Microchambers for inverted microscopes

The designs for two microchambers, non-flow and flow, for inverted microscopes are described. These chambers are intended to study dynamic processes in living cells. The chambers described are made completely of a glass. An advantage of the flow chamber is the possibility of making simple changes in its internal space. It is dismantled easily to allow careful cleaning. This is especially important when working with toxic substances.     

Use of a whirling cup rotor to stir benthic chambers

Realistic stirring is an important concern when using benthic chambers to measure processes at the sediment-water column interface. A simple whirling cup rotor device that is driven by external flow and stirs benthic chambers effectively is described here. Flow tank studies and field studies demonstrated that this device homogenizes benthic chambers rapidly and responds quickly and linearly to external flow conditions. The device is simple to build and use and avoids many of the disadvantages of conventional stirring motors and pumps.     

Ventilation of the branchial chambers in the amphibious West African freshwater crab,Sudanonautes (Convexonautes) aubryi monodi (Balss, 1929) (Brachyura,Potamonautidae)

The anatomy of the respiratory system of the savanna-zone African freshwater crab, Sudanonautes (Convexonautes) aubryi monodi [Balss, 1929], has been examined and has been found to be adapted for both aerial and aquatic gas exchange. The activities of the scaphognathites and the directions of flow of the ventilatory stream have been recorded in stressed, active and resting specimens during their exposure to a wide range of conditions from deep water to dry land.Ventilation of the branchial chambers during aquatic gas exchange in Sudanonautes kept in deep water is shown to consist of a rapid, predominantly forward water flow similar to that of fully-aquatic species. Ventilation of the branchial chambers during aerial gas exchange in Sudanonautes on land is shown to consist of a relatively slow forward air flow. This flow is continuous in post-operative crabs, pulsatile in active crabs and completely immobile in resting crabs.A second method of ventilation of the branchial chambers during aerial gas exchange is shown to consist of a pulsatile reversed air flow. This occurs (1) when Sudanonautes is kept in very shallow water and active or stressed; (2) when it has recently moved on to land; and (3) when it is completely immersed and exhibiting aerial gas exchange under water. The unusual phenomenon of aerial gas exchange under water is reported here for the first time in any species of crab.Bimodal ventilation of the branchial chambers occurs in stressed or active crabs partly immersed in shallow water. This consists of an alternation between forward water flow and reversed air flow.The morphology of the branchial chambers in Sudanonautes, and observational data on the patterns of ventilation of the branchial chambers, are discussed in relation to those described for other air-breathing decapod crustaceans.     

A portable flow chamber for in situ determination of benthic metabolism

1. Many stream ecologists are interested in determining the metabolic rates of benthic organisms, particularly those of production and respiration. It is often necessary to make these measurements on fresh material in the field at remote sites. Recirculating chambers are commonly used for this purpose.
2. A broad variety of recirculating chambers are described in the literature, but each design has inherent limitations. The most common are inability to control flow in the chamber and match it with external flow rates, and a lack of the power required to do this for extended periods. Alteration of spectral irradiance, temperature rise and elevated internal chamber pressures are additional limitations that have received little attention.
3. We have designed and constructed a flow chamber that eliminates some of these problems. The chamber utilizes a DC motor-driven propeller as an efficient recirculator (axial impeller), minimizing power requirements and it is constructed of UVB transparent acrylic to allow a full spectral complement of solar irradiance in the interior. Modular components allow the chamber to be taken apart quickly for cleaning and replacement of parts, making it more functional than some previous designs.
4. The axial impeller chamber was compared to a similar sized conventional chamber that had a small diameter return line and a high capacity centrifugal pump. The axial impeller chamber had less of a temperature rise during field incubations, lower power consumption and less internal pressure in the return line when producing equivalent water velocities.
5. The reported axial impeller design had relatively homogeneous velocity across the working section relative to other chambers and was capable of water velocities in excess of 1 m s^–1.     

Minitron II system for precise control of the plant growth environment

A transparent, cylindrical chamber system was developed to allow measurement of gas-exchange by small crop canopies in the undisturbed plant growth environment. The system is an elaboration of the Minitron system developed previously to compare growth of small plants in different environments within the same general growth area. The Minitron II system described herein accommodates hydroponic culture and separate control of atmospheric composition in individual chambers. Root and shoot environments are compartmented separately to accommodate atmospheres of different flow rate and/or gaseous composition. A series of 0-rings and tension-adjustable springs allow carbon dioxide in the flowing atmosphere to be analyzed without cross-contamination between chamber compartments or from external gas sources. Carbon dioxide has been maintained at set point +/- 9 g m-3 over a range of CO2 concentrations from 382 to 2725 g m-3 and with an atmosphere turnover rate of 136.7 cm3 s-1 by computer-assisted mass flow controllers. Each chamber has dimensions large enough (61 cm internal diameter, 0.151 m3 internal volume) to allow adequate replication of individual plants for statistical purposes (e.g., up to 36 equally-spaced plant holders). No significant variation in growth or photosynthetic rate of leaf lettuce occurred between chambers for a given set of environmental conditions. Gas-exchange rates in different chambers changed to a similar extent as CO2 concentration in the flowing atmosphere or chamber temperature were varied by the same amount. When coupled with appropriate control systems, Minitron II chambers can provide separate controlled environments for multiple small plants with adequate precision and at relatively low cost.     

