Embedded Files
Project Objective
Design a more reproducible and scalable system for engineering cartilage from genome-edited chondrocytes
This project was my first research project within Biomedical Engineering and was supported by the Abrams Scholar Award 2020-2021. The design of the device was a collaborative effort between me and my graduate student mentor, Matthew Rich.
Project Background
To study osteoarthritis (OA) gene variants, the lab obtains primary human chondrocytes, the cartilage cell type, from human donors and then creates the desired gene edits via CRISPR to produce isogenic clones. This process is relatively low yield, and these remaining edited cells are expanded and then cultured to form cartilage pellets and then analyzed. However, the current tissue engineering process has considerable variability due to chondrocyte de-differentiation and cell availability, which also limits the number of engineered cartilage that can be produced. The development of a platform that could more reproducibly create engineered cartilage on a larger scale would solve many of these issues.
Device Overview
Cost-effective, reusable, 3-D printed design
Snap-fit joint for easy clamping for centrifugation steps and simple removal for chondrocyte culture
Lid to preserve sterility when transferring apparatus between hood/centrifuge/incubator
Hydrogel layer for sealing between surfaces and cartilage adherence following surface treatment
11 wells for cartilage production in comparison to traditional 1 tube per cartilage pellet method
Design Inputs
Sufficient clamping force to prevent leakage of the cylinder contents
Prevent lateral and axial motion during centrifugation to preserve hydrogel integrity
Efficient hydrogel synthesis protocol
Desired cell seeding/density parameters post-centrifugation
Sterility and low cost
Hydrogel Production
Producing a hydrogel consistently with sufficient stiffness to withstand the pressure of the tissue carrier was a challenge. The hydrogel mixture we used required a mold to hold the mixture in liquid form, then UV photoactivation to cure it. Air exposure needed to be minimized for this mixture, which led to issues with air bubbles and leaks. My proposed initial design was a 3D print with a port for a needle to dispense the solution with clamped clear plates for a seal.
The 3D prints were not completely leak-proof so a different solution was needed. Laser-cut rubber gaskets sandwiched between glass plates proved to be the solution. I then ran multiple experiments with the hydrogel mixture at various UV mixture times and distances to find the optimal parameters for PEG cross-linking.
Gasket Design
Hydrogel testing setup
Uniform hydrogels
Body Design | Snap-Fit | Clamping Optimization
The initial design of the carrier was too heavy to go in a centrifuge and it was difficult to position the top half to form a seal with the hydrogel. The central feature of the redesign was the five prong top that would sit on top of the hydrogel inside a standard plastic tissue culture dish, which would then snap down onto the bottom piece, providing the clamping force needed to create a water-tight seal.
The longest part of the project was optimizing the clamping force of the snap-fit joint. Too little force would leak the contents of the wells, and too much force would crack the hydrogel. This required lots of adjustments factoring in the hydrogel height, the force required to insert and remove the top, and the joint height, among others. After numerous iterations of trial and error, I was able to produce consistent device clamping.
Summary
This project was my first foray into CAD, 3D printing, and design work. I was able to successfully streamline the hydrogel production process and produce a device that was watertight, easy to assemble, and kept the gel intact.
Challenges
Problem: Difficult to produce intact hydrogels of consistent stiffness and height
Solution: 3D print molds were tested before the successful laser-cut rubber gasket + glass design. After this design was solidified, I ran successive trials with the UV light source to determine the optimal UV exposure time.
Problem: Original design was too heavy to centrifuge
Solution: Redesigned the body of the device entirely to feature a five-pronged snap-fit design
Problem: Snap-fit was difficult to use and would either result in leaking or a damaged hydrogel
Solution: Optimized the height of the snap-fit joint and the dimensions of the prongs through successive prints and testing
Page updated
Google Sites
Report abuse