Team:Hamburg/Nanoscience

Nanoscience

chips

Applied Design for the advanced engineering of a lung-on-a-chip microfluidic device

Estimates from the World Health Organization predict a resurgence of bacteria to their long-lost top spot among the most devastating human diseases. How did it come to that? Since the discovery of Alexander Fleming, the first scientist that invented and proved the concept of antibiotics, over the years scientists have discovered a vast number of antibiotics to effectively treat bacterial infections. We could fight most bacterial threats for a long time which lead to a notion of false comfort that everything is under control! However, with the current overuse of antibiotics and acquired resistances among bacteria we are about to enter a new era, the post antibiotic era. We are now reaching the tipping point, at which new strategies are required to overcome multiresistance and prevent a resurgence of devastating infections. Our Project will help to combat multiresistant-infections with a Trojan-Horse-Approach utilizing gallium-loaded siderophores as a resistance-resistant therapy.

Animal testing has been the method of choice for simulating and predicting human responses to drugs, chemicals, pathogens and environmental toxins; however, animal studies are costly, lengthy and controversial, and their results often fail to predict human responses. These problems raise serious economical, ethical and scientific issues in areas ranging from environmental monitoring and biomedical devices to the development of new therapeutics and cosmetics.

With our method we will create a possible therapy to fight the imminent danger of devastating pandemics from multiresistant bacteria. We don’t want to rely on animal-testing, but on the in vitro simulation of grown human cells in physiological condition. Similar solutions would include living cells cultured in 2D monolayers, 3D extracellular matrix (ECM) gels or multicellular spheroids. But these conventional models lack the ability to fully recapitulate integrated physiological functions at the organ level.

Organs-on-chips’ are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications.

If we do not keep optimizing and honing such methods, resistant to the multi-resistant bacteria will pose a monumental danger to us. As such, this project offers a way to circumvent the use of antibiotics in future treatment of diseases, halting the development of omni-resistant germs.

For our design, we follow mostly the protocol from the Harvard Wyss Institute for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems.

Organs in the human body are complex living systems composed of different types of tissues that form complex tissue-tissue interfaces, such as between endothelium-lined blood vessels and parenchymal epithelial cells that exhibit organ-specific functions. Most organs are multimodular structures in that they consist of repeating smaller functional units that individually perform the major characteristic functions of the whole organ (e.g., gas exchange in the alveoli of the lung, absorption in the villi of the gut, metabolism in the hepatic triad of the liver, etc.). Typically, these functional units comprise different types of specialized tissues (e.g., epithelium, vascular endothelium, connective tissue, immune cells, nerves, etc.) that interface in organ-specific patterns and are subjected to dynamic changes in chemical and mechanical signals that vary depending on their particular spatial microenvironment.

To develop a useful organ surrogate device for in vitro analysis of complex human physiology, we mimic this complex physical microenvironment in which cells are normally situated. We recreated the critical alveolar-capillary interface of the lung air sac in our human lung-on-a-chip by developing a 3D microfluidic device that contains two parallel microchannels with the same dimensions separated by a thin flexible membrane made of poly(dimethylsiloxane) (PDMS) ECM-coated. We tried to approximate the width of our microchannels to the the average diameter of an alveolus in human lungs. Normally the membrane would be porous to allow for nutrient exchange between the cell tissues, but we did not have the time to make the silicon master for this. Also, we had to omit the adjacent vacuum channels and thus the breathing function, as it is stated, that the simulation of the human lung on a chip is pretty accurate, even without “breathing”.

Human alveolar epithelial cells and pulmonary microvascular endothelial cells are then introduced into the upper and lower microchannels, respectively, and grown on the ECM-coated membrane for at least 5 days to form two closely apposed tissue monolayers. During cell culture, medium is perfused continuously through both channels to provide cells with nutrients and to remove their metabolic wastes. Once confluence is reached, the culture medium is removed from the upper channel, and this channel is filled with air to form an air-liquid interface at the apical surface of the alveolar epithelium, which induces the cells to differentiate and express tissue-specific functions, such as surfactant production.

At the end, to test our device with its cells against toxicity, we infect them with multi-resistant microbial cells (Klebsiella pneumonia) for a 12 hour cycle. Next we introduce our modified Ga-siderophores so they hijack into the microbes and intoxicate them, while avoiding any human epi- and endothelial cells.

References:

  • [1] Dongeun Huh et al. (2013), "Microfabrication of human organs-on-chips", Nature Protocols. 2135, Vol. 8 No. 11.


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