Soluble factor signaling between immune cells and fibroblasts is critical in regulating biological processes. However, it is often dysregulated in diseases and leads to physiological changes, including airway inflammation in asthma and allergies. One immune cell type that can be attributed to airway inflammation is eosinophils (EOS). When activated by interleukin-3 and heat-aggregated immunoglobulin G, EOS release certain soluble factors associated with the activation of lung fibroblasts. To investigate the interactions between human lung fibroblasts (HLFs) and EOS, we used the open microfluidic coculture device. This device has two chambers, in which two types of cells can be cocultured in the shared media while being physically separated by a half wall. We found that HLFs in coculture with activated EOS had the highest levels of proinflammatory gene expressions and proinflammatory cytokines. However, the exact mediators responsible for promoting these biological processes are still uncertain. We hypothesize that EOS secrete a protein, transforming growth factor alpha (TGFa), to be consumed by HLFs, triggering proinflammatory responses of HLFs. The goal of this study was to elucidate the roles of TGFa in airway inflammation. HLF-EOS cocultures are seeded in the microfluidic coculture device, then TGFa and their respective cellular receptors are neutralized using antibodies. Then, reverse transcription quantitative-polymerase chain reactions are used to quantify gene expression levels relevant to proinflammatory responses of HLFs, in addition to multiplex immunoassays to analyze the secreted soluble factors from both cell types. We anticipate that HLF-EOS cocultures treated with neutralizing antibodies have lower expression levels of proinflammatory genes than cocultures without antibodies. Findings from this study will help us better understand the key regulators that promote proinflammatory behaviors of HLFs in airway inflammation.

We have observed that conventional respiratory pathogen sampling methods, such as pharyngeal swabs, elicit unpleasant experiences for both adults and children. Particularly for pediatric patients, having a non-invasive, enjoyable sampling approach is crucial to facilitate prompt diagnosis and treatment. In prior research, we introduced a novel saliva sampling device, the CandyCollect. This lollipop-inspired device, with its isomalt candy coating, is specially produced and surface-treated for capturing pathogens from saliva, providing a pleasant sampling experience to child patients. Clinical studies approved by multiple Institutional Review Boards (IRB) revealed an average candy dissolving time for CandyCollect (with a mass of 0.90g~1.10g) of 3.51 minutes, with a minimum of 1.25 minutes. To compete with original sampling methods which take up to 10 seconds, it is desirable for CandyCollect to have a shorter sampling time around 15–20 seconds. Therefore, this study aims to decrease the dissolving time by introducing a new CandyCollect recipe and design. For the new candy recipe, we replaced isomalt with a mix of glucose and sucrose. Additionally, baking soda (sodium bicarbonate) was added to increase the candy’s contact area with the tongue. An ongoing experiment will assess if baking soda affects PCR results for pathogen samples, and this modified recipe will be employed in a new clinical study. Concurrently, we have modified the CandyCollect design by placing the candy on the same side as the spiral, with a small candy reservoir beneath the spiral to decrease the mass to 0.06g-0.1g. To validate the efficiency of this new design, we plan to conduct another clinical study recruiting 30 younger participants and obtaining feedback about the new design. This collaborative study will provide valuable insights towards achieving a faster dissolving time, ultimately enhancing the viability of CandyCollect as an improved and more efficient replacement for conventional sampling methods.

