Designing Fibrosis Models 21-22
ÖffentlichFibrosis is a medical condition in which there is accumulation and abnormal distribution of extracellular matrix (ECM) in response to injury. Increased ECM stiffens the tissue leading to organ malfunction and even patient death. Fibrosis affects millions of people worldwide, but there is a notable lack of treatments available as current antifibrotic drugs are limited in therapeutic ability, and patients with highly progressed fibrosis often require invasive procedures such as organ transplantation. In vitro disease models can help streamline development of effective antifibrotic drugs, but current models are not representative of in vivo conditions as many only involve cells cultured in a monolayer on hard plastic substrates. Our team was tasked to create an in vitro model which was representative of in vivo conditions, had controllable parameters which could be used to modulate fibrotic pathologies, biocompatible, reproducible, amenable to drug testing, safe for the user, and cost-effective. The in vitro fibrosis model developed by the team utilized a hydrogel scaffold with cells encapsulated to introduce the dimensionality and stiffness of the in vivo fibrotic tissue microenvironment and macromolecular crowders (MMCs) in the cell culture media as a method of producing increased collagenous ECM protein deposition. Specifically, a 10% v/v gelatin hydrogel was chemically crosslinked with 70mM 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide (EDC) and 35mM N-hydroxysuccinimide (NHS). Rheological curing tests were performed to quantify the stiffness (elastic modulus) of the crosslinked gelatin hydrogels. It was found that the selected hydrogel composition was able to reach stiffness values over 3kPa as desired for modeling fibrotic tissue. Macromolecular crowders including Ficoll 70, Ficoll 400, PVP40, dextran sulfate, iota carrageenan, and kappa carrageenan were explored at different concentrations in 2D culture with cells on tissue culture plastic, 2.5D with cells on top of the hydrogel, and 3D with cells encapsulated within the hydrogel. AlamarBlue metabolic assay was used to measure the proliferation of NIH 3T3 embryonic mouse fibroblasts over time. Steady cell proliferation was observed overtime for 2D cell culture exposed to MMCs; however, presence of the hydrogel caused a sharp decline in proliferation after 24 hours. Sirius red/Fast green stain was used to qualitatively display changes in collagen deposition. 2D macromolecular crowding tests were able to display collagen deposition based on intensity of red stain. 2.5D cells on top of gelatin hydrogels showed inconsistent levels of Sirius red/Fast green staining and varying levels of cell attachment. Lastly, 3D cells encapsulated within the gelatin hydrogel showed drastic changes in cell morphology but were not able to properly convey collagen and ECM deposition due to issues with dye penetration.
- 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
- Identifier
- E-project-042822-102120
- 65041
- Stichwort
- Advisor
- Year
- 2022
- Date created
- 2022-04-28
- Resource type
- Major
- Rights statement
Beziehungen
- In Collection:
Objekte
Artikel
Miniaturansicht | Titel | Sichtbarkeit | Embargo Release Date | Aktionen |
---|---|---|---|---|
|
Fibrosis MQP D-Term Report.pdf | Öffentlich | Herunterladen |
Permanent link to this page: https://digital.wpi.edu/show/h989r654j