Parallel-plate fluid flow systems for bone cell stimulation

Bone responds to changes in its mechanical environment, but the mechanisms by which it does so are poorly understood. One hypothesis of mechanosensing in bone states that osteocytes can sense the flow of fluid through the canalicular system. To study this in vitro a number of fluid flow devices have been designed in which cells are placed between parallel plates in sealed chambers. Fluid flows through the chambers at controlled rates, most commonly driven by a peristaltic pump. In addition to fluid flow, high pressures have been observed in these chambers, but the effect of this on the cellular responses has generally been ignored or considered irrelevant, something challenged by recent cellular experiments using pressure only. We have, therefore, devised a system in which we can considerably reduce the pressure while maintaining the flow rate to enable study of their effects individually and in combination. As reducing pressure also reduces the risk of leaks in flow chambers, our system is suitable for real-time microscopical experiments. We present details of the new systems and of experiments with osteoblasts to illustrate the effects of fluid flow with and without additional pressure on the translocation of β-catenin to the nucleus.     

A low shear stress modular bioreactor for connected cell culture under high flow rates

A generic “system on a plate” modular multicompartmental bioreactor array which enables microwell protocols to be transferred directly to the bioreactor modules, without redesign of cell culture experiments or protocols is described. The modular bioreactors are simple to assemble and use and can be easily compared with standard controls since cell numbers and medium volumes are quite similar. Starting from fluid dynamic and mass transport considerations, a modular bioreactor chamber was first modeled and then fabricated using “milli‐molding,” a technique adapted from soft lithography. After confirming that the shear stress was extremely low in the system in the range of useful flow rates, the bioreactor chambers were tested using hepatocytes. The results show that the bioreactor chambers can increase or maintain cell viability and function when the flow rates are below 500 µL/min, corresponding to wall shear stresses of 10^?5 Pa or less at the cell culture surface. Biotechnol. Bioeng. 2010; 106: 127–137. © 2010 Wiley Periodicals, Inc.     

A new in vitro assay for cell motility and proliferation

We have described and characterized a new micropore membrane assay for migration and proliferation of cells of various tumourigenic potential. The assay was developed to facilitate analysis of some aspects of cancer invasion and metastasis. Tumorigenic and non-tumorigenic C3H/10 T 1/2 cells grow in and migrate out of a culture chamber during a 1-11 day period, the shorter periods are used for chambers with 6 micron thick polycarbonate membranes, the longer ones for 140 micron thick cellulose nitrate membranes. Cell growth within the chambers, in their micropore membranes and on the outside of the membranes, was assessed with microscopy, electronic cell counting, flow cytometry of propidium iodide (PI) stained cells, and 3H-thymidine [( 3H]TdR) incorporation. A complete retrieval of intact cells that have traversed the membraneous chamber wall is possible, and these cells can be recultured or used in other studies. The tumorigenic cells had a steeper growth curve in vitro than the non transformed cells, but the relative sizes of the emigrated subpopulations were not significantly different. The subpopulation of tumorigenic cells that emigrated spontaneously from the chambers was less able than the subpopulation retained to populate secondary chamber cultures, suggesting that the clonogenic (stem) tumour cells are 'slow movers'.     

Experimental Studies of the Factors Controlling Transpiration: I. APPARATUS AND EXPERIMENTAL TECHNIQUE

Apparatus and experimental techniques are discussed for usein the investigation of transpiration rate of leaves as controlledby stomatal aperture and leaf water content. The leaf chambers used and the methods adopted for the estimationof the water transpired are described. The designs of the porometer cups used for the different typesof leaves (Pelargonium and wheat) employed in the work are described.To obviate the difficulty that stomata within the cup behaveabnormally, the design employed makes possible a removal ofthe cup from the leaf except during the short periods requiredto estimate stomatal resistance to air flow at intervals duringthe course of an experiment. In these experiments the water content of the leaf is changedat will by interrupting the water-supply and re-establishingit to permit recovery from wilting. The methods used to carryout this cycle of operations are fully dealt with. Determinationsof the transpiration and absorption rates during the experimentand of the final leaf water content make it possible to followchanges in leaf water content throughout the experiment. An account is given of the methods used for varying the speedof flow, the humidity, and the CO₂ concentration of the airstreams.     

