利用電噴霧製備GelMA顆粒以及在三維細胞培養之應用

Abstract

人體是由許多精細且複雜的生物系統所組成的，而細胞為生物體組織結構以及功能的基本單位。以細胞培養進行的體外實驗提供了解人體運作的重要資訊。傳統的二維細胞培養有著缺乏細胞間和細胞與胞外基質間的交互作用、細胞受到不平均的拉力使其不自然生長等劣勢，使得二維細胞培養無法真實的模擬生物體內的運作，也因此，能更真實模擬體內生長環境的三維細胞培養技術在近年來越來越受到重視，也已經被廣泛的應用於再生醫學、癌症腫瘤研究、藥物研究等生醫工程領域。細胞球是常見的三維細胞培養技術之一，細胞在懸浮狀態下會聚集成球狀，以此可模擬胚胎生成(embryogenesis)、器官生成(organogenesis)等細胞自我聚集(Self assembly)的自然現象，但細胞球由於中心的養分以及氧氣不足而造成中心細胞會因此而死亡。因此，本研究將GelMA水凝膠顆粒與NIH-3T3纖維母細胞混合後，期望細胞藉由熱運動聚集更多顆粒並再聚集成細胞球，以此讓細胞球中有均勻的水凝膠顆粒，便可增加細胞中心的存活率。在製作顆粒方面，本研究使用電噴霧(Electrospray)技術製造GelMA水凝膠顆粒，有別於其餘方式，電噴霧具有簡單、高彈性、可控性高等特性。本研究詳細的記錄包含參數測試、去除光起始劑、乾燥方式等製造顆粒的過程，並對各步驟進行討論，以此尋找以及探討最穩定的製造方式。綜上所述，本研究目的主要分為兩個部分，第一部分是尋找並確定製程和參數以穩定製造GelMA水凝膠顆粒，第二部分是將GelMA顆粒應用於細胞球培養上，最後雖然實驗結果無法符合我們的預期，但我們根據結果討論分析失敗的可能原因，並提供可行的改進方式，期望之後能克服困難，達成實驗目的。

The human body is composed of many delicate and complex biological systems, and cells are the basic units of the tissue structure and function of the organism. In vitro studies using cell culture provide valuable information to understand the operation of the human body. Traditional two-dimensional cell culture technique has disadvantages such as lack of interaction between cells to cells and cells to extracellular matrix, and cell may grow unnaturally because of suffering unequal tension, which may be challenges for two-dimensional cell culture technique to truly simulate the operation of organisms. Therefore, the three-dimensional cell culture technology that can more realistically simulate the growth environment in vivo has received more and more attention in recent years, and has been widely used in biomedical engineering fields such as regenerative medicine, cancer tumor research, and drug research. Spheroid culturing are one of the common three-dimensional cell culture techniques. Cells will aggregate into spheres in a suspended state, which can simulate the natural phenomenon of cell self-assembly such as embryogenesis and organogenesis. Due to the lack of nutrients and oxygen in the center of the cell spheroid, the center cells don’t have high viability. Therefore, in our research, we propose a scenario to solve this problem. We mix GelMA hydrogel particles and NIH-3T3 fibroblasts cells, and expect multiple particles will adhere on cells and then aggregate into cell spheres, so as to make the cell spheres have uniform hydrogel particles inside, and we believed that can increase the viability of the cells in the cluster center. In terms of making particles, this study utilized electrospray technology to manufacture GelMA hydrogel particles. Different from other methods, electrospray has the characteristics of simplicity, high elasticity, and high controllability. The detailed records of this research include the process of manufacturing particles such as parameter testing, removal of photoinitiator, drying methods, etc., and discuss each step, so as to find and explore the most stable manufacturing method. In summary, the purpose of this research is divided into two parts. The first part is to find and determine the process and parameters to stably manufacture GelMA hydrogel particles. The second part is to apply GelMA particles to spheroid culturing. Finally, although the results cannot meet our scenario, but we discuss the possible reasons for the problems based on the results, and provide feasible improvement methods, hoping to overcome challenges and achieve the experimental purpose.