Ex vivo method

The antioxidant activities of carotenoids and other phytochemicals in the human body can be measured, or at least estimated, by a variety of techniques, in vitro, in vivo or ex vivo (Krinsky, 2001). Many studies describe the use of ex vivo methods to measure the oxidisability of low-density lipoprotein (LDL) particles after dietary intervention with carotene-rich foods. However, the difficulty with this approach is that complex plant foods usually also contain other carotenoids, ascorbate, flavonoids, and other compounds that have antioxidant activity, and it is difficult to attribute the results to any particular class of compounds. One study, in which subjects were given additional fruits and vegetables, demonstrated an increase in the resistance of LDL to oxidation (Hininger et al., 1997), but two other showed no effect (Chopra et al, 1996 van het Hof et al., 1999). These differing outcomes may have been due to systematic differences in the experimental protocols or in the populations studied (Krinsky, 2001), but the results do indicate the complexity of the problem, and the hazards of generalising too readily about the putative benefits of dietary antioxidants. 

The ex vivo methods lend themselves easily for the performance of mechanistic investigations. In order to optimize selection of drug candidates prior to further clinical development, it is important to decipher the contributive roles of permeation, metabolism, efflux, and toxicity. This will then make it possible to properly channel the optimization process, for instance, by permeation enhancement, mucoadhesion, modification of the physicochemical characteristics of the drug, or even change in the route of administration in case the drug and/or formulation turns out to be too toxic. Regarding permeability studies, it is possible not only to quantify passive diffusion but also to identify and characterize (compound)-specific carrier-mediated transport routes. These tools have been used to identify and characterize the relative contribution of... 

The laboratory evaluation of platelet response to aspirin therapy has demonstrated response variability and nonresponsiveness, Based on different ex vivo methods, studies have shown wide variability in the prevalence of aspirin resistance (< I-54,7%) (45,49-58) (Table I). Potential reasons for these discrepancies include (/) wide variability in the criteria to define aspirin resistance, (//) variability in the methods to measure responsiveness, (///) the timing of the laboratory test after aspirin treatment, (/V) the duration of aspirin treatment, and (v) the dose of aspirin administered,... 

Table 6 Quantitative In Vivo and Ex Vivo Methods Developed at the University of Ottawa and Their Application in Studies on the Survival and Germicide Inactivation of Viruses... 

Because of safety reasons, the in vivo methods mentioned above cannot be used for working with formulations where the safety of the chemical ingredients is unknown and/or with high-risk infectious agents such as HIV. To overcome this, we have developed ex vivo methods using either human skin removed during cosmetic surgery or pieces of human umbilical cord as detailed below and applied them to study the virucidal activity of topicals (Table 4). 

Cells are taken from the body of the genetically defective individual, treated, then returned to the body. An example of this ex vivo method is given below for the correction of severe combined immunodeficiency. If blood cells are used, and since blood cells have a limited lifespan, periodic cycles of ex vivo treatment and reinfusion are necessary. It may be more expedient to target the stem cells of the bone marrow, because these are apparently immortal. Neonates with SCID have now been treated by inserting genes into the stem cells, and at 2 years of age these were still thriving without the benefit of further treatment. 

Alternative in vitro or ex vivo methods have been developed and vahdated for assessing ocular irritation [60, 61]. 

In this experiment, gene expression was assessed by several methods. Liver biopsies found hepatocytes which expressed the LDL receptor. In addition, small changes in cholesterol metabolism were found. However, the clinical effect was negligible and the investigators concluded that the ex vivo method was limited by the low efficiency of genetic reconstitution . 

Koeneman BA, Lee KK, Singh A (2004) An ex vivo method for evaluating the biocompatibility of neural electrodes in rat brain slice cultures. Journal of Neuroscience Methods 137 257-263. 

The ex vivo IL-6 and TNF-inducing activities of fractionated and modified or unmodified poly(MA-CDA) were performed according to the method reported [26] and shown in Figs. 12 and 13, respectively. A similar tendency was shown in IL-6 and TNF induction from peripheral whole blood cells by those of poIy(M A-CDA). 

A major drawback of this study was that we measured lipid peroxidation ex vivo, but not in vivo using the latest and most promising methods such as F2 isoprostanes (Roberts and Morrow, 2000). However, we are planning to do that soon, so hopefully future studies will bring us more detailed information about the effects of phloem on lipid peroxidation. In conclusion, our study showed that lignans are bioavailable from the wood matrix, that long-term consumption of phloem is safe and that ingestion of phloem can inhibit lipid peroxidation in humans. 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

The initial approach to gene therapy involved manipulation of gene expression ex vivo. Toward this end, the desired target cells are identified and subsequently removed from the subject, transfected in vitro, then reintroduced into the patient. A number of protocols have been established for the ex vivo transfection of a wide variety of cell types. This method allows specific cell targeting and high transfection efficiency. However, the process is time consuming, complex, and costly. Additionally, the method is not applicable to all situations, such as those in which an immediate modification is required. 

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

Hermann, C. et al., A model of human whole blood lymphokine release for in vitro and ex vivo use, J. Immunol. Methods, 275, 69, 2003. 

However, others maintain that adipose tissue is an important contributor to VD. Bjorkman showed that adipose and muscle tissue partitioning are the two tissues that yield the best predictions of VDSS and that such data obtained in other tissues did not offer more accuracy [27]. (Note that the tissue partitioning data used to predict human VDSS were from rat or rabbit ex vivo measurements.) The emphasis on both adipose and muscle was also advocated by Poulin and Thiel in their prediction method that uses solvent/water partition coefficients [28] (see below). 

Fig. 9. Viscoelastic properties of ultrasound-treated ex vivo porcine muscle specimen. Muscle samples were coagulated with focused ultrasounds in selected regions. MRE using the method of Ref. 23 was carried out and shear moduli were calculated in normal and heated regions at different shear wave frequencies. Upper curve FUS-treated tissue. Lower curve normal tissue. Error bars are for standard deviations. (From Ref 47, reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley Sons, Inc.)...

Gene Therapies. The types of vectors that have been used or proposed for gene transduction include retrovirus, adenovirus, adeno-associated viruses, other viruses (e.g., herpes, vaccinia, etc.), and plasmid DNA. Methods for gene introduction include ex vivo replacement, drug delivery, marker studies, and others and in vivo, viral vectors, plasmid vectors, and vector producer cells. 

---