Lee-Barthel Ann, Baar Keith, West Daniel W D
Department of Neurobiology, Physiology, and Behavior, University of California, Davis.
Department of Neurobiology, Physiology, and Behavior, University of California, Davis; Department of Physiology and Membrane Biology, University of California, Davis;
J Vis Exp. 2017 Jun 11(124):55339. doi: 10.3791/55339.
In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been 'conditioned' by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.
体外实验对于理解生物学机制至关重要；然而，单层组织培养与人体生理学之间的差距很大，研究结果的转化往往不尽人意。因此，有大量机会采用替代实验方法。在此，我们介绍一种方法，即从人前交叉韧带组织残余物中分离人细胞，在培养中进行扩增，然后用于构建工程韧带。运动可改变血液中的生化环境，从而改善许多组织、器官和身体过程的功能。在本实验中，韧带构建体的培养基补充了经运动“预处理”的人实验血清。因此，这种干预在生物学上更具相关性，因为实验组织暴露于完整的内源性生化环境中，包括可能与未知感兴趣因子的活性协同改变的结合蛋白和辅助化合物。处理后，可对工程韧带的力学功能、胶原蛋白含量、形态和细胞生物化学进行分析。总体而言，本文提出的韧带组织生理模型相对于传统单层培养和动物模型具有四个主要优点。首先，韧带构建体是三维的，能够量化诸如极限拉伸应力、最大拉伸载荷和模量等力学性能（即功能）。其次，可以在功能背景下详细检查骨与肌腱成分之间的界面——附着点。第三，用运动后血清制备培养基能够以无偏倚的方式研究运动诱导的生化环境的影响，而这种生化环境是运动带来广泛健康益处的原因。最后，这个实验模型通过取代动物的使用，以人道和符合伦理的方式推进了科学研究，这是美国国立卫生研究院、疾病控制中心和食品药品监督管理局的一项核心任务。
