Ex-vivo approaches

Alternatively, gene therapy can be performed through in vivo manipulation of gene expression. In this case, the desired genes must be directly delivered to the appropriate cell population in the subject in vivo. This strategy offers several advantages over the ex vivo approach. For instance,... 

The in vivo approach overcomes some of these limitations because a single dosage form may be applicable to a large population of patients. Delivery in vivo, however, is fraught with considerably greater complexities and challenges than those encountered in the ex vivo approach. The in vivo approach must achieve delivery in the... 

A platelet aggregation inhibitor, nitric oxide (NO), for example, has shown promising results. Using an ex vivo approach, bovine smooth muscle cells... 

Boasquevisque CH, Mora BN, Boglione M, Ritter JK, Scheule RK, Yew N, Debruyne L, Qin L, Bromberg JS, Patterson GA. Liposome-mediated gene transfer in rat lung transplantation a comparison between the in vivo and ex vivo approaches. J Thorac Cardiovasc Surg 1999 117 8-15. 

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. 

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. 

With the exception of whole-animal host resistance assays, the actual testing approach can be described as ex vivo-in vitro in that exposure of the immune system to potential immunotoxicants takes place in vivo, with subsequent immunological evaluation taking place in vitro. Although this approach obviates many uncertainties (effect of xenobiotics on primary or secondary lymphoid tissue, potential requirements for metabolism/bio-transformation, etc.), the use of whole animals presents many secondary issues, such... 

An evaluation of the various studies reported in the literature for preclinical assessment of drugs for nasal administrations indicated the usefulness of in situ, ex vivo, and in vivo approaches. Evidence from the literature also indicates that the choice of a particular model or animal species by different... 

The degree of exposure of the fetus to a particular substance can be best assessed in human subjects, but concerns of fetal safety have restricted the use of this approach. Moreover, clinical studies cannot elucidate the various mechanisms that contribute to transplacental transport of a particular compound. There are many structural differences between the human placenta and the placenta of other mammalian species, which complicates extrapolation of data obtained from in vivo animal models to humans [7], Thus, several ex vivo and in vitro techniques have been developed to study the placental role in drug transfer and metabolism during pregnancy and there are some excellent articles that discuss these systems in detail [7], Both isolated tissues and various cell culture techniques are currently in use and these have been summarized below. 

Along with electronic transport improvements must come attention to substrate transport in such porous structures. As discussed above, introduction of gas-phase diffusion or liquid-phase convection of reactants is a feasible approach to enabling high-current-density operation in electrodes of thicknesses exceeding 100 jxm. Such a solution is application specific, in the sense that neither gas-phase reactants nor convection can be introduced in a subclass of applications, such as devices implanted in human, animal, or plant tissue. In the context of physiologically implanted devices, the choice becomes either milliwatt to watt scale devices implanted in a blood vessel, where velocities of up to 10 cm/s can be present, or microwatt-scale devices implanted in tissue. Ex vivo applications are more flexible, partially because gas-phase oxygen from ambient air will almost always be utilized on the cathode side, but also because pumps can be used to provide convective flow of any substrate. However, power requirements for pump operation must be minimized to prevent substantial lowering of net power output. 

Long term ex vivo maintenance of human hematopoietic stem cells requires growth promoting cytokines , factors that inhibit proliferation of clonogenic cells (such as MIP-la) stroma-derived growth factors and direct contact with stroma. The need for direct cell-to-stroma contact is still a matter of many scientific debates and its resolution depends on the development of new scientific approaches. 

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