Controlled-Environment Sunlit Plant Growth Chambers

Controlled environment sunlit plant growth chambers have been built because of a great interest in plant responses to environmental variables under light intensities approaching those of natural sunlight conditions. Individual research projects have designed sunlit chambers that differ in size, structure, material, and environmental control systems dependent on the goals of the projects. Most literature describes plant organism responses to environmental variables, whereas reports of system design and performance are few. The objective of this article is to present a review of the engineering aspects of the design, environmental control, and performance of sunlit growth chambers that have been described in the literature. Most controlled environment sunlit growth chambers have been constructed with experimental plants grown in either pots or soil bins. Although a few sunlit growth chambers were designed for field grown plants, precise environmental control was not available. Further progress in the development of precise controlled environment sunlit growth chambers should include portability (or movability) so these chambers can be used in multiple field sites for greater cost-effectiveness. Modifications for improvements in guidelines for the design and operation of controlled environment growth chamber studies are also proposed.     

A new in vitro assay for cell motility and proliferation

Abstract. We have described and characterized a new micropore membrane assay for migration and proliferation of cells of various tumourigenic potential. The assay was developed to facilitate analysis of some aspects of cancer invasion and metastasis. Tumorigenic and non-tumorigenic C3H/10 T1/2 cells grow in and migrate out of a culture chamber during a 1–11 day period, the shorter periods are used for chambers with 6 μ m thick polycarbonate membranes, the longer ones for 140 μ m thick cellulose nitrate membranes. Cell growth within the chambers, in their micropore membranes and on the outside of the membranes, was assessed with microscopy, electronic cell counting, flow cytometry of propidium iodide (PI) stained cells, and ³H-thymidine ([³H]TdR) incorporation. A complete retrieval of intact cells that have traversed the membraneous chamber wall is possible, and these cells can be recultured or used in other studies. The tumorigenic cells had a steeper growth curve in vitro than the non transformed cells, but the relative sizes of the emigrated subpopulations were not significantly different. The subpopulation of tumorigenic cells that emigrated spontaneously from the chambers was less able than the subpopulation retained to populate secondary chamber cultures, suggesting that the clonogenic (stem) tumour cells are 'slow movers'.     

Automatic lipid extraction and thin-layer chromatography application with a prgrammed flow system

An eight-channel programmed flow system for automatic lipid extraction and TLC application is described. Each channel has a container for lipid extraction connected by Acidflex tubing through an AutoAnalyzer pump to a TLC applicator needle. Extraction containers are prepared from disposable Oxford sampler pipet tips by inserting a small cotton filter into their lower, narrower end, which is connected to the pump tubing. The applicator needles are supported vertically in a manifold, and their tips rest on a TLC plate placed on a hot plate. Serum is added to isopropanol in each extraction container, and proteins are completely precipitated in 2 min and retained in the extraction chambers by the cotton filters; lipid extracts are then transferred on to the heated TLC plate by intermittent pumping at a rate allowing for continuous evaporation of isopropanol under streams of warmed air or nitrogen. The lipids accumulate on the plate in eight small spots, one for each channel. Solvent is proportionally added into the extraction chambers from a common reservoir through Acidflex tubing in a second AutoAnalyzer pump. During the extraction procedure, both pump motors are automatically operated by a programmed timer with a solid-state switch. Of several different solvents tested, isopropanol is the fastest for protein precipitation and lipid extraction and does not extract substances from the Acidflex tubing which interfere with chromatographic separation.     

Ion exposure chambers for small animals

Ion exposure chambers that have been designed and tested for use in biological and behavioral research with small animals are described in this report. The chambers exhibit an acceptable degree of uniformity in ion concentration, current density, and electric field within the exposure area. Gaseous by-products of corona discharge (O3 and NO2) have been measured and found to be less than .01 ppm and less than .1 ppm, respectively. Filtered air is fed to the individual exposure chambers, and temperature and humidity are well controlled. Noise due to corona and the air delivery system has been measured.     

