Y13 - Evaluating Effects of Macromolecular Crowders on the Structure and Mechanical Integrity of In-Vitro Tissue Ring Models of Uterine Myometrium

Introduction: : Uterine fibroids, which are benign tumors within the uterine tissue layers, are one of the most common diseases that affect people with uteruses. Most fibroid research utilizes animal models, which are expensive and often do not accurately model human fibroid pathophysiology. In-vitro modeling is emerging, but there is a significant unmet need for robust, accurate in-vitro models of uterine fibroids. Our long-term goal is to develop an in-vitro intramural fibroid model to replicate fibroids that embed in myometrial tissue. In support of this model development, the current study uses self-assembled human uterine smooth muscle cell (HUtSMC) tissue rings to model myometrial tissue using a tissue fabrication method leveraged by other researchers for vascular applications. Here, we build upon previous work from our lab that showed macromolecular crowding with Ficoll 70, Ficoll 400, and ascorbic acid significantly increase collagen production in 2D HUtSMC. After confirming that HUtSMCs can form tissue rings, we extend the model development to identify growth conditions that balance self-assembly with collagen production to support ring integrity. We characterized the mechanical properties and cell morphology of HUtSMC rings grown with or without macromolecular crowders through uniaxial testing and histological staining. We hypothesized that rings incubated in media with macromolecular crowders would have a higher ultimate tensile strength and elastic modulus while exhibiting more collagen deposition compared to regular growth media. Overall, this work advances research into fibroids by creating a myometrial tissue ring as the base tissue for a novel in-vitro model of intramural fibroids.

Materials and

Methods: : We used primary human uterine smooth muscle cells (HUtSMCs; ATCC) expanded from a low-passage stock (P3). The two media formulations used were: 1) Vascular Cell Growth Media (VCGM) prepared using vascular cell basal media supplemented with recombinant human FGF (5 ng/mL), Insulin (5 ug/mL), EGF (5 ng/mL), ascorbic acid (50 ug/mL), FBS (5%), and L-glutamine (10 mM); and 2) Ficoll Media prepared by supplementing VCGM with Ficoll 70 (37.5 mg/mL), Ficoll 400 (25 mg/mL), and ascorbic acid (0.17mM).
To generate tissue rings, HUtSMCs were seeded into agarose ring molds (described in Strobel et al., Vis Exp. 2018, 134:56618) at 250,000 cells/ring and overlaid with VCGM or Ficoll Media. Mechanical testing was performed on Day 7 rings using a calibrated CellScale testing apparatus fitted with a 2.5N load cell and specialized test arms to hold the ring in place while submerged in DPBS. The ring was manually pretensioned to 5 mN then ran through eight pre-tensioning cycles. The ring was pulled to failure at a strain rate of 10 mm/minute while pull length and force were recorded (n=14 VCGM and n=8 rings over N=2 trials). ImageJ was used to determine the cross-sectional area of pre-strained rings for calculations of ultimate tensile strength (UTS) and elastic modulus (E). For histology, Day 7 rings were transferred to sponges soaked in buffered formalin and fixed according to established protocols. Fixed rings were embedded in Paraffin then sectioned and stained using Hematoxylin/Eosin (H&E). Stained rings were imaged to assess cellular alignment and structure.

Results, Conclusions, and Discussions::

Results:
Two trials totaling 22 rings determined there was no statistically significant difference (P=0.71) between the UTS or E of Ficoll Media and VCGM rings after seven days. Figure 1 compares the ultimate tensile strengths of VCGM (142 kPa ± 60 kPa) and Ficoll Media rings (122 ± 42 kPa). Although the UTS range was larger for the VCGM rings, the VCGM and Ficoll Media rings share similar average and median UTS. Figure 2 shows the elastic modulus comparisons of the VCGM rings (583 ± 116 kPa) and Ficoll Media rings (561 ± 146 kPa), indicating similar variance, means, and medians.
The histological data of 2D tissues cultured in Ficoll media and VCGM media shown in Figure 3 indicate both rings produce similar amounts of extracellular matrix. Previous histological analysis of the Ficoll Media and VCGM rings showed that Ficoll Media rings had significantly higher nuclear eccentricity (p < 0.0001) compared to VCGM.
Discussions:
Since we did not observe significant differences between VCGM rings and the Ficoll Media rings on ultimate tensile strengths or elastic moduli, we must reject the initial hypothesis. It is possible that the early usage of macromolecular crowders interferes with cell junction formation since Ficoll Media was added at the start of culture. Nevertheless, these results suggest that the tissue rings likely do not require additional macromolecular crowders to maintain sufficient integrity and accurately model myometrial tissue. This simplification of the culture and fabrication reduces the number of non-physiologically-relevant molecules in culture, allowing for a more straightforward approach to modeling the tissue.

Conclusions:
Overall, we formed HUtSMCs self-assembled tissue rings that retain their shape and have sufficient integrity to sustain mechanical loading. These data will be bolstered by additional UTS and E mechanical trials and H&E/ Immunohistochemistry staining currently in progress. Future results will inform the next stages of the project that build towards a model of intramural fibroids to ask biological questions about fibroids initiation, growth, and therapeutic response. Ultimately, this project contributes to improving the standard of modeling for uterine fibroids by informing the basis of the first in-vitro model of intramural fibroids.

Acknowledgements and/or References (Optional)::