Designing an In-Vitro Model of Uterine Myometrium to Study Intramural Uterine Fibroids

Hicks, Elizabeth; Alkaff, Zara; Wilde, Micah

Student Work

Designing an In-Vitro Model of Uterine Myometrium to Study Intramural Uterine Fibroids

Public

I. Introduction Uterine fibroids (UF) are benign tumors found in the uterus that form from a single-cell clonal expansion. There are several types of fibroids in the uterus, the most common being intramural, subserosal, and submucosal (Stewart et al., 2016). They have a disorganized internal structure with a lot of extracellular matrix (ECM) and can range in size from 1 mm to 20 cm (Bulun, 2013). They are found in approximately 70% of people with uteruses at reproductive age and disappear after menopause. This number may be far greater because many cases go unreported due to a lack of symptoms, which can include pelvic pain, heavy bleeding, and infertility (McWilliams & Chennathukuzhi, 2017). There is currently no curative treatment for UF. Many treatments have serious complications and high recurrence. The most common clinical models used for UF currently are animal models, and current in-vitro 3D models are limited and not independently representative of an in-vivo fibroid (Weiswald et al., 2015). This project focused on designing and assessing the feasibility of tissue model components to mimic features of intramural UFs within the myometrium (muscular wall). II. Design Process Overview A successful model for this purpose must replicate the structures and functions of UF. The design objectives for this project were to create an effective modeling system that replicated fibroid/tissue interface, replicated in situ tissue composition, was reproducible, ensured cell viability, and was affordable. A pairwise comparison chart determined the best of several modeling approaches to address these objectives and to prioritize the replication of the fibroid/tissue interface as the most important objective. III. Methodology Different alternative designs were tested to ensure the components of the model could accurately mimic the structures and properties of intramural uterine fibroids. The designs of 3D spheroids, 3D rings, and alginate beads with encapsulated cells were established and tested to identify the most promising model components. These designs were first used with rat smooth muscle cells (RaSMCs) to establish techniques before using uterine smooth muscle cells (uSMCs) to represent fibroids or the myometrium. A. Spheroid Formation Spheroids were selected as a way to represent the intramural fibroids. They were formed via the agarose mold (AM) and hanging drop (HD) methods. In the AM method, two 2% (w/v) agarose molds of the same diameter but different volumes (190 μL and 75 μL) were used to form spheroids with different initial numbers of cells per well. In the HD method, 10 μL of a concentrated cell suspension was pipetted onto the top plate of a petri dish, then inverted to form the spheroid. Cell suspensions of different densities were tested via the hanging drop method to see if initial seeding density had an impact on circularity, spheroid size, and viability. Spheroid size and viability were measured for each method at Days 1, 3, and 5. The size was measured using ImageJ and the viability was measured using the CellTiter 96® AQueousOne Solution Cell Proliferation Assay (MTS). B. Ring Formation The ring model was selected to represent the myometrial wall as they portray a similar structure and tissue composition. 2% (w/v) agarose ring molds were made and set to rest for a day in media (Gwyther et al., 2011). Both RaSMCs and uSMCs were seeded into each well of the molds and incubated for 8, 10, or 14 days in their respective culture media. For uSMCs, supplemented vascular basal media with macromolecular crowders, Ficoll PM70, Ficoll PM400, and ascorbic acid, was used to support cell growth and increase ECM deposition for enhanced ring integrity. C. Bead Formation Alginate beads were selected to represent intramural fibroids. Alginic acid sodium salt was combined with a HEPES and NaCl buffer to reach a desired concentration, and gelatin was added at 0.5% w/v to aid cell adhesion. Alginate solution was extruded with a 27g needle into CaCl2 for alginate crosslinking. Cells were encapsulated by suspending in an alginate solution, forming as described above, and incubating. Bead diameters were measured for average size at Day 0 and Day 2 and a degradation study was completed. IV. Results A. Spheroid Formation HD spheroids were larger than both sizes of AM spheroids. The HD spheroids had average diameters at D3 of 161.27 ± 69.9 μm, and the AM spheroids had D3 diameters of 118.97 ± 16.40 μm in the larger molds and 119.94 ± 12.32 μm in the smaller molds. AM spheroids had an aspect ratio of 0.94 at D3, while HD spheroids had a ratio of 0.87. The aspect ratio decreased as seeding density decreased. The sizes of the hanging drop spheroids were highly variable because larger (>150 μm) and smaller spheroids (< 100 μm) had formed within one hanging drop. There was some size variation for AM spheroids as well because spheroids towards the center of the mold were larger than at the edges, as expected. Though the AM spheroids were larger and had a higher variability, they were unexpectedly more viable than the AM spheroids. They had a D3 viability of 73.9 ± 5.06%, which is not above the benchmark, but is significantly closer than the AM spheroids, which had a