Porous membrane systems

The previous discussions have been based on products like dishes and flasks that have a solid substrate modified for tissue culture. Cells growing on a solid substrate, however, can only receive nutrient and actively transport from the exposed (top) layer. 

A system that more closely resembles the in vivo state is the porous membrane system. Because cells can receive nutrient from both sides of the membrane, the cell layer has an active basal and apical (or luminal) surface that allows the selective uptake and transport of nutrients and secreted products. A variety of biological functions can now be studied, such as active transport, better differentiation and co-culture of different cell types (Pitt Gabriels, 1986 Millipore, 1990 Halleux Schneider, 1991). 

There are several commercially available products that use this principle. The device is commonly a plastic ring with a membrane attached, which can be placed into a multiwell plate or dish. Cells can be grown on one side of the membrane surface, on both sides of the membrane (transmembrane co-culture) or on both the membrane and plate well bottom for co-culture. This membrane device is commonly used in a static system (e.g. multiwell plate or culture dish) but it may also be mounted in an appropriate chamber for continuous perfusion culture. Examples of these products are the Falcon Cell Insert (Becton Dickinson Labware, NJ, USA), Millicell (MiUipore, MA, USA) and Transwell (Costar, MA, USA). 

There are several factors that need to be taken into consideration when using cell inserts  

The membrane material is important for cell attachment and growth. Common materials are polycarbonate, cellulose esters (acetate and nitrate) and PET (polyester). These materials sometimes need to be coated for cells to grow and differentiate. The different membrane materials have very different optical and mechanical properties, which must be considered when planning any experiment. 

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Micrographs of Albatros membrane by W. L Gore and Associates (a) Optical picture showing membrane and support and (b) SEM of nano-porous membrane system. (Source Reprinted based on reference 43.)... 

Membranes. Membranes comprised of activated alumina films less than 20 )J.m thick have been reported (46). These films are initially deposited via sol—gel technology (qv) from pseudoboehmite sols and are subsequently calcined to produce controlled pore sizes in the 2 to 10-nm range. Inorganic membrane systems based on this type of film and supported on soHd porous substrates have been introduced commercially. They are said to have better mechanical and thermal stabiUty than organic membranes (47). The activated alumina film comprises only a miniscule part of the total system (see Mel rane technology). 

The teehniques of membrane extraetion permit an effieient and modern applieation of elassieal liquid-liquid extraetion (LLE) ehemistry to instmmental and automated operation. Various shorteomings of LLE are overeome by membrane extraetion teehniques as they use none or very little organie solvents, high enriehment faetors ean be obtained and there ai e no problems with emulsions. A three phase SLM system (aq/org/aq), where analytes are extraeted from the aqueous sample into an organie liquid, immobilized in a porous hydrophobie membrane support, and further to a seeond aqueous phase, is suitable for the extraetion of polar eompounds (aeidie or basie, ehai ged, metals, ete.) and it is eompatible with reversed phase HPLC. A two-phase system (aq/org) where analytes ai e extraeted into an organie solvent sepai ated from the aqueous sample by a hydrophobie porous membrane is more suitable for hydrophobie analytes and is eompatible with gas ehromatography. 

Another possibility of constructing a chiral membrane system is to prepare a solution of the chiral selector which is retained between two porous membranes, acting as an enantioselective liquid carrier for the transport of one of the enantiomers from the feed solution of the racemate to the receiving side (Fig. 1-5). This system is often referred to as membrane-assisted separation. The selector should not be soluble in the solvent used for the elution of the enantiomers, whose transport is driven by a gradient in concentration or pH between the feed and receiving phases. As a drawback common to all these systems, it should be mentioned that the transport of one enantiomer usually decreases when the enantiomer ratio in the permeate diminishes. Nevertheless, this can be overcome by designing a system where two opposite selectors are used to transport the two enantiomers of a racemic solution simultaneously, as it was already applied in W-tube experiments [171]. 

As the main disadvantage of liquid membrane systems is the instability over a longer period of time, another approach would be to perform separation through a solid membrane [22]. Enantioselective polymer membranes typically consist of a nonse-lective porous support coated with a thin layer of an enantioselective polymer. This... 

The ion pair extraction by flow injection analysis (FIA) has been used to analyze sodium dodecyl sulfate and sodium dodecyl ether (3 EO) sulfate among other anionic surfactants. The solvating agent was methanol and the phase-separating system was designed with a PTFE porous membrane permeable to chloroform but impermeable to the aqueous solution. The method is applicable to concentrations up to 1.25 mM with a detection limit of 15 pM [304]. 

Therefore, the following method was suggested and realized (the scheme is shown in Fig. 17). A 1.5 M solution of KCl or NaCl (the effect of preventing BR solubility of these salts is practically the same) was used as a subphase. A platinum electrode was placed in the subphase. A flat metal electrode, with an area of about 70% of the open barriered area, was placed about 1.5-2 mm above the subphase surface. A positive potential of +50 -60 V was applied to this electrode with respect to the platinum one. Then BR solution was injected with a syringe into the water subphase in dark conditions. The system was left in the same conditions for electric field-induced self-assembly of the membrane fragments for 1 hour. After this, the monolayer was compressed to 25 mN/m surface pressure and transferred onto the substrate (porous membrane). The residual salt was washed with water. The water was removed with a nitrogen jet. 

TABLE 3 Photocurent Observed in a System Using Porous Membranes Covered with BR Film Deposited by the Usual LB Technique and Electric Field-Assisted Technique... 

Tape >System of Analysis. A tape system which is used widely for analysis in the Pediatric Laboratory is a system whose principle was developed by the author. A reagent is placed on a paper tape. The paper is covered with a membrane, such as cellophane, cellulose nitrate or cellulose acetate, porous to low molecular weight substances. Finally, the serum is placed above the porous membrane, so that diffusion of the components of serum take place and a stain is produced on the paper (60). This principle has been incorporated for example, with glucose oxidase, in the conmercially available Dextro-Sticks. In addition, a similar principle is being applied by some for the analysis of components in urine (Ames Co., South Bend, Indiana). 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Consider a system in which both solutions contain various ions for which the membrane is permeable (diffusible ions) and one type of ion that, for some reason (e.g. a macromolecular ion for a porous membrane), cannot pass through the membrane (non-diffusible ion). The membrane is permeable for the solvent. 

The main emphasis in this chapter is on the use of membranes for separations in liquid systems. As discussed by Koros and Chern(30) and Kesting and Fritzsche(31), gas mixtures may also be separated by membranes and both porous and non-porous membranes may be used. In the former case, Knudsen flow can result in separation, though the effect is relatively small. Much better separation is achieved with non-porous polymer membranes where the transport mechanism is based on sorption and diffusion. As for reverse osmosis and pervaporation, the transport equations for gas permeation through dense polymer membranes are based on Fick s Law, material transport being a function of the partial pressure difference across the membrane. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

A common method to slip-cast ceramic membranes is to start with a colloidal suspension or polymeric solution as described in the previous section. This is called a slip . The porous support system is dipped in the slip and the dispersion medium (in most cases water or alcohol-water mixtures) is forced into the pores of the support by a pressure drop (APJ created by capillary action of the microporous support. At the interface the solid particles are retained and concentrated at the entrance of pores to form a gel layer as in the case of sol-gel processes. It is important that formation of the gel layer starts... 

The passage of most foreign compounds from the blood into the liver normally is not restricted because the endothelium of the hepatic blood sinusoids behaves as a porous membrane. Hence, drugs with molecular weights lower than those of most protein molecules readily reach the hepatic extracellular fluid from the plasma. A number of compounds are taken up into the liver by carrier-mediated systems, while more lipophilic... 

A cumulative success of artificial ion-channel functions by simple molecules may disclose a wide gate for the design of ion channels and possible applications to ionics devices. Incorporation of these channels into bilayer lipid membrane systems may trigger the developments towards ionics devices. The conventional BLM system, however, is not very stable, one major drawback for the practical applications, and some stabilization methods, such as impregnating the material in micro-porous polycarbonate or polyester filters, are required. On the other hand,... 

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