Can we engineer pancreatic beta cells to function properly and safely increase insulin production for diabetics?

Methods

Summary

Methods

This project is an in vitro (cell culture style) study observing the effects of different combinations of mitochondrial enzymes present at various stages of the TCA cycle each with DNA nucleotide bases and anti-metastasizing agents, at specific time intervals on Mus musculus (Mouse cells) in comparison to diabetic and cancerous beta cell growth to normalize rates of insulin production and beta cell function and overall lifespan. 

1. Sonication, to break open cells that release key substances 

2. Transfection, with the use of CaCl2 solution containing phosphate to intrude mtDNA into the beta cell. 

3. Liposomal treatment, with the use of a lipid solution to create an amphipathic surrounded vehicle to transport the enzymes combinations to the mitochondria. 

4. Anti-cancer liquids would be added into complete medium solution before insertion of substances (act as cell bonds [when cells touch they have a small chance of creating cancerous cells])
5. Flow cytometry would be used to observe changes in weight, size, and granule level ranges within the cells. 

6. GSIS (glucose-stimulated insulin secretion) would be detected with ELISA (enzyme-linked immunosorbent assay) kit to detect and measure the amount the the target between matched antibody pairs for insulin in mice. 

Challenges

Cell culture can sometimes seem daunting with all of the complications that could arise including contamination, low amounts of substance to test, using the wrong cell type, accidentally tripping and ruining the whole experiment (a possibility), and other factors all could easily produce faulty results. My efforts in preventing these possibilities are in how I organize my plans: I plan to use 70% ethyl alchohol (the best anti-contamination protocol) on all items that I plan to use before using them; measure equal amounts of medium and substance; labeling and organizing all of my samples; and making sure the floor is not slippery to fall :). Overall though, I believe that I can overcome these obstacles and successfully conduct my research. 

Pre Analysis Plan

The preliminary stages of this experiment involve basic observational tests on the beta cell lines (NIT-1 (ATCC® CRL-2055™) and other cell lines from ATCC) under the same culture environments (37 degrees Celsius incubator with constant 5% CO2 levels)  (all set up at same time periods)

1. Observe non-diabetic beta cell growth of Mus musculus over a 1-5 week period with day to day checks and 2-hour checks on first day(islets in non-diabetic cells can grow within 48 hours, but development can occur after) 

2. Observe cancerous beta cell growth of Mus musculus over a 1-5 week period with same checkups (islets with grow <48 hours, but proliferation of islets should be noted for many days)

3. Observe diabetic beta cell growth of Mus musculus for 1-5 week period with same checkup times (islets would grow >48 hours but under 7 days) 

4. Record results and retest under same guidelines if needed. 

After this stage, a new set of unimmortalized cells would be used in safely extracting key mitochondrial enzymes and nucleotide-specific isoforms with methods such as sonification, glycosylation, and tranfection as explained previously.

After examining and recording average rates of growth and insulin production (with an ELISA [enzyme immunosorbent assay] kit), I would then use cells from all of same cell lines (being the same type not same sample as they are regular, unimmortilized growing cells and it could result in faulty results) in sonification at a local ASU lab to break the membranes and release the inner fluids. Next, I would use transfection and glycosylation (a nucleotide magnet per se) to pick up the specific DNA coming in a white mixture, and specific set liposomes (or little membranes) to pick up the enzymes (depending on on various factors of charge, weight, etc.). I would then set a separate microscope study/interaction between the two kinds of substances (at various amounts). Once I find a few clear matches (a group of substances that does not quickly denature or produce other complications) I would then create the isoform groups by having them be inserted into the cell DNA through liposomes (again). Lastly I would repeats steps 1-4 as shown above to analyze my results and form conclusions.