D3 viability of 45.62 ± 12.03% (large molds) and 39.32 ± 12.80% (small molds). B. Ring Formation RaSMC rings produced a thickness over the 10 days of culture of 344 ± 22.5 μm, and a successful removal percent yield of 75%. uSMC rings produced a thickness over the 10 days of culture of 959 ± 5 μm, and a successful removal percent yield of 0%. uSMC rings crowded with macromolecular crowders of Ficoll 70 and Ficoll 400 produced a thickness over the 10 days of culture of 843 ± 6.5 μm, and a successful removal percent yield of 79%. uSMC rings crowded with macromolecular crowders of Ficoll 70, Ficoll 400, and Ascorbic Acid produced a thickness over the 10 days of culture of 487 ± 5.5 μm, and a successful removal percent yield of 100%. Histology testing of Gomori Trichrome stain was conducted with both the uSMC rings crowded with Ficolls and the uSMC rings crowded with Ficolls and Ascorbic Acids, and resulted in successful stains that mimicked the collagen produced in the myometrial tissue. C. Alginate Beads The alginate beads formed were mostly spherically shaped and many beads could be formed during each trial. The alginate gelatin beads both with and without cells were more transparent and teardrop shaped than those with only alginate. Cell distribution was inconsistent between beads. Average bead sizes used alginate concentrations of 2%, 1.2%, and 0.8% w/v and gelatin concentration of 0.5% w/v for all except the 0.8% alginate that failed to form beads. The largest bead size was 2% alginate gelatin and the smallest was 1.2% alginate. The average bead diameters from Day 2 produced the same results as above with reduced sizes. The 0.8% alginate beads were the most consistent in size and stability with the lowest standard deviation in diameter of 82.56 µm at Day 0 and the smallest change in size over 5 days. V. Discussion and Future Directions A. Discussion Spheroids formed rapidly and consistently in agarose molds. The AM spheroids were consistently under the maximum benchmark size of 150μm, whereas HD spheroids were larger. HD spheroid shape was dependent on the shape of the initial seeding drop and became less circular as seeding density decreased. AM spheroids formed in a mold with set dimensions (400 μm diameter, 800 μm depth), and so were more consistent in size and shape. The spheroids formed by D1, however, did not hit all benchmark viabilities. HD was closer to hitting all benchmark viabilities than AM, though they were larger and more variable in size. Overall, spheroids are a quick way to form 3D cell cultures, but may not be an effective model of fibroids due to issues with viability. Because the uSMCs without any crowders were not successful in forming, it was hypothesized adding macromolecular crowders would increase the ECM deposition, resulting in more structurally compact rings. So, two more experiments were conducted using Ficoll 70, Ficoll 400, and Ascorbic Acid as crowders. The uSMC rings crowded with Ficolls-only were within the benchmark thickness after 10 days, 0.84-0.87 mm, and demonstrated that this ring is most structurally similar to the myometrial wall. However, the uSMC rings crowded with Ficolls and Ascorbic Acid produced the highest successful removal rate at 100%. This is due to the increased ECM deposition from all the macromolecular crowders. The Ficoll and Ascorbic Acid-crowded uSMC rings proved to have the most similar tissue composition to the myometrial wall. This stain showed the collagen dispersed throughout the whole ring which is representative of human myometrial tissue. Overall, it was determined that uSMC rings are representative of myometrium tissue when combined with Ficoll and Ascorbic Acid crowders. It is recommended that more tests be conducted to accurately determine the best way to mimic the myometrium tissue. The alginate beads were highly reproducible with minimal variability in size. The bead diameter decreased as expected when a smaller needle gauge was used for extrusion. Alginate gelatin crosslinked beads with cells encapsulated had a higher degradation rate than alginate-only beads. Cell encapsulation in the beads resulted in an inconsistent distribution of cells across the beads with some beads having no cells. The alginate gelatin beads had an unexpected result in morphology with many teardrop-shaped beads forming and transparent appearance. The cause of the difference in shape between the alginate and alginate gelatin beads is unknown. The 0.8% alginate beads were the most consistent in size and had the least degradation. In the degradation study the 2% alginate beads had the largest change in diameter with a difference of over 100 µm from 0 to 120 hours. B. General Takeaways and Potential Future Applications The use of rings and alginate beads in the predictive model seem most advantageous to the longevity and predictability of the model, however, more research is needed to confirm this. Future research and applications can include formulations of the three components working together in the same modeling system. Next steps to create this model would include attaching cells to alginate beads, attaching alginate beads to rings and spheroids, and adding fibroid cells and tissues into all models. These future steps would allow the team to achieve their main objective of replicating the fibroid/tissue interface and create a fully functioning model of a uterine fibroid.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

Creator

Publisher