Use of a Silicone Rubber (Clear Seal) as a Section Adhesive Resistant to Acid, Alkali and Heat

An optically clear silicone rubber adhesive is recommended for use in histochemical procedures in which detachment of tissue sections is likely. Procedure: Cut paraffin sections and float on a 45-50 C water bath; leave frozen sections on the microtome knife in the cryostat; spread the silicone rubber thinly and evenly over 2/3 of the slide (Clear Seal—General Electric, was used); pick up paraffin sections directly from the floatation water and frozen sections from the microtome knife with a warm slide; dry for 1.5 hr at 25 C; place paraffin sections in a 60 C oven for 0.5 hr, deparaffinize through xylene and hydrate through alcohols to water. Stain sections as desired, but avoid clearing agents before mounting after strong acid or alkaline treatment, and mount rapidly if a synthetic resin is used because of the solvent effect on the silicone rubber. Of the adhesives tried, silicone rubber is the only one capable of withstanding boiling 10% HCl for any period of time without detachment of sections.     

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO_2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO_2gas and that experienced by the cells (pO_2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen‐permeable silicone rubber membranes can be used to control pO_2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene‐propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte‐like increases with decreasing pO₂ only when using oxygen‐permeable silicone membrane‐based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO_2cell being equal to pO_2gas at the tissue‐membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO₂ distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO_2cell, and these results suggest they are a valuable tool for investigating pO₂ effects in many applications, such as stem cell differentiation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A Method for Processing Light Microscopy Sections for Oriented Ultramicrotomy

A simple technique is described for processing optical microscopy sections attached to glass slides for ultramicrotomy in any desired plane. A silicone rubber mold with a central orifice is clamped onto the slide so that the orifice overlies the section. Routine processing and embedding procedures are carried out in the well formed by the orifice.     

Use of oxygen-permeable silicone rubber pouches for growing mass cultures of bacteria

Growth of most bacteria often involves the use of expensive incubated shaker systems. In this report, oxygen-permeable silicone rubber pouches, with oxygen permeability over 100 times higher than other polymers, were employed for growing bacterial cultures. With little, if any, agitation oxygen-permeable silicone rubber pouches produced bacterial growth rates equivalent to growth rates obtained in shaker flasks. The silicone rubber pouch described has a glass cuvette integrated into its design that permits readings of bacterial density without opening the pouch. One can sterilize and store powdered bacterial culture medium in silicone rubber pouches ; therefore, bacterial cultures can be initiated by simply adding water and bacteria.     

Silicone rubber membrane as a support for long-term cultivation and electron microscopic processing of nervous tissue

Silicone rubber membrane can be successfully used as a support for cultivation of nervous tissue; its processing for electron microscopy is described: The advantages of silicone rubber membrane as support, compared with glass, are. its biological inertness, the ability to withstand dry-heat sterilization at 160°C, transparency, easy processing for electron microscopy. Spatial configuration of cells and explants in specimens is preserved.     

Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

This article describes how a combination of an ultra scale‐down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Σ_M) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell‐plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO‐S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development. Biotechnol. Bioeng. 2009; 104: 321–331 © 2009 Wiley Periodicals, Inc.     

Quantitative Histochemistry; Design and Use of a Simple Microcell for Standardized Incubation on the Slide

Avoidance of stain variation among mounted sections incubated in small volumes of fluid can be overcome by the construction and use of a microcell. Perspex rings of appropriate diameter are attached to small Perspex rectangles to form wells, small trip bars being also attached. to the rectangles to simplify subsequent manipulation. The wells are filled with incubation fluid, and slides bearing frozen sections are held in position with small rubber bands. With certain dehydrogenases formazan production by this method is uniformly greater than that produced by incubation in drops of solution. In the case of the moderately active enzyme, 6-phosphogluconate dehydrogenase, the volume of the incubation mixture was not critical between 65 and 270 μl.     

