Two fields of study, immunology and neurobiology, may be particularly affected by a reliance on in vitro experimentation. Both fields study complex systems that are difficult, or impossible, to replicate in vitro, but for that reason it is even more important to consider biases that may be introduced during in vitro experiments and the consequence of these bias on the models predicted. In addition, these two fields of investigations have been chosen because they display the unique characteristics of utilizing generally primary non-immortalized cell cultures which are more sensitive to possible artificia conditioning of the in vitro systems.
“Ninety-six-well plate immunology” has been instrumental in shedding light on fundamental mechanisms of immune cell function including antigen recognition, cytokine secretion, and immune tolerance, to name just a few. A vast majority of in vitro
data in immunology comes from cultures in the presence of bovine sera, as discussed above. In addition, cell isolation techniques based on bead separation or flow cytometry (FACS) employ antibodies that bind to cell surface antigens [10
]. Antibody binding to cells significantly affects cell signalling and immune responses. In the case of positive selection of cells via
magnetic beads or flow cytometers, the binding of antibodies to the cell surface followed by passage through columns or FACS-sorters with high levels of pressure (60 psi
, pounds per square inch; 15 psi
= 1 atmosphere, therefore 4 Atm), speed (90 km/h), electric shock (varying from 2000–4000 V), and laser lights at various wavelengths (i.e. 488nm, 640nm, and 405nm) can significantly alter the intracellular signalling events that may be under investigation [10
Neurobiological experimental approaches also utilize isolated cells, immortalized cell lines, and organotypic cultures [12
]. The pitfalls described above regarding immunobiological approaches apply to these techniques as well [12
]. Over the past 50 years, important insights have been gained into neuronal functions through the use of slice electrophysiology. This tool, which remains widely used and respected by the field, hinges on decapitation of animals followed by labor-intensive and time-sensitive preparation of slices in a an artificial cerebrospinal fluid as a medium to maintain “a living state” [12
]. After hours of preparation subsequent to decapitation, electrical recordings are made from the slices and inferences are drawn regarding neuronal characteristics of synapses, circuits, and overall brain functions. Undoubtedly, this tool has generated extremely useful information on fine parameters of neuronal functions. By now, very few laboratories rely exclusively on slice electrophysiology, although numerous high impact publications can still be found revolving around this approach. However, the extent to which that information is transferable to the better understanding of in vivo
functioning of healthy and diseased brain remains unknown.
On the other side of the spectrum of contemporary neurobiological tools are recently developed techniques for the functional imaging of the intact human brain. These remarkable approaches suggest that there may come a time when rudimentary and ambiguous techniques (e.g., slice electrophysiology or electron microscopy) for the examination of the central nervous system become obsolete in the quest to understand the human brain and mind. Until then, however, much needs to be improved regarding functional imaging. Despite efforts to suggest otherwise, all of these tools provide descriptive information that can in no way explain mechanism, circuit involvement, or aetiology of altered function. These different techniques, such as fMRI and PET, rely on detecting specific molecular shifts as an indication of altered neuronal function in a given area. The fact that these arbitrary measurements and changes can be derived in a precise area during a task compared to other regions does not confirm the specific involvement of that region to that task. Beyond the fact that no experimental data exists to directly connect specific neuronal activity to the altered signal in any of these approaches, the complicated and cumbersome nature of data analyses makes the current imaging techniques more like art than an objective scientific tool. Of course, these methods have the potential to revolutionize human neurobiology as they improve and evolve into a quantifiable descriptive tool but one that can test the role of various brain regions and neuronal populations in brain functions.