The Study Of Construction And Application Of Tissue-stimulated Hollow Fiber Bioreactor In Food Toxicology Evaluation

Food safety toxicological evaluation is a kind of food safety evaluation, such as acute toxicity, genetic toxicity, chronic toxicity and the evaluation of the carcinogenic. The objects of these study include new resource food, functional food, food additives, food packaging materials and various chemical and biological contaminants through food processing and so on. Nowadays, people mainly use animal experiments to complete the toxicological evaluation of toxic substances in functional food, but there are several inevitable problems such as low efficiency of the evaluation method, long experimental period, and the existence of ethical issues. Thus, the international trend is using the in-vitro cells model to replace the animal experiment. But as a two-dimensional culture model, traditional monolayer cultivation cannot provide a similar micro environment for cells, which results in a large discrepancy of cell morphology and signal transduction between in vivo and in vitro model. Therefore, there is urgently need to develop a new method of food toxicology evaluation which can not only reflect the actual metabolism of the cells but also easy to use.The objective of this dissertation is to construct a three-dimensional cell models by culturing cells in hollow fiber, and to explore the feasibility of using these bioreactors for further functional ingredients study appraisal. In this study, use of rat tail collagen was help to distribute and adhere uniformly in the surface of hollow fiber membrane during cell culture. Hollow fiber membrane bioreactors are capable of offering an in vivo-like microenvironment for adherent cell types, such as Caco-2 and HepG2 cells, and enable a high quantity of the desired cellular product with less population variation and favorable operating costs.As mentioned above, the main research results are listed as follow:1. the Caco-2 cells were cultivated in polyether sulfone (PES) and polyvinylidene fluoride (PVDF) hollow fiber membranes as well as the classic Transwell culture plate. According to morphological observation and functional analysis, we compared and evaluated the proliferation and differentiation of Caco-2 cells between three-dimensional hollow fiber reactor and two-dimensional models. Briefly, time-dependent morphology observation of cell layers indicated that the Caco-2 cells in hollow fiber, gradually formed confluent monolayer within 10 days, spread well and showed a clear outline. The brush border enzymes were well differentiated with higher expressed function within 8-14 days compared to the traditional model. All of them tended to be consistent in the late cultivation. Therefore, both of the PES and PVDF hollow fiber membrane can effectively accelerate the proliferation and differentiation of Caco-2 cells during 21 days culture period, thereby come to the formation of a highly stable 3D cell model.2. Based on the formation of successfully constructing of Caco-2 cells bioreactor, we further attempted to build hepatocyte hollow fiber reactors. This study also incubated HepG2 (Human hepatoblastoma cell lines) and LO2 (human normal liver cells) cells in PES and PVDF hollow fiber membrane by mixing with rat tail collagen. HepG2 cells embedded in hollow fiber reactor can survive for more than 10 days, and maintain hepatocyte function.3. We selected acrylamide for HepG2 cell bioreactor to verify its accuracy and practicability in terms of toxicity studies. Furthermore, mulberry anthocyanins also been used to explore the feasibility of the hepatocyte bioreactor in toxicological evaluation of functional ingredients.In summary, this study aims to construct a novel bioreactor for Caco-2 cells and HepG2 cells culture, and to explore the feasibility and applicability of this model in absorption and toxicological evaluation of functional ingredients. According to these experiments, we successfully constructed three-dimensional cell models by optimizing experiment conditions and functional analysis. These bioreactors can be used for absorption and toxicity evaluation of ingredients, which might provide a novel method for the in vitro research of functional foods.