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Tissue Engineering

Tissue Engineering - Poster Session D

Poster K20 - Phenotypic Modulation of Lymphatic Endothelial Cells by Oscillatory Flow on Gel Substrate

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Lymphatic diseases such as lymphedema and lymphoma affect millions globally, causing significant morbidity1. These conditions can lead to severe swelling, impaired immune function, among other debilitating functions. Lymphatic tissue engineering can provide a solution for those affected by lymphatic disruption. Though our lab has previously demonstrated to have developed lymphatic capillary-like vessels from lymphatic endothelial cells (LECs) in a 3D gel2, there is still a need to engineer a lymphatic vessel with a functional lymphatic-valve. Since oscillatory flow has been identified to influence lymphatic valve morphogenesis in vivo3, we will be investigating how oscillatory flow can impact LEC phenotype when cultured on a gel. Understanding the impact of oscillatory flow on LECs in a controlled environment has the potential to provide valuable insights into valve formation and function.

1. Alderfer L, Wei A, Hanjaya-Putra D. Lymphatic Tissue Engineering and Regeneration. Journal of Biological Engineering. 2018;12(1):32. doi:10.1186/s13036-018-0122-7

2. Alderfer L, Russo E, Archilla A, Coe B, Hanjaya-Putra D. Matrix stiffness primes lymphatic tube formation directed by vascular endothelial growth factor-C. FASEB J. 2021;35(5):e21498. doi:10.1096/fj.202002426RR

3. Sabine A, Agalarov Y, Maby-El Hajjami H, et al. Mechanotransduction, PROX1, and FOXC2 Cooperate to Control Connexin37 and Calcineurin during Lymphatic-Valve Formation. Developmental Cell. 2012;22(2):430-445. doi:10.1016/j.devcel.2011.12.020

Materials and

Methods:  The Ibidi μ-Slide I Luer 3D was coated with 40 µg/mL fibronectin, washed with phosphate buffer saline, and filled with 2.5 mg/mL of collagen. After 30 minutes of polymerization, it was seeded with juvenile dermal LECs at a density of approximately 8x105/mL and incubated at 37ºC with 5% CO2 for 24 hours. The chip was connected to the Ibidi Pump System and oscillatory shear stress was exposed to the slide at 4 dyne/cm2 for 48 hours inside an incubator. Immunostaining was conducted and imaging was done on a fluorescence microscope.

Results, Conclusions, and Discussions: 

Results: Fluorescent images of the mechanosensitive protein, VE-cadherin, on LECs reveal a distinctive arrangement where the LECs begin to align with one another, especially when present under flow. This indicates an adaptive response of LECs to mechanical stimuli. These specific cellular configurations of LECs on collagen are features found in early LEC lymphatic-valve formation stages.

Conclusion: LECs respond to the oscillatory flow in the presence of collagen which demonstrates that these cells can undergo a critical process in the development of functional and mature lymphatic vessels while being cultured on a biomaterial. The mechanical stimuli mimics the natural conditions experienced by LECs in vivo. Collagen was used due to its ability to support cell survival without the need for additional growth factors.

Discussion: This study supports the idea that lymphatic cells maintain their capability of responding to physical changes in their environment while being cultured on a biomaterial. Specifically, the application of oscillatory shear stress highlighted the cells’ undisturbed potential for growth under mechanical stimulation. Future research includes further characterization of the 3D gel interaction with LECs under flow and using a norbornene modified hyaluronic acid (NorHA) gel instead of collagen due to its ability to mimic the natural behavior of the extracellular matrix.

Acknowledgements (Optional):  Thank you to the Hanjaya-Putra Lab and the SMASH REU.