure at small spatial scales (<100m²) in tallgrass prairies was defined by a diverse array of infrequent species because dominant species occurred in all samples at this scale. Also, patch structure was not significantly different from that derived from random species associations. Based on these results, we hypothesized that remo val of a dominant species would have no effect on patch structure in these prairies. We tested this hypothesis by removing a dominant grass, Schizachyrium scoparium (Poaceae), from half of each of four 10 m × 10 m study blocks, and comparing differences in patch structure between control and removal halves before and after removal. The minimum resolution in our study was 1 m². Patches of similar species composition were defined by cluster analysis of presence/absence data and cover data. Patch sizes ranged from 1 to 34 m². Following the removal of S. scoparium there was an overall increase in the number of species in the removal half of each block compared to pre-treatment levels. However, the number of patch types and number of spatially mapped groups, based on presence/absence or cover data, did not change between control and removal plots after the removal of S. scoparium. This supports the hypothesis that removal of a large, dominant species would have no effect on patch structure at this scale of resolution in these prairies. Thus, patch structure, as defined here, is an emergent property in these grasslands that is not predictable from changes in species composition. This property of stochastic patch structure results from interactions of processes operating at scales both larger and smaller than our scale of resolution. Stochastic models may provide a reasonable approach to modelling small-scale patch dynamics in tallgrass prairie communities.     

A perfusion air-lift bioreactor for high density plant cell cultivation and secreted protein production

A new bioreactor design that allows continuous perfusion cultivation of plant cell suspensions is described in this paper. This design incorporates an internal cell settling zone with an external-loop air-lift bioreactor. The settling zone is created by inserting a baffle plate into the upper portion of the downcomer. Using this bioreactor, Anchusa officinalis suspension culture was cultivated to a cell density of 27.2 g l⁻¹ DW in 14 days at a perfusion rate of 0.123 per day. The maximum total extracellular protein concentration attained 1.11 g l⁻¹. Complete cell retention was achieved throughout the culture during which the maximum packed cell volume (PCV) exceeded 80%. In comparison, the maximum cell density and extracellular protein concentration in the batch culture were 12.6 g l⁻¹ DW and 0.47 g l⁻¹, respectively. SDS-PAGE of the extracellular protein samples revealed two major bands at 58 and 47 kDa, each accounted for approximately 45% of the total secreted proteins.     

Reconstitution of the native mitochondrial outer membrane in planar bilayers. Comparison with the outer membrane in a patch pipette and effect of aluminum compounds

Summary Detergent-free rat brain outer mitochondrial membranes were incorporated in planar lipid bilayers in the presence of an osmotic gradient, and studied at high (1 m KCl) and low (150 mm KCl) ionic strength solutions. By comparison, the main outer mitochondrial membrane protein, VDAC, extracted from rat liver with Triton X-100, was also studied in 150 mm KCl. In 1 m KCl, brain outer membranes gave rise to electrical patterns which resembled very closely those widely described for detergent-extracted VDAC, with transitions to several subconducting states upon increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, however, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mm KCl, bilayers containing only one channel had a conductance of 700 ± 23 pS for rat brain outer membranes, and 890 ± 29 pS for rat liver VDAC. Use of a fast time resolution setup allowed demonstration of open-close transitions in the millisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference higher than approx. ± 60 mV induced an almost irreversible decrease of the single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the behavior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Biophys. J. 20:311–319).The neurotoxicological action of aluminum was studied in single outer membrane channels from rat brain mitochondria. We found that m concentrations of Al Cl₃ and aluminum lactate decreased the conductance by about 50%, when the applied potential difference was positive relative to the side of the metal addition.The authors thank Dr. O. Moran for helpful discussions, Dr. M. Colombini for a sample of polyanion, and the Sharing Company for financial support to Dr. T. M. This work was partly supported by funds from the Ministero dell' Universitá e della Ricerca Scientifica e Tecnologica of Italy.     

Microfluidic perfusion for regulating diffusible signaling in stem cells

Background

Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands.

Methodology/Principal Findings

We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs.

Conclusions/Significance

Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell