Liquid-covered culture

Keywords Nasal absorption Air-interfaced culture Absorption enhancer Liquid-covered culture Mucociliary clearance Nasal epithelial cell mono-layer Paracellular absorption Tight junction Transcelluar absorption Drug transport... 

Source Data from Ref. [46]. AIC, Air-interfaced culture. LCC, Liquid-covered culture. 

For AIC conditions, the apical surface of the epithelial cell layer is exposed to air after the nasal cells reached confluence on the Transwell insert, while the basolateral side is fed with culture fluid. Figure 9.3 shows TEER changes in epithelial cell layers cultured up to 20 days in LCC versus AIC methods [46], In AIC condition (initiated from day 3 after seeding), TEER peaked on day 5 and maintained above the TEER values observed for LCC counterparts. By contrast, TEER observed for LCC conditions peaked on day 2 and declined toward zero by day 15. These data indicate that human nasal epithelial cells at an air interface culture exhibit better electrophysiological characteristics than those cultured by the conventional liquid-covered conditions. 

Figure 13.3 A flowchart illustrating various steps for the preparation and maintenance of primary rabbit conjunctival epithelial cell layers, cultured under liquid-covered and air-interfaced conditions (See also color insert).

Vaginal material is best submitted as liquid in a tube, although swabs submitted in a small amount of saline may be used. A drop of the material is covered with a cover slip and examined with reduced light. To culture, 1 or 2 drops of urine sediment or vaginal exudate are inoculated into tubes of warmed, modified Diamond medium. If vaginal swabs are submitted, the swab is immersed in the medium and pressed against the side of the tube to express material. Tubes are incubated at 35°C, and drops of culture are examined by wet mount at 48 and 72 h for motile trophozoites. 

A schematic of the experimental system is shown in Fig. 1. One liter of culture liquid was added to a 2.8-L cylindrical glass column reactor, and 300 mL of cylindrical carrier was packed with rock wool. All of the tubing connections, stoppers, and seals in the column were made of butyl rubber. To prevent photodecomposition of vitamin B12, the outside of the whole device was covered with a vinyl sheet to shut out light. The digestion was carried out at 55°C and 20 d of hydraulic retention time (HRT). 

Liquid Culture Medium Dissolve 15 g of peptonized milk, 5 g of water-soluble yeast extract, 10 g of anhydrous glucose, and 2 g of anhydrous potassium dihydrogen phosphate in about 600 mL of water. Add 100 mL of filtered tomato juice (filtered through Whatman No. 1 filter paper, or equivalent), and adjust to pH 6.5 by the dropwise addition of 1.0 N sodium hydroxide. Add, with mixing, 10 mL of the Polysorbate 80 Solution. Dilute with water to a final volume of 1000 mL. Add 10-mL portions of this Liquid Culture Medium to test tubes, cover to prevent contamination, and sterilize by heating in an autoclave at 121° for 15 min. Cool the tubes rapidly to keep color formation to a minimum, and store at 10° in the dark. 

The second method incoporates a blender with an autoclavable container-stirrer assembly. (Several companies sell aluminum and stainless steel units specifically manufactured for liquid culture techniques—refer to the sources listed in the Appends). Fill with water until two thirds to three quarters full, cover with aluminum foil (if a Tight fitting metal top is not handy), sterilize and allow to cool to room temperature. 

Figure 59 Eberbach container manufactured for liquid culture. Note bolt covering inoculation hole.

Effective plasma treatment of bacteria, cells, and other biomaterials is possible not only if they are in contact with plasma but even when they are separated from plasma and protected by a layer of intermediate substance, in particular submerged into a culture medium (Kieft et al., 2004, 2005 Fridman et al., 2007). Detachment of mammalian vascular cells was observed after indirect treatment by a non-thermal atmospheric-pressure plasma needle when the thickness of the liquid layer covering the cells was about 0.1 mm (Kieft et al.,... 

Remove the culture medium by aspiration and add 2 ml PBS per well to wells that contain a cover slip. Incubate for 1 min at room temperature and wash once more with 2 ml PBS. Aspire all liquid, add 1 ml freshly prepared fixative, and incubate 15 min at room temperature. Remove the fixative and wash three times for 1 min with 2 ml PBS. To reduce autofiuorescence, incubate the cover slips after fixation in 2 ml freshly prepared NaBHi solution and incubate 5 min at room temperature. Remove the solution and add another 2 ml NaBH4 solution and incubate again 5 min. Next, wash two time for 1 min with 2 ml PBS. Cover slips with fixed cells can be stored in PBS at 4°C. Add a preservative to prevent bacterial and fungal growth. Do not allow the cover slips to dry. 

At least 1 h before collecting the eggs, set up four 35-mm tissue-culture dishes containing 2-3 mL M16 medium and two 35-mm culture dishes containing M16 microdrops (40 pL) covered with liquid parafifm. Allow these to equilibrate in a 3TC incubator gassed with 5% (v/v) CO. At the same time, prepare live 35-mm tissue culture dishes containing 2-3 mL M2 medium and leave at room tanpeiature. 

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