Biocompatibility and fatigue properties of polystyrene-polyisobutylene-polystyrene, an emerging thermoplastic elastomeric biomaterial

This paper will discuss the biocompatibility and dynamic fatigue properties of polystyrene-b-polyisobutylene-b-polystyrene thermoplastic elastomer with 30 wt % polystyrene (SIBS30), an emerging FDA-approved biomaterial. SIBS30 is a very soft, transparent biomaterial resembling silicone rubber, with superior mechanical properties. Using the hysteresis method adopted for soft biomaterials, the dynamic fatigue properties of SIBS30 were found to be between those of polyurethane and silicone rubber, with fatigue life twice as long as that of silicone. Under single load testing (SLT, 1.25 MPa), SIBS30 displayed less than half the dynamic creep compared to silicone, both in air and in vitro (37 degrees C, simulated body fluid). Hemolysis and 30- and 180-day implantation studies revealed excellent biocompatibility of the new biomaterial. The results presented in this paper indicate that, in comparison with silicone rubber, SIBS30 has similar biocompatibility and superior dynamic fatigue properties.     

Fabrication and selective functionalization of amine-reactive polymer multilayers on topographically patterned microwell cell culture arrays

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. "Reactive" layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts.     

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.     

A new silicone rubber duplicating material advantageous for teaching and research in physical anthropology

Room temperature vulcanizing silicone rubber is an excellent duplicating material which has many advantages when compared to other available materials. A silicone mold of an object is flexible for an indefinite period of time, dimensionally stable, remarkably heat stable, and resists weathering and oxidation. A silicone mold is simply prepared without incurring damage to the duplicated object. Successive casts of various types of materials can be made from the mold. The physical anthropologist may use this material in preparing casts for display or as a teaching aid, and for research purposes.     

A New Time-Saving Device for Embedding in Paraffin

A simple device to be used in place of the usual paper boxes or watch crystals used for embedding in paraffin in the preparation of histological sections is described. Short lengths of cooled rubber tubing are placed on a glass surface, which has previously been painted with glycerin, and are filled with melted paraffin. The rubber tubing sections are best cut by stretching the tubing on a wooden rod and cutting while the wood is turning in a lathe.     

Gelatin-glutaraldehyde cross-linking on silicone rubber to increase endothelial cell adhesion and growth

Silicone is a biomaterial that is widely used in many areas because of its high optical clarity, its durability, and the ease with which it can be cast. However, these advantages are counterbalanced by strong hydrophobicity. Gelatin cross-linking has been used as a hydrophilic coating on many biomaterials but not on silicone rubber. In this study, two gelatin glutaraldehyde (GA) cross-linking methods were used to coat a hydrophilic membrane on silicone rubber. In method I, gelatin and GA were mixed in three different proportions (64:1, 128:1, and 256:1) before coating. In method II, a newly formed 5% gelatin membrane was cross-linked with a 2.5% GA solution. All coatings were hydrophilic, as determined from the measurement of contact angle for a drop of water on the surface. Bovine coronary arterial endothelial cells were shown to grow well on the surface modified by method II at 72 h. In method I, the cells grew well for gelatin-GA proportions of 64:1 and 128:1 at 72 h. No cell attachment on untreated silicone rubber was observed by the third d of seeding. The results indicated that both methods of gelatin-GA cross-linking provided a hydrophilic surface on silicone for endothelial cell adhesion and growth in vitro.     

Four Years' Experience with Indwelling Silastic Cannulae for Long-term Peritoneal Dialysis

Indwelling silicone rubber cannulae have been used for peritoneal dialysis in 41 uraemic patients for periods of up to 46 months. The simplicity of this treatment is particularly suited to patients awaiting transplantation.     

Silicone rubber membrane bioreactors for bacterial cellulose production

Cellulose production byAcetobacter pasteurianus was investigated in static culture using four bioreactors with silicone rubber membrane submerged in the medium. The shape of the membrane was flat sheet, flat sack, tube and cylindrical balloon. Production rate of cellulose as well as its yield on consumed glucose by the bacteria grown on the flat type membranes was approximately ten-fold greater than those on the non-flat ones in spite of the same membrane thickness. The membrane reactor using flat sacks of silicone rubber membrane as support of bacterial pellicle can supply greater ratio of surface to volume than a conventional liquid surface culture and is promising for industrial production of bacterial cellulose in large scale.     

