Adaptation of Explanted Primary Cells to 2D and 3D Culture Environments

人们普遍认为，细胞通过对局部信号和物理提示做出反应来适应环境。例如，在传统的二维(2D)聚苯乙烯基质上生长的培养细胞采用了不自然的几何结构，重塑了它们的细胞骨架，改变了它们的生长、分化和功能特征，成为了天然细胞的拙劣替代品。在单层培养中生长的单个细胞的绝大多数表面积要么暴露在塑料基质中，要么暴露在孵化介质中，与相邻细胞相互作用的机会最少，这与真实组织中的情况形成了鲜明对比。这些因素对细胞性能有很大影响，从而影响生物检测的代表性。人们认识到，今天研究中使用的许多现有的和受欢迎的细胞系已经远离它们的来源，不再作为一个有效的模型真正具有代表性。三维(3D)细胞培养模型已被证明克服了许多这些限制，使细胞能够以更现实的方式生长和发挥功能。生物技术公司Reinnerate Limited(www.reenerate.com)开发了Alvetex，这是一种新型的多孔聚苯乙烯支架，为细胞提供了一个三维生长空间。支架被设计成200微米厚的薄膜，安装在现有的细胞培养板和培养皿中。细胞占据支架，与相邻细胞紧密结合形成3D结构，在体外基本上产生一层薄的组织层。Alvetex是作为一种广泛通用的平台技术开发的，并通过共同开发设备(如井插件)进行了优化，为用户提供了设计自己的3D文化系统的灵活性。在这项研究中，我们建议研究和比较原代细胞在2D(传统塑料器皿)和3D(Alvetex)格式下移植和维护到聚苯乙烯基质上时的生长、分化和功能。我们将使用鸡胚胎的外植体作为已建立的模型来研究不同环境下的组织形成。在一定的体外条件下，颅神经脊外植体通过正常的分化顺序被证明发生了软骨形成和成骨的相关过程。例如，当不同面部突起的外植体在体外保持时，软骨形成的形态与它们衍生出来的面部突起相关。胚胎肢体的外植体经历了类似的软骨形成的空间模式。我们的目标是在采用Alvetex技术的3D培养系统中从这些来源获得原代培养物，并与传统塑料器皿上的外植体进行比较。我们试图建立在2D和3D原代培养中保持的细胞类型之间的差异，在不同空间模式中发生相同的分化过程的系统中。这些差异将突出限制现有2D培养技术潜力的因素，并表明如何提高细胞在培养中的能力。为了进一步开发和测试培养系统，学生将致力于以下目标：(1)开发在新型聚合物基质中建立鸡胚胎软骨细胞、肢芽间充质和面部突起间充质的原代培养方案；(2)通过微阵列分析来自2D和3D培养的RNA以及每种培养类型的特征标记。(3)比较在2D和3D培养中维持已建立的细胞系的效果以及两种相同类型的细胞的发育潜力。小鼠胚胎细胞系ATDC5能够形成软骨，其分化能力也将在2D和3D培养中进行研究，然后在这些环境中长期培养。

It is widely recognised that cells adapt to their environments through responding to local signals and physical cues. For example, cultured cells grown on conventional two dimensional (2D) polystyrene substrates adopt an unnatural geometry, remodel their cytoskeleton and change their growth, differentiation and functional characteristics, and become a poor proxy of their native counterparts. The vast majority of the surface area of individual cells grown in monolayer cultures is either exposed to the plastic substrate or incubating medium, with minimal opportunity for interaction with adjacent cells, which is in contrast to what occurs in a real tissue. These factors have a significant impact on cell performance and consequently influence the representation of the biological assay. It is recognised that many of the existing and popular cell lines used in research today have become far removed from their source of origin and are no longer truly representative as an effective model. Three-dimensional (3D) cell culture models have been shown to overcome many of these limitations and enable cells to grow and function in a more realistic manner. The biotechnology company, Reinnervate Limited (www.reinnervate.com), has developed Alvetex, a novel porous polystyrene scaffold that provides a 3D space in which cells can grow. The scaffold is engineered into a 200 micron thick membrane that is mounted within existing cell culture plates and dishes. Cells occupy the scaffold and form 3D structures in close union with adjacent cells and essentially produce a thin tissue layer in vitro. Alvetex is developed as a platform technology for widespread generic use and has been optimised through the co-development of devices such as well inserts to provide users with flexibility to design their own 3D culture systems. In this study, we propose to investigate and compare the growth, differentiation and function of primary cells when explanted and maintained onto polystyrene substrates in 2D (conventional plasticware) and 3D (Alvetex) formats. We will use explants of chick embryo as an established model to study tissue formation in different environments. The linked processes of chondrogenesis and osteogenesis have been demonstrated to occur from cranial neural crest explants under certain ex vivo conditions via the normal sequence of differentiation. For example, when explants of different facial processes are maintained ex vivo, chondrogenesis occurs with a morphology that correlates with the facial process from which they derive. Explants of embryonic limbs undergo similar spatial patterns of chondrogenesis. Our aim is to derive primary cultures from these sources in a 3D culture system employing Alvetex technology in comparison to explants on conventional plasticware. We seek to establish the differences between cell types maintained in primary cultures in 2D and 3D in systems where the same differentiation process occurs in different spatial patterns. These differences will highlight the factors limiting the potential of existing 2D culture techniques and indicate how the capabilities of cells in culture can be improved. To develop and test the culture system further the student will work on the following objectives: (1) Develop protocols for establishing primary cultures of chick embryo chondrocytes, limb bud mesenchyme and facial process mesenchyme in the novel polymer matrix; (2) By microarray analyse RNA from 2D and 3D cultures and markers that are characteristic of each culture type. (3) Compare the effects of maintaining established cell lines in 2D and 3D culture and the developmental potential of both equivalent cell types. The mouse embryonic cell line, ATDC5, is capable of cartilage formation and its capacity to differentiate will also be investigated in 2D and 3D culture subsequent to long term culture in these environments.