MLII cell culture

2D in vitro cell culture

  • Airway smooth muscle cells, lung fibroblasts, airway epithelial cells, endothelial cells, mast cells
  • All from human origin
  • Analysis of anti-inflammatory efficacy and anti-remodelling efficacy

We use, amongst others, human airway smooth muscle cells, lung fibroblasts and airway epithelial cells, and analyse the anti-inflammatory and anti-remodelling efficacy of your compound. The exact assays and read-outs of choice will be discussed dependent on your needs, and can include;

Anti-inflammatory efficacy

Inhibition of cytokine (IL-1β, IL-13, TNF-α, others) or cigarette smoke induced responses, including:

  • ELISA or Luminex analysis of pro-inflammatory cytokine release.
  • Mucus seceretion (airway epithelial cells).
  • Gene and protein expression for inflammatory markers such as COX-2.
  • Activation of transcriptional mechanisms (e.g. STAT).
Anti-remodelling efficacy

Inhibition of growth factor (TGF-β, PDGF, WNT, others) induced responses, including:

  • Cell proliferation.
  • Cell migration or adhesion (xCELLigence).
  • Myofibroblast and smooth muscle differentiation (lung fibroblasts, airway smooth muscle).
  • Epithelial mesencymal transition (EMT; epithelial cells).
  • Angiogenesis.
  • Gene and protein expression for matrix proteins, proliferation markers.
  • Activation of transcriptional mechanisms (e.g. Smad, β-catenin, Sp1, others)

In addition, we can offer several possibilities for target validation in human samples. Please contact us to see how we can help you.

3D in vitro cell culture

  • Air-liquid interface culture of airway epithelial cells
  • Co-culture with fibroblasts or other cell types on request

Adequate study of epithelial cell function requires studies of human airway epithelial cells in air-liquid interface. Using this system, human airway epithelial cells are cultured on transwell inserts and air-exposed to allow differentiation into pseudostratified epithelia. The system is particularly suitable for the study of investigational compounds into epithelial cell differentiation, goblet cell metaplasia and epithelial barrier function. Co-cultures with fibroblasts (healthy and diseased) is possible. Typical read-outs include (immune)histochemistry, gene expression and ELISA of secreted factors in the medium. We have shown efficacy of tiotropium against IL-13 induced goblet cell metaplasia

Lung on chip models

  • Airway cholinergic neurons
  • Airway smooth muscle cells
  • Other cell types on request

Lung on chip represents a new tool to study dynamic responses and interactions between different airway or alveolar cell types in vitro. We have implemented a microfluidic lung on chip model to study the interaction between airway smooth muscle cells and airway cholinergic neurons. Other cell types can also be cultured on this chip, such as epithelial cells. Lung on chip enables to study more complex human physiology and pathophysiology, including asthma, using primary human cells, and to evaluate drug effect for preclinical drug development. 

The lung on chip is a microfluidic cell culture device, fabricated at the facility of the University of Groningen, adapted from the design of Peyrin et al. (doi: 10.1039/c1lc20014c). It contains hollow microchannels that are lined by neurons, with outgrowth to the axonal compartment to interact with the effector cell type airway smooth muscle cells. We have for the first time developed protocols to differentiate vagal cholinergic neurons from human pluripotent stem cells (hPSCs) and have thereby overcome the limitation of the in vitro availability of peripheral neurons (unique in the world).

This model is suitable for studies on neuro-effector interactions such as neuro-immune interactions and neuron-smooth muscle interactions and for studies into neuroplasticity in asthma. Such studies would not be feasible using commonly used cell culture models. Furthermore, it overcomes the limitation of translational challenges caused by species differences, which are relevant in particular with respect to the nervous system in mice.

Read-outs
  • Neuronal density
  • Calcium imaging
  • Gene expression in neurons or ASM cells

This model was developed in collaboration with the University of Groningen and funded by a grant (114021505) from the ZonMW/Proefdiervrij Meer Kennis met Minder Dieren (Animal free – More Knowledge with Fewer Animals) program with co-financing from Boehringer Ingelheim, Lung Foundation Netherlands and Aquilo.

Previous publications

–        Wu X, Verschut V, Woest ME, Ng-Blichfeldt JP, Matias A, Villetti G, Accetta A, Facchinetti F, Gosens R, Kistemaker LEM. Rho-Kinase 1/2 Inhibition Prevents Transforming Growth Factor-β-Induced Effects on Pulmonary Remodeling and Repair. Front Pharmacol. 2021 Jan 20;11:609509.

–        Kistemaker LEM, Hiemstra PS, Bos IST, Bouwman S, van den Berge M, Hylkema MN, Meurs H, Kerstjens HAM, Gosens R. Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells. Thorax 2015; 70:668-76.

–        Oenema TA, Mensink G, Smedinga L, Halayko AJ, Zaagsma J, Meurs H, Gosens R, Dekkers BG. Cross-Talk between Transforming Growth Factor-beta1 and Muscarinic M2 Receptors Augments Airway Smooth Muscle Proliferation. Am J Respir Cell Mol Biol 2013;49:18-27.

–        Baarsma HA, Spanjer AI, Haitsma G, Engelbertink LH, Meurs H, Jonker MR, Timens W, Postma DS, Kerstjens HA, Gosens R. Activation of WNT/beta-catenin signaling in pulmonary fibroblasts by TGF-beta(1) is increased in chronic obstructive pulmonary disease. PLoS One 2011;6:e25450.

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