Methods for stable recording of short-circuit current in a Na+-transporting epithelium

Epithelial Na(+) transport as measured by a variety of techniques, including the short-circuit current technique, has been described to exhibit a "rundown" phenomenon. This phenomenon manifests as time-dependent decrease of current and resistance and precludes the ability to carry out prolonged experiments aimed at examining the regulation of this transport. We developed methods for prolonged stable recordings of epithelial Na(+) transport using modifications of the short-circuit current technique and commercial Ussing-type chambers. We utilize the polarized MDCK cell line expressing the epithelial Na(+) channel (ENaC) to describe these methods. Briefly, existing commercial chambers were modified to allow continuous flow of Ringer solution and precise control of such flow. Chamber manifolds and associated plumbing were modified to allow precise temperature clamp preventing temperature oscillations. Recording electrodes were modified to eliminate the use of KCl and prevent membrane depolarization from KCl leakage. Solutions utilized standard bicarbonate-based buffers, but all gasses were prehydrated to clamp buffer osmolarity. We demonstrate that these modifications result in measurements of current and resistance that are stable for at least 2 h. We further demonstrate that drifts in osmolarity similar to those obtained before prior to our modifications can lead to a decrease of current and resistance similar to those attributed to rundown.     

Analytical techniques for cell fractions. XVI. Preparation of protein-free supernatants with a "Z"-path rotor

A new “Z”-path centrifuge rotor is described for the separation of pellets from supernatants in up to 15 samples simultaneously. The samples and precipitating solutions are stored in separate chambers at rest, and are transferred to a sedimentation chamber by centrifugal force during rotation, where mixing occurs during rapid changes in speed, followed by sedimentation of the precipitate. The supernatant decants itself at rest into a third series of chambers, where it may be transferred centrifugally into radially disposed measuring devices, transferred with suitable pipettes, or used as a sample ring for conventional analyzers.     

The circumfusion system for multipurpose culture chambers. I. Introduction to the mechanics, techniques, and basic results of a 12-chamber (in vitro) closed circulatory system

A self-contained mechanical system for circulating nutrient fluid through 12 tissue culture chambers is described in detail. This system utilizes nonperforated cellophane membranes in the chambers which separate the circulating nutrient from the tissue culture environments. The nutrient, therefore, is dialyzed through the cellophane of each chamber; some cell products are retained in the microenvironment between the closely apposed cellophane and cover slip, whereas the other cell products move from chamber to chamber in the circulating nutrient. The resultant environmental conditions directed by the circumfusion systems are highly favorable for maintaining the differentiation of chick embryo tissues over protracted periods; a number of micrographs are shown.     

A multichamber equilibrium dialysis apparatus

A method for inexpensively producing large quantities of equilibrium dialysis cells as well as two types of cell rotators is described. The method of production is simple enough that several hundred chambers can be produced in a single day. The assay procedure is flexible enough that any number of assays from one to several hundred may be completed in a short time. The chambers have proved extremely useful in the isolation and kinetic characterization of proteins which appear to be associated with membrane transport systems. They will also suffice for any of the other many uses for equilibrium dialysis. The large number of chambers available also provides an ideal means for determining the proper conditions for the growth of protein crystals by dialysis against the crystallizing agent. Microgram quantities of protein are easily crystallized by this technique.     

Temperature gradient chambers for research on global environment change. I. Portable chambers for research on short-stature vegetation

Field temperature gradient chambers designed for experiments on short-stature plants such as wheat are deseribed. The chambers are portable, easily erected and dismantled, and are self-contained for control and measuring equipment. The design is modular, the modules being bolted together longitudinally although separated by slotted transparent septa which divide the chamber into zones of different temperature. Fresh air, which is blown in horizontally into one end of the chamber by two fans and extracted by a fan mounted vertically at the other end, passes sequentially through the modules. The air stream progressively heats when the sun is shining. Fans are automatically speed-controlled in 100 steps between 20 and 100% of full output to keep the end-to-end temperature difference to within 5°C. During darkness, when the fans are running at minimum speed, heaters mounted in the outlet module are turned on. The chambers in the configuration described enclose 6 × 8m rows of crop, are l-25m high and have side walls which are entirely composed of rigid, vertically sliding doors for crop access.     

Full factorial screening of human embryonic stem cell maintenance with multiplexed microbioreactor arrays

Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine/paracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.     

A Microflow Respirometer for Measuring the Oxygen Consumption of Small Aquatic Organisms

A microrespiration device is decribed which uses a Clark electrode to measure the oxygen consumption or production of small and microscopic aquatic organisms in an open flow system. The construction and working principles of the device, which can measure oxygen consumptions as low as 0.5 nl · h⁻¹, are described. The design of the apparatus permits parallel measurements under identical conditions with a single electrode. The device can be matched to various sizes of animal and oxygen consumption rates by means of specimen chambers of different volumes (6 μl, 35 μl, 140 μl) and a variable water flow rate. The microflow respiration device has been used successfully to measure the respiration of zooplankton and meiobenthos organisms as well as protozoans and has also been used successfully on board a research vessel.