Ethylene loss from the gas phase of container-seal systems

Ethylene losses from the gas phase of various container-seal systems were studied to develop acceptable methods for containing ethylene during experiments. Ethylene at an initial amount of 104 μI I^-1 was stored in glass vials at near atmospheric pressure for 20 h at 25–27°C and at 35% relative humidity external to the vials. Crimped serum vials sealed with saturated (NH₄)₂SO₄ solution, neoprene rubber septa, nitrile rubber (Hycar) septa, butyl rubber septa, and brown translucent silicone rubber septa lost ethylene at the rate of 1.8, 10.2, 16.2, 16.5, and 40.2 nl m^-2s^-1, respectively, over the 20-h period. Screw-capped reaction vials sealed with white silicone rubber septa lost ethylene at the rate of 30.2 nl m^-2s^-1. The (NH4)₂SO₄ solution was utilized as a seal by inverting a vial so that the salt solution covered the internal surface of the vial septum. Saturated (NH₄)²SO₄ solution is an effective seal. Silicone rubber should be avoided as a seal in systems for containing ethylene. Ethylene production values in the literature may be underestimates where silicone rubber seals have been used.     

A membrane bioreactor for biotransformations of hydrophobic molecules

The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases. This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification. The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation. Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane. Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1. The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system. The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction. Copyright 1998 John Wiley & Sons, Inc.     

The Mounting of Carbowax Sections with Lighter-Fluid and Rubber Cement

Serial sections cut from plant tissues embedded in Carbowax have been affixed to slides with rubber cement. A rather thick layer of undiluted rubber cement was first spread on the slides. The Carbowax ribbons were added next. Lighter-fluid, essentially petroleum ether which can be substituted for it, was then run under the sections to dissolve the rubber cement and to float the ribbons. This notation medium did not dissolve the Carbowax and the ribbons could be manipulated in it for accurate location. The slides were dried on a 45° C warming table which also helped to flatten the sections. Adhesion was best when drying times were held to 4 hr or less. All excess rubber cement was washed away with xylene immediately prior to covering and the cover slips were carefully applied with a very thin resinous mounting medium to prevent dislodging the sections. Both aqueous and alcoholic stains have been used successfully and the slides have been left in them for as long as 3 days without loss of sections. The method was developed for fluorescence microscopy but serves equally well for visible light microscopy. Slides stained with a safranin-fast green combination have been used for both purposes, the safranin staining and fluorescing in a manner similar to rhodamine B.     

Use of operating windows in the assessment of integrated robotic systems for the measurement of bioprocess kinetics

This study examines the utility of an automated liquid handling robot integrated with a microwell plate reader to enable the rapid acquisition of bioprocess kinetic data. The relationship between the key parameters for liquid handling accuracy and precision and the sample detection period has been characterized for typical low-viscosity (<2.0 mPa x s) aqueous and organic phases and for a high-viscosity aqueous phase (60 mPa x s), all exhibiting Newtonian rheology. The use of a simple graphical method enables the suitability of a given automation platform to be assessed once the user has determined the minimum sample detection period and the minimum accurate and precise dispense volume. This provides for a reduction in the duration of any experiment by maximizing well usage within each microwell plate. The suitability of employing an integrated automation platform to gather kinetic data for systems typical of those encountered in bioprocessing is analyzed via a series of case studies. Application to alkaline cell lysis, where disruption is complete within 120 s, showed that the range of available dispense volumes and the number of wells that can be utilized is limited. In contrast, analysis of a system exhibiting slow process kinetics, the fermentation of Escherichia coli TOP10 pQR239 in microwell plates, demonstrated that, for a typical sample detection period of 30 min, the only restrictions on the degree of well utilization are the liquid handling accuracy and precision and the volume capacity of the liquid handling robot. Finally, liquid-liquid extraction, an example of a kinetically independent operation, was also examined. In this case, only a single equilibrium measurement is required, which means that the only restrictions to the utilization of the integrated devices are the liquid handling accuracy and precision. Integrated automation platforms represent a powerful process development tool over traditional experimental methods used for bioprocess development. Smaller volumes of reagent and sample can be used to achieve greater throughput, while high levels of reproducibility and sensitivity are maintained.     

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.