Poster Presentation Flashcards
Why did we choose adipose tissue?
- Higher success rate of MSC isolation over umbilical cord tissue
- Adipose tissue had the highest colony frequency of the three
- Can be cultured longer and has a higher proliferation capacity than bone marrow
- Shows the same differentiation capacity as bone marrow
How many cells can we get from 1 cc of adipose tissue?
~600,000
How do we isolate adipose tissue?
- Centrifugation with multiple resuspensions in Ficoll-Paque density gradient media
- Filtering through size exclusive mesh
- Magnetic cell sorting
What markers can be used for magnetic cell sorting?
CD105, CD106, CD146, CD161, and STRO-1
Does magnetic tagging effect cellular proliferation or viability?
No
How fast can magnetic cell sorting sort cells?
6.67*10^7 cells/min
How can we identify our cells?
Immunophenotyping, using markers on the cellular surface for identification
Try pan blue allows for discrimination between live and dead cells
What kind of flask are we going to grow our cells in?
T75
What conditions will our cells be grown in?
37C, pH 7.4, 5% CO2, 21% O2
When will be subculture our cells?
80% confluence
How often will the media be replaced?
Every 2 days
How many cells will be seeded?
~6000 cells/cm^2 or 450,000 cells per T75 flask
How many cells will be grown at 80 % confluency?
9.4 million cells
What is our desired amount of cells?
10 to 30 million cells
What characteristics do we want our biomaterial to have?
- Biodegradable
- Biocompatible
- Ability to transmit mechanical inputs
- Adequate pore structure
What size do we want our pores to be?
400 microns
What mechanical properties should we test?
- Storage (dynamic mechanical thermal analysis)
- Compressive strength
- Cross link density (swelling tests)
What is hyaluronic acid?
A disaccharide polymer with an alternating pattern of Beta 1, 3 glucuronic acid and Beta 1,4 N-acetylglucosamine
How are we going to increase the length it takes to degrade?
Esterification with a benzyl group on the carboxyl group of the beta 1,3 glucuronic acid.
What process are we going to use to produce our crosslinked HA?
- HA tetrabutlyammohnium salt solubilized in dimethylsulfoxide at 25C
- Add benzyl bromide and tetrabutlyammonium iodide and kept at 30C for 12 hours
- Poured into ethyl acetate under constant agitation
- Filter precipitate and wash 4 times with ethyl acetate
- Vacuum dry for 24 hours at 30C
How long will our 100% esterified HA take to biodegrade?
Two to three months
Where is HA naturally found?
ECM of skin, umbilical cord tissue, embryonic tissue, and malignant tissues
What are the biproducts of HA degradation?
Ammonia, carbon dioxide, and water via the Kreb’s cycle
Why did we choose HA?
- Cell adhesion
- Growth
- Migration of keratinocytes
- Regulating ECM organization and metabolism
- Non-immunogenicity
- Reduce scaring
How does HA help with organization of the ECM?
Interacts with surface cell receptors CD44 and RHAMM which sustain basal mobility
What process are we using to form our HA derivative fibers?
Melt blowing developed by Dr. Shambaugh, Dr. Papapvassilou, and Mr. Shambaugh
How many grams/hole/min are used during melt blowing?
.2-.8 grams
What will the velocity of air be to produce a fiber that is 100 microns wide be?
110 m/s
Where will our facilities be located?
Oklahoma City, Louisville KY, Reno NV, and Hartford CT.
Why did we choose these cities?
Economic feasibility, coverage, proximity to metropolitan areas, shipping within 48 hours
What will be our total man hours per week?
4240
What OSHA requirements must we meet?
- Initial hazard evaluation and complete a compilation of written safety information before any process is performed
- Develop and implement written operating procedures that contain “clear instructions for safely conducting activities in each covered process”
- Provide continuous training to employees
- Documents covering equipment maintenance
- Emergency plans and response
- Proper Chemical storage
Properly labeled
Large containers kept on ground level
Flammable liquids stored separately from other flammable liquids
No more than 25 gallons of flammable liquid can be stored outside an approved safety cabinet
No more than 60 gallons of flammable or 120 gallons of combustible liquids can be stored in a single cabinet
Each storage area can contain no more than 3 storage cabinets
Gas cylinders with combustible gas must be stored in a dry, well-ventilated, and well-protected area that is at least 20 feet from highly combustible materials. They should never be stored near elevators, stairs, gangways or where they could become part of an electrical circuit.
What FDA and CDC requirements must be met?
BSL2 requirements
PPE
Detailed lab procedures
Sink and eye wash stations
Restricted lab access
Use of biological safety cabinets
Autoclave
Self-closing doors
Safety signsWhat will we follow for shipping regulations and what does it entail?
International Air Transport Association (IATA)
1. Biological Materials Training Certificate obtained and renewed every 2 years 2. Must be in double containers that are leak proof and made of plastic with a sturdy outer package for a total of three packages 3. Containers must be disinfected before leaving the lab 4. Absorbent material and padding should be included to minimize leaks and movement. 5. Biohazard sticker must be on packaging 6. Safety Data sheet must be provided with initial shipment 7. Company name and contact information must be on the outer package
How much did we build into our risk assessment for clinical trials?
20%
How many sheets of biomaterial can be made from 9g?
500
How many devices can be made from 9 grams of polymer?
7.5
How many people do we plan to help treat our first year?
81,120
How many people do we plan to treat after the first year?
28,036
What is our cost in materials, shipping, misc per patient?
$208.10
How do we prepare our solution to be made into threads?
Resolubolized in dimethylsulfoxide at a concentration of 20mg/ml
