Optimising cell-based bioassays via integrated design of experiments (ixDoE) - A practical guide.

For process optimisation Design of Experiments (DoE) has long been established as a more powerful strategy than a One Factor at a Time approach. Nevertheless, DoE is not widely used especially in the field of cell-based bioassay development although it is known that complex interactions often exist. We believe that biopharmaceutical manufacturers are reluctant to move beyond standard practices due to the perceived costs, efforts, and complexity. We therefore introduce the integrated DoE (ixDoE) approach to target a smarter use of DoEs in the bioassay setting, specifically in optimising resources and time. Where in a standard practice 3 to 4 separate DoEs would be performed, our ixDoE approach includes the necessary statistical inference from only a single experimental set. Hence, we advocate for an innovative, ixDoE approach accompanied by a suitable statistical analysis strategy and present this as a practical guide for a typical bioassay development from basic research to biopharmaceutical industry.

Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling.

Sudden Ca2+ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152-2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (approximately 10 ms) after Ca2+ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (approximately 24 ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca2+ removal until troponin C is switched off. However, this switch off occurs approximately 5 times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.