Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons. ProtocolPrior to any manipulation, you must identify the group of tendons to be used depending on the experiment you are conducting and the apparatus at your disposition. For our purposes, ventral tendons were chosen because they are smaller and thus easier to manipulate when measuring the cross-sectional area and fitting them into the bioreactor chamber.Please note that all instruments are autoclaved or sterilized with 70% ethanol. Moreover, a spray bottle containing 70% ethanol is placed beside each work station to sterilize the experimenter's gloves before each operation.

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