Macrophage Polarization

Author: Daniel Maxenberger | Major: Biomedical Engineering | Semester: Spring 2022

Over the course of the 2022 Spring grant term, I continued to work in the lab of my honors mentor, Dr. Christopher Nelson, researching the possible polarization of macrophages using CRISPR gene editing to eventually be used as a potential therapy for diabetic wound healing complications which is the continuation of the project I have been working on since the beginning of the grant term in the Fall of 2021. Macrophages are a class of cells that can be widely divided into two phases: M1 and M2. M1 cells are pro-inflammatory cells that activate to clear out harmful bacteria and debris from wound sites by irritating it. After M1 cells activate and fulfill their role, they are polarized into their M2 phase. M2 cells are anti-inflammatory cells that begin the closing and healing process at the wound site.

The reason healing can be problematic for those with diabetes is that there is a delay between the polarization between the M1 phase and the M2 phase. This keeps the wound from closing properly and causes further inflammation delaying the wound closure process and allowing for the beginning of chronic wounds. The goal of our project is to use CRISPR gene editing to alter the malfunctioning macrophages to allow them to properly polarize into the M2 state.

This semester, I needed to restart the process of designing, cloning, and sequencing the plasmids with the mouse genome refence. Doing this, I started the cloning process, inserting the oligos with the desired sequence into the lentiviral backbone that would serve as my transfection agent for the experiment. Afterwards, I transformed and plated them on Agar plates.

This process was fairly lengthy. My first few attempts didn’t have any colonies form after plating, so I began it again each time. After each attempt didn’t work, I would have to restart the process from the beginning. After I finally had colonies form, I would then inoculate and make glycerol stock of each sample for further use. Once done, I would do miniprep on the samples to prepare for sequencing, which the first two times did not come out correctly. With the incorrect sequences, that means that there was an error in the beginning of the process, and it would need to be restarted.

While waiting for the overall process to work, I was also trained in lentiviral transfections so as to perform the next tasks more easily. This will allow the process to go fairly smoothly once the new guides are ready to be transfected into the cells.

Upon returning to the lab in August, I have a few tasks to undertake. First, I will correct the issues that occurred at the beginning of the cloning process, going through and rechecking each of my steps along the way to make sure minimal issues occur and that my sequencing will come out correctly. Afterwards, I will be able to transfect Raw 264.7 cells with the guides and culture them so as to perform an ELISA to check what the effect the transfection had on their state. This will allow us to know whether the transfection itself further irritated the cells, causing more of them to be categorized as M1, if they began polarizing more towards M2, or whether there is no justifiable difference in their state.

This Honors College Research Grant allowed me to focus on working on my Honors Thesis more consistently. I love the subject my project revolves around, and the opportunity to do hands-on research I am passionate about has been phenomenal. The project has never failed to interest me, and the more I work on it the more respect and love I have for research. I am excited to see where this goes over the next two semesters.