The process of designing, building, and validating a cell stretching platform and using this device to study the effect of mechanical stimulation on different cell types requires the development of several skills, which formed the basis of the learning outcomes required for the successful completion of the project. Through participating in this project, undergraduate students should have developed knowledge and skills in engineering design, basic cell biology, and experimental design. This required students to gain experience in programming, using computer aided design -related software, such as SOLIDWORKS, and cell culture. Students were also encouraged to apply for either individual or project related funding through the Edwards Life sciences Summer Undergraduate Research Program and Undergraduate Research Opportunities Program at the University of California, Irvine , respectively. Through these programs, students gained experience not only in reading and writing scientific articles,rolling grow benches but also presentation of research findings in formal settings including at the annual Undergraduate Research Symposium at UCI. The learning outcomes associated with this project were similar to those of other experiential learning modules as key knowledge and skills that promote learning and individual development are gained through student involvement.
Over the next four years, junior undergraduate students were recruited and mentored by their senior counterparts. This approach created a continuity of knowledge over multiple years and provided the students with mentoring experiences. The undergraduate students were from biomedical and mechanical engineering programs in their sophomore or junior years. As the project progressed, some of the students wanted to continue and stayed to pursue graduate degrees and participated more directly in recruitment. After working on this project, the students were given surveys to gauge learning and to provide feedback on how the project can be improved for a better learning experience.The uniaxial cell stretching device is composed of two experimental substrates housed in a 10.16 cm 15.24 cm 6061-T6 aluminum channel. The substrates, made by joining silicone sheets and 2.54 cm inner diameter silicone tubing, are held in place by a movable center clamp and fixed outer clamps, with top clamps and wing nuts used to apply pressure and maintain substrate tension during application of cyclic stretch. Cyclic strain is generated by using a programmable servomotor to move the center clamp, which is coupled to a gear and gear rack system. This center clamp slides on two 0.635 cm rails and is aided by bronze bushings to reduce friction and wear. Once the device is assembled, the experimental substrate has a length of 3.81 cm in the direction parallel to the uniaxial stretch. Different amplitudes of strain can be generated by programming the servomotor to rotate a circumferential distance corresponding to a fraction of the experimental substrate length. For example, 5, 10, 15, and 20% strain amplitudes can be generated through rotating the servogear 0.191, 0.381, 0.572, and 0.762 cm , respectively. In addition, an aluminum block is used to either extend both experimental substrates and generate static strain, or extend one experimental substrate and create a temporary static strain until the servomotor is powered resulting in equal cyclic strain in both experimental substrates.
A 1 Hz cyclic stretch was used for all experiments in this study. The CAD files and drawings have been made publicly available. The parts and their costs as well as detailed assembly instructions are provided in the Appendix, which is available under the “Supplemental Materials” tab for this paper on the ASME Digital Collection.The experimental substrates were fabricated through sealing silicone tubing to a 0.05 cm thick silicone sheet using polydimethylsiloxane , which was then cured at 60 C. To validate the strain profiles generated by the cell stretcher, videos were captured of the servo gear rotating and the experimental substrate stretching and deforming. The videos were processed through IMAGEJ software to track either a single point of interest on the servogear or a 5 5 matrix of markers on the surface of the substrate using the MTrack2 plugin. The data obtained were analyzed using a custom python code to validate the waveform output by the servo or the resulting strains parallel and perpendicular to the direction of stretch, respectively.Experimental substrates were sterilized by autoclave, several 70% ethanol and phosphate buffered saline washes, and then coated with a 10 lg/mL fibronectin solution and incubated at 4 C overnight. The substrates were further rinsed with phosphate buffered saline before cells were seeded onto the surface. Bone marrow derived macrophages were obtained by flushing the bone marrow from the femurs of 6–12 week old female C57BL/ 6J mice . This was accomplished using Dulbecco’s modified eagle medium supplemented with 10% heat-inactivated fetal bovine serum , 1% penicillin/ streptomycin, 2 mM L-glutamine , and a 10% conditioned media, produced from CMG 14–12 cells that express recombinant mouse macrophage colony stimulating factor, which differentiates bone marrow cells to macrophages. Red blood cells were removed by treating the collected bone marrow cells with a red cell lysis buffer.
