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

外植原代细胞对 2D 和 3D 培养环境的适应

• 批准号: BB/I015817/1
• 金额: $ 11.71万
• 依托单位: Durham University
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI015817%2F1
• 关键词: Adaptation; Explanted; Primary; Cells; 2D

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.

人们普遍认为细胞通过响应局部信号和物理线索来适应环境。例如，在传统二维 (2D) 聚苯乙烯基质上生长的培养细胞采用不自然的几何形状，重塑其细胞骨架并改变其生长、分化和功能特征，并成为其天然对应物的不良代表。在单层培养物中生长的单个细胞的绝大多数表面积要么暴露于塑料基质，要么暴露于培养介质，与相邻细胞相互作用的机会极小，这与真实组织中发生的情况相反。这些因素对细胞性能有重大影响，从而影响生物测定的代表性。人们认识到，当今研究中使用的许多现有和流行的细胞系已经远离其起源，并且不再作为有效模型真正具有代表性。三维 (3D) 细胞培养模型已被证明可以克服许多这些限制，并使细胞能够以更真实的方式生长和发挥作用。生物技术公司 Reinnervate Limited (www.reinnervate.com) 开发了 Alvetex，这是一种新型多孔聚苯乙烯支架，可提供细胞生长的 3D 空间。该支架被设计成 200 微米厚的膜，安装在现有的细胞培养板和培养皿中。细胞占据支架并与相邻细胞紧密结合形成 3D 结构，并在体外基本上产生薄的组织层。 Alvetex 是作为一种广泛通用的平台技术而开发的，并通过孔插入件等设备的共同开发进行了优化，为用户提供设计自己的 3D 培养系统的灵活性。在这项研究中，我们打算研究和比较原代细胞以 2D（传统塑料器皿）和 3D（Alvetex）格式移植并维持在聚苯乙烯基质上时的生长、分化和功能。我们将使用鸡胚外植体作为既定模型来研究不同环境中的组织形成。软骨生成和骨生成的相关过程已被证明是在某些离体条件下通过正常的分化顺序从颅神经嵴外植体发生的。例如，当不同面部突起的外植体在体外保存时，软骨发生的形态与它们所源自的面部突起相关。胚胎肢体的外植体经历类似的软骨形成空间模式。我们的目标是在采用 Alvetex 技术的 3D 培养系统中从这些来源中获得原代培养物，与传统塑料器皿上的外植体进行比较。我们试图确定在 2D 和 3D 系统中原代培养物中维持的细胞类型之间的差异，其中相同的分化过程发生在不同的空间模式中。这些差异将凸显限制现有二维培养技术潜力的因素，并表明如何提高培养细胞的能力。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）制定在新型聚合物基质中建立鸡胚软骨细胞、肢芽间质和面部过程间质的原代培养物的方案； (2) 通过微阵列分析来自 2D 和 3D 培养物的 RNA 以及每种培养物类型特征的标记。 (3) 比较在 2D 和 3D 培养中维持已建立细胞系的效果以及两种等效细胞类型的发育潜力。小鼠胚胎细胞系 ATDC5 能够形成软骨，其分化能力也将在这些环境中长期培养后在 2D 和 3D 培养中进行研究。