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Lungs for Living Research Centre

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Techniques

The Lungs for Living centre covers a range of molecular and cell biology techniques including 2D and 3D culture of epithelial cells, and models of cancer and other lung diseases, both in vitro and in vivo. 

As part of our airway epithelial regeneration work we have developed methods to expand human airway epithelial cells from small endobronchial biopsy samples. After expansion, we differentiate these towards the mature airway cell types, including ciliated and mucosecretory cells, using either air-liquid interface or 3D organoid (or 'tracheosphere') assays (shown in the image below). You can read more about our group's work on the expansion of human epithelial stem cells for tissue engineering here (Butler et al. 2016).

Human airway organoids

We are investigating the relationship between cancer cells and the stroma using spheroids embedded in tunable stiffness collagen matrices, containing stromal cells or PDMS gel. There is compelling evidence for the role of stroma and its physical interaction with epithelial cells in the acts of metastasis. Stiffer tissues and increased extracellular matrix (ECM) stiffness have been shown to affect several processes associated with tumour progression. The images below show three dimensional cultures of normal human bronchial epithelial cells (left) and lung squamous carcinoma cells (right), in co-culture with fibroblast cells and in a collagen matrix.

Human bronchial epithelial cells and fibroblasts in 3D culture

Wild type fibroblasts stably expressing a red fluorescent protein embedded into collagen gel. HBEC cells expressing nuclear GFP were seeded on top of the gel and organized accordingly.

Lung cancer cells and fibroblasts in 3D culture

Wildtype fibroblasts stably expressing a red fluorescent protein embedded into collagen gel. Spheroids of lung cancer cell line H2170 expressing nuclear GFP were seeded on top of the gel.

 

Our group is also interested in the signalling changes taking place during cellular transformation. We use both lentiviral and CRISPR/Cas9 methods of genetic manipulation to study the effects of specifically changing the expression of genes thought to be critical in lung cancer development. The left image below shows normal lung epithelial cells, expressing tumour suppressor protein p53, stained in white. This compares to the absence of p53 in the cells shown in the right image, which underwent a CRISPR/Cas9 targeted deletion of p53.

Control cells

p53-negative cells

Our group has also developed improved methods of imaging of the airways, and in 2013 published the first paper using a whole mount technique on lung tissue (images shown below).

Wholemount

Wholemount image of lung tissue

Wholemount - zoomed

Zoomed in area of the wholemount