Analyzing freshly isolated ATII cells from rodent injury models or human diseased tissue using microarray technology is a powerful tool to determine disease related altered phenotypes in lung injury and repair [27, 40]. However, for functional analysis, the culture of primary ATII cells is of utmost importance. Challenged by the fact that ATII cells possess the intrinsic properties to differentiate into ATI cells when placed into normal cell culture, a wide range of culture conditions and media compositions for ATII cells are described. A careful selection of culture conditions is crucial to obtain meaningful results. Depending on the applied assay, culture vessels as well as the presence of specific media supplements might influence experimental outcomes. Plating fresh ATII cells on plastic dishes induces the gradual loss of ATII cell characteristic [62, 63]. Coating cell culture dishes with extracellular matrix (ECM) components such as fibronectin, collagen or laminin or a combination thereof, will lead to differences in the dynamics of trans-differentiation processes. Additionally, culturing of ATII cells on trans-well filter inserts has been described to result in stabilized monolayers of ATII cells and allow cultures at the air liquid interphase [61]. Furthermore, the supplementation of commonly used cell culture media (e.g., DMEM or DMEM/F12) with KGF [64–67] and glucocorticoids in the combination with cAMP [68, 69] has been described to promote ATII cell phenotype in culture.

Due to their role as progenitor cells, the proliferative capacity of ATII cells is a critical feature in lung injury and repair processes within the lung. Thus, the assessment of proliferative behavior of this cell population is one of the most assessed cell characteristics. For the determination of in vitro proliferation capacity several different methods can be applied. Determination of gene and protein expression of genes related to cell cycle progression such as Ki67, Ccng1, and Ccng2 [27], are widely used to compare proliferative capacities of injured versus non-injured ATII cells and furthermore their response to different stimuli. The analysis of protein expression of proliferation markers Ki67 [27, 70], PCNA [71, 72] and phosphorylated histone H3 [27, 73] in cells in vitro as well as in in vivo models by immunofluorescence/immunohistochemistry represents a complementary approach. Direct functional assays for the detection of proliferating cells include the use of metabolic activity assays such as the WST-1 assay [74, 75] where a tetrazolium salt is converted in a colored formazan by endogenous dehydrogenates displaying a proportional relationship to cell number. Furthermore, the incorporation of bromodeoxyuridine (BrdU) [21] or [3H]thymidine [27] into the DNA of proliferating cells represents the gold standard for determining proliferation. Usage of several of these techniques provided insight in the reprogrammed phenotype and aberrant proliferative capacity of fibrotic ATII cells and the observation that targeting this phenotype attenuates pulmonary fibrosis in different models [27, 70].

Besides changes in the proliferative behavior of injured ATII cells, the presence of apoptosis is another important parameter when analyzing injury and repair processes in the lung epithelium. The most commonly applied strategies are the analysis of caspase activity [76, 77] and the caspase mediated cleavage of endogenous substrates such as PARP [77], a crucial step in the apoptotic process. Furthermore, early apoptotic changes such as the flip of phosphatidylserine from the inside to the outside of the cell plasma membrane is used as a surrogate marker for apoptosis and detected by Annexin V binding and further analysis by flow cytometry [77]. TdT-mediated dUTP-biotin nick end labeling (TUNEL), a method to detect DNA fragmentation occurring in apoptotic cells, can be used for in vitro studies as well as for the detection of apoptotic cells in tissues of in vivo models of lung injury [70, 78]. The use of TUNEL staining provided data on the presence of increased numbers of apoptotic alveolar epithelial cells in fibrotic mouse models, as well as IPF tissue [70, 78, 79].

Besides the described imbalance of proliferation and apoptosis in models of epithelial injury, the occurrence of EMT is widely discussed in the context of attempted alveolar repair processes. In vitro studies of EMT of cultured epithelial cells regularly use the cytokine TGF-β1 for EMT induction, which has been demonstrated in several organs including the lung [80–82]. Monitoring of decreased expression of epithelial marker genes such as E-Cadherin, cytokeratin and TJ-proteins is performed for the characterization of epithelial integrity on gene expression level as well as on protein level. Moreover, analysis should include the expression of EMT transcription factors, such as Snail, Slug, Zeb, or Twist as well as mesenchymal markers. Several mesenchymal markers, including αSMA, Calponin, and ECM related proteins such as collagen1, fibronectin and vimentin are used to describe the gain of mesenchymal cell characteristics [27, 80, 83, 84]. Importantly, co-expression of epithelial and mesenchymal markers by immunofluorescence/immunohistochemistry staining should be analyzed and has been demonstrated in human tissue of different lung diseases [28, 85, 86]. These descriptive investigations should be further complemented with functional cell assays, such as cell migration, which is a prominent feature of EMT.

For studying the in vivo relevance of findings generated by in vitro cultures, lineage tracing animals can be used to determine the cell fate in the context of lung injury. These studies utilize different transgene mouse strains, which express traceable markers under the control of the surfactant protein C promotor [19, 86, 87].

ATII cells expressing surfactant proteins are able to self-renew and trans-differentiate into ATI cells [20, 22, 88–90]. Depending on the type of injury applied, other epithelial cell populations, negative for SPC, can contribute to the attenuation of lung injury [58, 91]. Therefore, understanding how ATII cells differentiate into an ATI cell phenotype is under intense investigation including respective gene expression signatures as well as the morphological conversion from a cuboidal to a squamous cell shape. Early studies described the trans-differentiation of ATII cells into ATI-like cells in primary culture. These observations were based on the gradual loss of gene and protein expression of surfactant proteins as well as the loss of lamellar bodies, investigated by the use of electron microscopy [92]. Furthermore, the gain of features of ATI cells such as a flattened cell morphology and the expression of ATI cell-associated markers T1α (podoplanin) [93–96], aquaporin 5 (AQP5) [64, 97], receptor for advanced glycosylation end products (RAGE), and caveolin [98–100] were described. An overview of ATII and ATI cell specific markers is displayed in Table 6.2. Applying freshly isolated ATII cells to standard cell culture conditions is now widely used to mimic differentiation and repair mechanisms to investigate molecular cues in response to lung injury. The model has been utilized to study ATII cell trans-differentiation potential in various species including rat, mouse and human. Monitoring of epithelial cell identity and trans-differentiation is mainly achieved by gene and protein expression analysis of the respective markers in combination with microscopic evaluation of cell morphology.