Poster 79 - From Error to Insight: Revisiting DRG Cell Experiments with Corrected DMSO Dosage for Accurate Pain Signaling Analysis

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

SK

Sharon Kodali

Intern
UCSD
San Diego, California, United States

Co-Author(s)

XW

Xinyan Wei

Student Mentor
University of California, San Diego, United States

Co-Author(s)

NZ

Nicole Zhang

Student
OPALS, United States
AL

Angela Li (she/her/hers)

Volunteer Research Intern
University of California, San Diego: Institute of Engineering in Medicine
San Diego, California, United States

Co-Author(s)

MY

Max Yang

Intern
UCSD
San Diego, California, United States
Cindy Li (she/her/hers)

Intern
UCSD, United States
KS

Karylle Salileng

Intern
UCSD, United States
RM

Rizan Muhammad

Intern
UCSD, United States
NP

Nikhil Patel

INtern
UCSD, United States
CW

Chengbiao Wu

Professor
UCSD School of Medicine Dept of Neuroscience, Outreach Program for Advancing Learning in STEM (OPALS), United States

Primary Investigator(s)

Linda Shi, PhD (she/her/hers)

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Last Author(s)

VG

Veronica Gomez-Godinez

Scientist
University of California, San Diego
La Jolla, California, United States

Introduction: Dimethyl Sulfoxide (DMSO) is widely used in biological research as a solvent for drugs and compounds due to its unique ability to dissolve both polar and nonpolar substances. It also serves as a cryoprotectant to protect biological tissue from freezing damage. DMSO is also commonly used in control experiments to ensure that observed effects are due to the compound being tested rather than the solvent. In our study, DMSO was initially added to facilitate the delivery of a drug intended to inhibit Nav 1.8 function, thereby lowering calcium intensity in Dorsal Root Ganglion (DRG) cells—clusters of sensory neurons located in the spinal cord which are derived from newborn mice. Additionally, the pH of the media was altered to activate Acid-Sensing Ion Channels (ASICs) to induce pain signaling in DRG cells that are highly sensitive to low pH. This research is to define how solvents like high concentration of DMSO alters cellular functions and experimental outcomes.

Materials and

Methods: DRG cells were cultured from E18 rat embryos and maintained under standard conditions at pH 7.4. Initially, DMSO was mistakenly added to the media at a concentration 10 times higher than intended, at a concentration of 1% instead of 0.1%, with the media’s pH adjusted to 4.95 to examine the combined effects of acidity and high DMSO concentration. Calcium signaling in the DRG cells was monitored using biophotonic imaging techniques, with data processed and analyzed using ImageJ software. Control DRG cells were maintained in a pH 7.4 environment without DMSO.

Results, Conclusions, and Discussions: In the experiment, DRG cells showed normal spontaneous responses, with normalized calcium intensity ranging from 1 to 3 in the first 15 minutes (as shown in Figure 1). Upon exposure to 10 times the intended DMSO concentration at pH 4.95, the calcium signal spiked by 1.95 times within 3 seconds, likely due to cell bursting. This was followed by a gradual decrease in intensity over the next 15 minutes, indicating cell death. The phase images confirm that the high DMSO concentration caused all the cells to die, as the decrease in calcium intensity aligned with the observed cellular demise. In summary, our findings reveal the significant cytotoxic effects of DMSO at high concentrations, particularly in acidic conditions, which can rapidly lead to cell death. This underscores the importance of meticulous solvent handling and strict adherence to protocols to prevent unintended outcomes. Understanding the interaction between DMSO and acidic environments is vital for accurately studying ASICs, Nav. 1.8, and pain signaling. Moving forward, we aim to refine our experiments by using precisely measured DMSO concentrations on DRG cell lines and systematically varying one variable at a time. This approach will help us isolate the specific effects of DMSO and acidic pH on DRG cell viability, leading to more reliable and meaningful results, and ensuring the validity of our findings.

Acknowledgements (Optional): This work was supported by a generous gift from Beckman Laser Institute Inc. to LS. The high school students (*) participated in the UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional):