Priya Gajendiran

Rationale:
The liver is composed of four main types of cells: Hepatocytes, Kupffer cells, sinusoidal
endothelial cells, and hepatic stellate cells. When the liver is injured, hepatic stellate cells
activate and repair the injured tissue and in the process they produce extracellular matrix (ECM).
However, after repeated injury, mostly due to alcoholism or hepatitis C, the hepatic stellate cells
remain in a constant activated form and secrete an excessive amount of ECM. These ECM
proteins can create thread-like structures, which can lead to liver fibrosis and, if left untreated, to
cirrhosis and hepatocellular carcinoma. If there is a qualitative or quantitative difference between
an active and inactive hepatic stellate cell, then liver fibrosis can be detected early and drugs can
be used to induce an inactive state of the hepatic stellate cells. This would lead to a decrease in
cases of cirrhosis and hepatic carcinoma.
Research Question:
Is there a qualitative and quantitative difference in the mitochondrial membrane potential
between active and inactive hepatic stellate cells?
Hypothesis:
Using a fluorescent stain that indicates mitochondrial membrane potential, there should be a
qualitative difference between the membrane potential of an active and an inactive hepatic
stellate cell and this should support qualitative analyses of ATP, glucose, and lactic acid.
Engineering Goals:
Create an experiment that definitively shows a difference between the mitochondrial membrane
potential of an active and an inactive hepatic stellate cell.
Expected Outcomes:
A qualitative difference between the mitochondrial membrane potential of an active and an
inactive hepatic stellate cell from the fluorescent stain is expected and the qualitative analyses of
ATP, glucose, and lactic should support this.
Procedures:
Cell Removal and Replating-
1. Remove growth medium from LX2 cells.
2. Wash cells with Phosphate Buffer Saline (PBS)
3. Add enough Acutase to completely cover the cells and incubate until the cells are free
from the bottom of the culture dish.
4. Add twice the amount of growth medium as Acutase to neutralize the remaining Acutase.
5. Put the cell-growth medium-Acutase solution into a plastic tube and centrifuge at
1000rpm for 5 minutes.
6. Remove the growth medium-Acutase solution from the tube leaving the cell pellet.
7. Re-suspend the cell-pellet in growth medium.
8. Count the number of cells in the solution.
9. Allocate 20000 LX2 cells for plating in a four chamber well.
Priya Gajendiran

10. Dilute Geltrex in a 1:1 ratio with Optimum medium (without growth serum)
11. Cover two chambers of the four-chamber well with the Geltrex solution and incubate for
at least 2 hours to solidify.
12. Plate 10000 LX2 cells in a chamber without the geltrex coating and plate 10000 cells in a
chamber with the geltrex coating.
13. Repeat steps 1-12 for the Rat-HSC cells.
TMRM Staining with DAPI-
1. Prepare 1 mL of 250 nM TMRM staining solution in complete medium.
2. Remove media from live cells.
3. Add the TMRM staining solution.
4. Incubate for 30 minutes at 37°C.
5. Wash 3 times with PBS.
6. Take 42 μL of the DAPI and make up to 2ml with PBS.
7. Add 400 μL of the solution to each chamber of the four chamber well.
8. Incubate for 5 mins.
9. Wash 3 times with PBS.
10. Add prewarmed growth medium.
MitoTracker Green Staining with DAPI
1. Dilute 1 mM MitoTracker stock solution to the final working concentration of 200nM in
PBS.
2. Centrifuge to obtain a cell pellet and aspirate the supernatant.
3. Re-suspend the cells gently in prewarmed (37°C) staining solution containing the
MitoTracker probe.
4. Incubate for 45 minutes.
5. After staining is complete, re-pellet the cells by centrifugation and re-suspend cells in
fresh prewarmed medium.
6. Take 42 μL of the DAPI and make up to 2ml with PBS.
7. Add 400 μL of the solution to each chamber of the four chamber well.
8. Incubate for 5 mins.
9. Wash 3 times with PBS.
10. Add prewarmed growth medium.
Risks and Safety:
Acutase, TMRM, DMSO, MitoTracker, and Acetone are all potential skin irritants and be
harmful if ingested. In addition, hepatic stellate cells may be a potential source of pathological
contamination. Precautions that will be taken to reduce risk are that all experimental procedures
will be conducted inside a fume hood to prevent any contamination and gloves will be used to
prevent exposure to pathogens and irritating chemicals.

Data Analysis:
Priya Gajendiran

The fluorescent stains will be analyzed by imaging cells with a spinning 3i confocal microscope.
After taking images with the microscope, the images will be edited and analyzed further using
ImageJ and Adobe Photoshop.
The ATP analysis will be done using the ELISA method and the Lactic Acid and glucose assay
will be done using a kit. Data from these analyses will be analyzed using Microsoft excel.
Bibliography:
Thermo Fischer Scientific . (n.d.). Functional Mitochondrial Staining Protocol. Retrieved
January 18, 2017, from https://www.thermofisher.com/content/dam/LifeTech/global/life-
sciences/cellanalysis/Files/1014/Functional%20Mitochondria%20staining%20Step%20by
%20Step%20protocols_D3.pdf
Thermo Fischer Scientific . (n.d.). MitoTracker Mitochondrion Selective Probes. Retrieved
January 18, 2017, from https://tools.thermofisher.com/content/sfs/manuals/mp07510.pdf
Ganapathy-Kanniappan S, Karthikeyan S, Geschwind JF, Mezey E. Is the pathway of energy
metabolism modified in advanced cirrhosis? J Hepatol. 2014 Aug;61(2):452. PubMed
PMID:24810232.
Ganapathy-Kanniappan S, Kunjithapatham R, Geschwind JF. Glyceraldehyde-3-phosphate
dehydrogenase: a promising target for molecular therapy in hepatocellular
carcinoma. Oncotarget. 2012 Sep;3(9):940-53. Review. PubMed PMID: 22964488.