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，组织特异性结构、机械和生物化学线索以及细胞-细胞相互作用被改变或丢失。三维（3D）细胞培养物试图通过保留单个细胞的3D完整性并使细胞能够与细胞外基质结合产生其自身的小生境来模拟体内情况。有很好的证据表明，3D模型可用于研究生理学，其中组织样功能可以保持。然而，通常的做法是在将此类细胞接种到3D培养模型中之前最初扩增2D培养物中的细胞群体（特别是细胞系）。在这种情况下，细胞同时：a）适应它们的新环境（2D到3D生长转变）;和B）作为3D细胞培养测定的一部分被激发和/或检查（例如，药物细胞毒性测试）。这使得它很难破译的精确结果的测定细胞同时暴露于两个这样的主要variable.We假设，最初繁殖和扩增的细胞群体在3D环境中更有可能适应他们的周围环境时，接种到一个3D培养模型或移植在体内（即不需要2D到3D的过渡）。在这个项目中，我们将与ECACC Culture Collections合作，这是一个为研究人员提供细胞系的组织。常规生长为贴壁2D单层的流行细胞系（例如A549肺上皮细胞; MCF 7乳腺肿瘤;已建立和广泛了解）的繁殖将在新系统中维持，该系统能够在3D中连续传代细胞，以将其表型转化为更天然的3D状态。具体而言，学生将：1）（0-18个月）优化方法以在3D培养中连续传代A549和MCF 7人细胞系; 2）（6-24个月）通过详细检查细胞形态、细胞骨架结构、整合素/钙粘蛋白和黏着斑蛋白的表达、已知在2D培养中丢失的与3D表型相关的标志物的表达来表征细胞表型;第三章（18-30个月）进行2D与3D与体内的3向比较（例如MCF 7肿瘤组织）检查已知在2D和3D培养中差异调节的关键结构细胞蛋白;四、（24- 36个月）测试2D与3D繁殖细胞在设计用于支持体外3D细胞生长的替代技术中生长时产生3D结构的能力，例如支架和球状体培养物; 5）（30-36个月）评估2D和3D繁殖的细胞在移植到免疫缺陷小鼠中时形成异种移植肿瘤的能力。将比较生长速率、大小和组织组成。该项目将提供有关生长环境如何在培养细胞的基础生物学中发挥重要作用的新信息。学生将获得细胞科学的知识，并与学术和工业合作伙伴一起进行细胞和分子技术培训。预计这些维持细胞的新方法将立即有益于希望改善其体外模型的生物学家。

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.