Boubriak Olga A, Urban Jill P G, Cui Zhanfeng
Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK.
J R Soc Interface. 2006 Oct 22;3(10):637-48. doi: 10.1098/rsif.2006.0118.
At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.
目前，对组织工程构建体的评估几乎总是通过生化或组织学方法进行破坏性检测，以确定整个构建体内的细胞数量、活力和组织生长情况。由于许多此类实验耗时较长，需要数周甚至数月才能完成，因此，简单且易于应用的非破坏性方法来监测构建体内细胞代谢、活力和组织沉积的变化将非常宝贵；此类方法可以在培养早期指出不良反应。在此，我们描述了使用微透析检测组织工程构建体内细胞代谢的局部变化。将包裹在藻酸盐凝胶中的牛关节软骨细胞组成的三维构建体在生物反应器中培养两周。分别将葡萄糖和乳酸作为主要营养物质和代谢产物，通过微透析进行监测。构建体内浓度梯度明显，构建体中心乳酸浓度最高。局部乳酸浓度是细胞代谢活性的指标，随着细胞活性下降而降低，随着细胞活性受到刺激而升高。在故意在不利条件下培养的构建体中，可以很容易地检测到构建体中心的营养饥饿和细胞死亡。结果表明，探针测量可以对不适当的局部代谢变化发出早期预警。在组织工程构建体生长过程中获得此类信息将有助于采取纠正措施或尽早终止不成功的试验。
