Impact of long term propagation in 3D culture on the phenotype of existing human cell lines

在二维(2D)细胞培养的简化条件下，组织特定的结构、机械和生化线索以及细胞与细胞的相互作用被改变或丢失。三维(3D)细胞培养试图通过保持单个细胞的3D完整性来模拟体内的情况，并使细胞能够与细胞外基质一起创建自己的利基环境。有很好的证据表明，3D模型可以用于研究生理学，在那里可以保持组织样功能。然而，在将这些细胞种植到3D培养模型之前，最初在2D培养中扩大细胞群体(特别是细胞系)是常见的做法。在这种情况下，细胞同时：a)适应它们的新环境(2D到3D生长过渡)；以及b)作为3D细胞培养试验的一部分接受挑战和/或检查(例如，药物细胞毒性试验)。这使得很难破译测试的准确结果，因为细胞同时暴露在两个这样的主要变量中。我们假设，最初在3D环境中繁殖和扩展的细胞群在种植到3D培养模型或移植到体内时更有可能适应环境(即不需要2D到3D的转换)。在这个项目中，我们将与为研究人员提供细胞系的组织ECACC培养收集公司合作。常规生长为贴壁2D单层的流行细胞系(例如，A549肺上皮；MCF7乳腺肿瘤；既已建立，也被广泛了解)的增殖将在一种新的系统中保持，该系统使细胞能够在3D中连续传代，将其表型转换为更自然的3D状态。具体地说，学生将：1)(0-18个月)优化方法，在3D培养中连续传代A549和MCF7人类细胞系；2)(6-24个月)通过详细检查细胞形态、细胞骨架结构、整合素/粘附素和粘着斑蛋白的表达以及已知在2D培养中丢失的3D表型相关标记的表达来表征细胞表型；3)(18-30个月)进行2D与3D与体内(例如MCF7肿瘤组织)的3种方式的比较，检查已知在2D和3D培养中差异调控的关键结构细胞蛋白；4)(24-36个月)测试2D和3D繁殖细胞在支持3D细胞体外生长的替代技术中生长时产生3D结构的能力；5)(30-36个月)评估2D和3D繁殖细胞在移植到免疫缺陷小鼠体内时形成异种移植瘤的能力。将对生长速度、大小和组织组成进行比较。该项目将提供有关生长环境如何在培养细胞的基本生物学中发挥重要作用的新信息。学生将获得细胞科学知识，并与学术和工业伙伴一起接受细胞和分子技术方面的培训。预计这些维持细胞的新方法将立即对寻求改进他们的体外模型的生物学家有利。

Tissue-specific architecture, mechanical and biochemical cues and cell-cell interactions are altered or lost under the simplified conditions of two-dimensional (2D) cell culture. Three-dimensional (3D) cell cultures attempt to mimic the in vivo situation by preserving the 3D integrity of individual cells and enabling cells to create their own niche in conjunction with the extracellular matrix. There is good evidence to show that 3D models can be used to study physiology where tissue-like function can be maintained. It is common practice however to expand populations of cells in 2D culture initially (particularly cell lines) prior to seeding such cells into 3D culture models. In such circumstances, cells are simultaneously: a) adapting to their new surroundings (2D to 3D growth transition); and b) being challenged and/or examined as part of the 3D cell culture assay (for example, a drug cytotoxicity test). This makes it difficult to decipher the precise outcome of the assay given that cells are concurrently exposed to two such major variables.We hypothesise that cell populations initially propagated and expanded within a 3D environment are more likely to adapt to their surroundings when seeded into a 3D culture model or transplanted in vivo (i.e. without the need for 2D to 3D transition). In this project we will collaborate with ECACC Culture Collections, an organization that supplies cell lines to researchers. The propagation of popular cell lines that are grown routinely as adherent 2D monolayers (e.g. A549 lung epithelium; MCF7 breast tumour; both established and widely understood), will be maintained in a novel system that enables continual cell passaging in 3D to convert their phenotype to a more native 3D state. Specifically, the student will: 1) (0-18 mths) optimize methods to continually passage A549 and MCF7 human cell lines in 3D culture; 2) (6-24 mths) Characterize the cell phenotype by detailed examination of cell morphology, cytoskeletal structure, expression of integrin/caherins and focal adhesion proteins, expression of markers associated with 3D phenotype known to be lost in 2D culture; 3) (18-30 mths) conduct a 3-way comparison of 2D vs 3D vs in vivo (e.g. MCF7 tumour tissue) examining key structural cell proteins known to be differentially regulated in 2D and 3D culture; 4) (24-36mths) test the ability of the 2D vs 3D propagated cells to produce 3D structures when grown in alternative technologies designed to support 3D cell growth in vitro, e.g. scaffolds, and spheroid cultures; 5) (30-36 mths) Assess the ability of 2D & 3D propagated cells to form xenograft tumours when transplanted into immune deficient mice. The growth rate, size and tissue composition will be compared. The project will provide new information of how the growth environment plays an important role in the basic biology of cultured cells. The student will gain knowledge of cell science and training in cellular and molecular techniques with academic and industrial partners. It is anticipated that these new approaches for maintaining cells will be immediately beneficial to biologists looking to improve their in vitro model.