The cells were then centrifuged, resuspended in the culture media, and seeded onto non-tissue culture treated petri dishes for 7 days, before being harvested using an enzyme-free dissociation buffer and seeded onto experimental substrates. The resulting macrophages were seeded at a density of 2 105 cells per substrate. Following 4 h of incubation, the media was replaced with either regular or 0.3 ng/mL interferon-gamma and 0.3 ng/mL lipopolysaccharide containing media then cyclically stretched at a 10% stretch amplitude for a period of 18 h. Following stretch, supernatants were collected and analyzed for the presence of tumor necrosis factor-alpha , interleukin-6 and monocyte chemoattractant protein- 1 cytokine secretion using ELISA kits following the manufacturer’s instructions.The designed, low cost, uniaxial cell stretching device was subjected to a number of tests to ensure adequate and repeatable mechanical function. For example, by tracking and analyzing the rotation of the servogear and positional markers on the experimental substrates, the waveform and the strain profiles output by the servo were computed. The servo was capable of generating sine, triangle, and square waves through fine adjustments in the rotation speed and acceleration of the servo gear. Similarly, changing the degree of rotation of the servo gear generated different strain amplitudes. The parallel and perpendicular cyclic strains generated by the device were similar to theoretical 10% and 20% stretch amplitudes, respectively. This device is, therefore, able to generate uniform strain profiles in the center of the well that is comparable to other, more expensive, cell stretching systems. However, decreases in strain were observed toward the transverse boundaries on the stretchable membrane . Once the mechanical functions were validated, the uniaxial system was used to mechanically stimulate macrophages and cardiomyocytes.When subjected to cyclic uniaxial stretch, bone marrow derived macrophages were observed to alter their cell morphology. Following stretch,drying cannabis the degree of macrophage alignment was quantified by calculating the OOP. The OOP ranges from 0 for a completely isotropic arrangement to 1 for perfectly aligned organization. Significantly higher OOP values were obtained for unstimulated macrophages in response to cyclic stretch when compared to static controls. This alignment of macrophages in response to a 1 Hz uniaxial strain at a 10% amplitude was previously observed. IFN-c/LPS stimulated macrophages are also aligned in the direction of stretch and displayed significant elongation in response to cyclic stretch. However, no significant differences were observed in inflammatory cytokine secretion for IFN-c/LPS stimulated macrophages subjected to cyclic strain at a 10% amplitude , which has also been previously reported.This project was initiated as a collaboration between two labs with different biological interest , but a common goal of promoting undergraduate research projects. The initial group of students focused mainly on device design and manufacturing and worked under the supervision of both principal investigator’s as a team. They were given a wide degree of autonomy to research and design the stretchers. For example, it was through their independent research that the possibility of using low-cost servomotors was discovered. Beyond mentoring the newly recruited team-members, these students also mentored high school students who were part of the center’s CardioStart high school summer program.
As the project progressed, the students were more involved in the biological experiments that aligned with the research programs of each lab. However, they continued to work as a team on optimizing the device design. Thus, the students gained experience in design and manufacturing, biological experimental design, and working on an interdisciplinary team, all of which are valuable for careers in industry or academia. Overall, the students indicated that the project had a positive impact on their educational growth and helped to influence their career decisions . Through working on this project, the students perceived that they gained valuable knowledge and skills in engineering design, experimental design, and basic cell biology. They also indicated that they gained experience in reading and writing scientific articles, programming, using CAD-related software, and cell culture. In addition, working in interdisciplinary teams and presenting their work to a wide range of audiences resulted in improved communication skills. The students were also asked to assess the impact the project had on their careers. They indicated that their direct involvement with this project helped to influence their chosen career paths and the experience also helped to develop the skills necessary for their career decisions. The first two students on the project originally intended to pursue industry careers, but both chose to also acquire master’s degrees . Of the latter group of students, one went to industry and two are pursuing doctoral degrees.This study describes a low cost uniaxial cell stretcher that produces consistent strain profiles, similar to alternative commercially available systems. While the mechanical functions are similar, the fabrication and maintenance costs of this device are only a fraction of those required for other similar systems, thus potentially increasing the widespread availability of this apparatus. The proposed device is composed of readily available materials and utilizes a low cost servomotor to perform the mechanical motions necessary to elicit a uniaxial strain. The device is capable of multiple strain profiles with differing amplitudes and frequencies, and can maintain applied strains for a minimum of three days under continuous use, thus validating the clamping mechanism. In addition, no heating issues resulting from the continuous operation of the servomotor were observed. When compared to other commercially available stretchers, the proposed design is considerably less expensive to maintain, but has comparable functions . However, this uniaxial cell stretcher has several limitations that can be addressed with minor modifications. For example, the servomotor used is unable to generate strains greater than a 20% amplitude. In addition, at maximum amplitude, the motor is capable of generating strains up to a frequency of 3 Hz, whereas higher frequency waves can be generated at lower amplitudes. These limitations can be addressed by substituting the given servomotor for another higher specification Hitech standard servo, which nominally increases the total cost. The standard servos typically have the same frame size, as a result, no additional modifications to the overall design of the cell stretcher should be needed. The designed cell stretcher was used to verify the alignment of macrophages and cardiomyocytes in response to cyclic uniaxial stretch. Macrophages were subjected to a 1 Hz strain at a 10% amplitude for a period of 18 h, whereas cardiomyocytes were subjected to a 1 Hz strain at a 20% amplitude for a period of 6 h. The chosen frequencies and strain amplitudes have previously been described as within normal physiological ranges for stretch experienced by macrophages recruited to blood vessels and cardiomyocytes in the heart. In the experiments performed, cardiomyocytes were seeded at a higher density to form a confluent monolayer that is more representative of cardiac tissues and analysis of alignment was dependent on actin organization, as determined by immunofluorescence. Macrophages were seeded moresparsely to allow ample space for cell spreading in response to cyclic stretch and to distinguish between individual cells using phase contrast images, which was necessary for the analysis of alignment and elongation, since macrophages do not display actin stress fibers. The stretch duration is also unique to each cell type but is typically long enough to allow for cytoskeletal rearrangement. Although the experimental setups were somewhat different, both macrophages and cardiomyocytes displayed alignment in response to stretch.