Traditional methods of sampling for respiratory illnesses can be uncomfortable and possibly act as a deterrent for children and adults. The CandyCollect is a lollipop-inspired device developed to aid in the testing for respiratory infections. Fabricated by a computer numerically controlled (CNC) mill, the device contains a plasma-treated microfluidic channel that collects pathogens from saliva; the addition of strawberry-flavored isomalt candy on the head of the stick aids in the resemblance, feel, and taste of a traditional lollipop. In previous remote human subjects studies, approved by the Institutional Review Board (IRB), we demonstrated the ability of CandyCollects to detect commensal bacteria, Streptococcus mutans and Staphylococcus aureus, in healthy adults using qPCR and detect infections in adults displaying symptoms of respiratory infections. From these and IRB-exempt device improvement studies, we aimed to shorten the dissolve time of the candy to make it comparable to the sampling time of nasal and oral swabs. In order to investigate the usability and accuracy of the CandyCollect for children, our team launched the Parent-Child Dyad Study. The IRB-approved study recruited parents with children displaying symptoms of respiratory illness via online forums. The 40 eligible dyads, consisting of children aged 5-15 and their parents, received a kit including a nasal swab, two mouth swabs, and three CandyCollects. After using the CandyCollects, the children and parents completed their respective surveys asking them about their comfort and experience with the different testing methods. As with all remote sampling methods, limitations included potential biases towards nasal and/or mouth swabs. However, the CandyCollect is no more susceptible than currently employed methods. User feedback data demonstrates children preferring the CandyCollect over mouth and nasal swabs, reporting better taste and comfort. The parent survey results also show preference for the CandyCollect. Here we will present the results of their user feedback.

Cell co-culture systems are used to study intracellular interactions by culturing distinct cell-type populations within a shared environment. This in vitro method is more representative of the highly complex and diverse processes that occur in organisms, allowing accurate insight into mechanisms of cell signaling pathways, disease, drug interactions, etc. Existing designs can be two- or three-dimensional, with or without cell-cell contact, and use systems like microfluidics, solid supports, or transwells to control contact. Methods that physically partition individual cell-type populations often allow soluble factor transmission by using permeable material or flooding the compartments so the solvent is shared. However, this is less representative of the human body, where physical partitions do not divide different cell types. Thus, there is a need for a co-culture system with a partition allowing initial separation, that can later be removed to allow interaction without a physical barrier. We are developing this system by utilizing open microfluidic gel patterning techniques to test if a removable partition can be formed with enzyme-degradable polyethylene glycol (PEG). I first use computer-aided design to engineer a rail scaffold outlining two compartments, and fabricate these devices using 3D printing. I pipette PEG into an inlet in the rail, flowing along the scaffold channel due to spontaneous capillary flow and patterning the insert. The PEG polymerizes to form the insert with two distinct cell chambers on the well’s bottom surface, and the cells are seeded into their corresponding compartments. After the cell culture period is complete, sortase (SrtA) is added to completely degrade the PEG insert, allowing the cell populations to interact. We expect the PEG inserts to polymerize similarly to agarose, and leave no residue in the well once degraded. Future work will include utilization of this device for experiments using functionalized beads to monitor soluble factor signaling in co-cultures.

Biological tissues are a group of cells that have similar structure and that function together as a unit. In between these cells is the extracellular matrix (ECM), which provides structural support for resident cells. The makeup of the ECM consists of fibrous proteins, such as collagen, that are interlocked and cross-linked, following a nonlinear stress/strain curve and is considered viscoelastic. The dominant mechanism behind this response is the presence of largely elastic, spring-like straightening/uncrimping of fibrils. This can be thought of like applying a force to springs in parallel. Overall, this mechanism allows the ECM fibrils to align and elongate significantly under small loads, thereby aligning the cells. This, in turn, affects the overall tissue structure and its mechanical properties. We have developed a method for patterning a cell-infused collagen mixture as a three-dimensional tissue, and subsequently stretching it, in order to observe how the cells develop in a strained environment. Specifically, we have engineered two devices that fit within a 6-well plate: the tissue is patterned on the first device, and then transferred to the second for stretching. During each phase, the suspended tissue is incubated for a period of time in order to facilitate cell development and hydrogel gelling. Once the tissue has been stretched for a certain period of time, it is then removed from the device and imaged. Our modular design designates strain as an known and adjustable value, allowing us to relate it to the internal stresses of the tissue via Hooke's Law. We are able to identify the quantitative conditions that promote tissue alignment and maturation within the suspended tissue.