(340h) Fluids-Based in Vitro Models for Disease and Development

My scientific passion is addressing questions at the intersection of engineering and human health by creating in vitro models of development and disease. Such models offer higher throughput and control than corresponding in vivo models, but often at the expense of being less physiologically relevant. To enhance my models’ physiological relevance and provided insight, I apply principles of fluid mechanics to control and observe the spatiotemporal evolution of the system.

In my doctoral work, I’ve developed two simple yet robust platforms for investigating (i) how biochemical gradients pattern stem cell differentiation in the developing gut and (ii) the effect of a mucin-like glycoprotein on the stability of the thin liquid film that covers the eye. In creating these systems, I emphasize designs that make these protocols accessible to other scientists in the field as well as enable collaboration with biologists, clinicians and industry. Moving forward in my career, I hope to continue using my interdisciplinary knowledge to positively impact ongoing efforts in advancing human health.

A microfluidics-based in vitro model of anterior-posterior gut patterning

In this work, we demonstrate, for the first time, spatially controlled differentiation of human pluripotent stem cells (hPSCs) into the anterior foregut (AFG) and mid/hindgut (MHG) cell types within a single cell monolayer using chemical gradient-generating microfluidics. This result represents an advance in the ongoing efforts to harness the processes by which complex tissues arise during embryonic development in vitro—a long-standing goal of tissue engineering and regenerative medicine. In embryos, uniform populations of stem cells are exposed to spatial gradients of diffusible extracellular signaling proteins, known as morphogens. Varying levels of these signaling proteins induce stem cells to differentiate into distinct cell types at different positions along the gradient, thus creating spatially patterned tissues.

To accomplish this spatially controlled differentiation, or patterning, we combined principles of engineering and biology to develop a novel, reproducible, and easily accessible method for the anterior-posterior patterning of hPSCs. We performed a 6-day, on-chip differentiation protocol within a commercially available microfluidic chip. We used finite element analysis to model the distribution of morphogens within the microfluidic device and determined that the chip can generate two stable and opposing linear morphogen gradients. Quantitative analysis of immunofluorescence data showed that expression of AFG and MHG markers is localized to their respective morphogen sources and that both display a decreasing linear profile with increasing distance from their sources. This platform thus allows us to explore fundamental questions about how a single population of stem cells differentiate into multiple cell types along a body axis in response to exposure to morphogen gradients, such as whether the response in marker expression is graded or discretized as well as the influence of neighboring cells on cell fate decisions. Our in vitro model contributes to the stem cell and developmental biology toolkit and may eventually pave the way to create increasingly spatially patterned tissue-like constructs in vitro.

Quantifying the effects of recombinant human lubricin on model tear film stability

Dry eye disease (DED) is an ocular pathology affecting hundreds of millions of patients worldwide, and the lack of available treatments can be in part owed to the difficulty of studying the complexity of the human tear film. The tear film is a several micron thick multilayer structure that resides above the corneal epithelial surface of the eye and consists of an innermost mucin layer, an aqueous layer containing mucins in solution, and an outermost lipid layer that resists evaporation. These components act together to maintain tear film hydration and stability, and this interplay is key to maintaining clear vision and ocular health.

Though few effective treatments exist for DED patients, one molecule that has shown promise in addressing DED symptoms in clinical trials is recombinant human lubricin, a mucin-like glycoprotein that protects the ocular surface. Using a previously developed interferometry-based instrument called the Interfacial Dewetting and Drainage Optical Platform (i-DDrOP), we can create acellular model tear films in vitro to study the effect of recombinant human lubricin on tear film stability. Measurements made using the i-DDrOP capture the rich spatiotemporal behavior of these thin films, and from these data we extract important yet simple-to-use parameters through a robust analysis pipeline. Specifically, from the wetted area fraction evolution plot, we can obtain (i) the evaporative break-up time (EBUT), which represents how quickly the onset of evaporation-driven film instability occurs, and (ii) the wetted area fraction at ten minutes (A10), which represents the thin film’s ability to keep the model ocular surface hydrated over time.

To demonstrate the method’s ability to assess tear film stability in the presence of DED treatments, we quantified the effect of recombinant human lubricin on wetted area fraction evolution. Our data corroborates previously published trends showing that higher lubricin concentrations are better able to stabilize the tear film against evaporation and breakup. We then compared the efficacy of unstressed recombinant human lubricin samples to that of stressed lubricin samples in response to variations in pH and temperature. We were able to capture differences in thin film evaporative breakup and ability to maintain hydration between these samples using the EBUT and A10 parameters described above. In addition to elucidating differences in performance among samples, we show that there exists a threshold concentration at which thin films of recombinant human lubricin solution are able to resist evaporation-driven instability and maintain a wetted curved surface. Finally, we describe ongoing work in creating a supported lipid bilayer-based system capable of mimicking various levels of mucin deficiency in the tear film and thus provide a tunable and more physiologically relevant model system. By providing a facile way to quantify and compare the effect of recombinant human lubricin samples on thin film stability, we hope that these methods will ultimately help elucidate the mechanism by which recombinant human lubricin protects the ocular surface.