Amedica

Krissy Peterson
What is Amedica?
What: They produce Silicon
Nitride, which is a product that can
be used as a bone implant

Where: Amedica is based in Salt

Lake City, Utah.

Why: They are working to create a

strong and helpful bone implant,
that also had anti-bacterial
qualities.
What Makes Silicon Nitride
Different than Other
Implant Materials?
Objectives of the Amedica Project

Vs.
Methods for Finding Growth and Standard Curve

1: Cell Breakout from 2: Passage cells in 3: Seed cells into 24

the -80° Freezer flasks well plates at varying
seeding densities.

4: fix cells to plates for

crystal violet assay

6: Count cells with a 5: Prep cells for

Hemocytometer counting
 Step 1-3: Seeding Density of Plates for
Growth Curve

Cell Counting 0 0 0 80 80 80

20 20 20 100 100 100

1: prepare cells on 24 well
40 40 40 120 120 120
plates
60 60 60 140 140 140
Step 4: 10 uL Sample + 10 uL Trypan Blue

2: Grow cells Seeding Density of Plates for Standard

Curve

0 0 0 250 250 250

3: Trypsinize wells
100 100 100 300 300 300

150 150 150 350 350 350

4: Trypan Blue
200 200 200 400 400 400

5: Count Cells
Step 5: Counting Live Cells on Primaries
Data- Growth Curve From Cell Counting
Data- Standard Curve From Cell Counting
Crystal Violet Assay

1: prepare cells on 24 well

plates

2: Grow cells

3: fix cells

4: stain with Crystal Violet

5: measure absorbance
 Crystal Violet
Assays
Data Sets: Normalized
Absorbance Growth Curve
 Crystal Violet
Assays
Data Sets: Normalized Standard
Curve
Moving Forward
● Seeding density- Possibly too high?
● Lab Technique- Improve cell counting technique

0 0 0 50 50 50

20 20 20 60 60 60

30 30 30 70 70 70

40 40 40 80 80 80
Acknowledgements to the amazing Amedica
Project team!
Miranda, Cody and Marianne, also Kate and
Mary :)
Slide Presentation Notes:
Slide 3: Silicon nitride is a ceramic substance, that has a crystal-like surface. It is unique because it has the ability to resist bacteria
infection.

Slide 4:
Peek: a plastic- the medical standard implant
Silicon nitride
Mg-63 osteoblast cells

Slide 5: basic flowchart- the work done for this data--

Slide 6: Cell Counting Specifics

1. Prepare cells in four, 24 well plates
2. Grow cells, removing one plate after 0 hours(24 hours), 24 hours, 48 hours, and 72 hours.
3. When plate is removed wash and trypsinize each well with .5 ml.
4. Add .5 ml of complete media to each well, take 10 ul samples of each well, add 10 ul of Trypan Blue to the sample
5. Place on Hemocytometer and count primaries under a microscope.

Slide 7: Data, Growth Curve: Turns out our cell counting data show us we have some lessons to learn --point out axis and time
color boxes
inconsistent counting- explain the super high bar in the graph

Slide 8: Data, Standard Curve: point out axis and trendline- R^2 value-- Sucks explain inconsistency again
Learning Everywhere!
Slide 9: Crystal Violet Specifics

1. Prepare cells in four 24 well plates

2. Grow cells, removing one plate after, 0 hours(24 hours), 24 hours, 48 hours and 72 hours
3. Fix cells to plate buy washing and the adding .5 mL Methanol to each well, allow cells to die, then wash the plate 5 times
with dH2O and let dry
4. Add crystal violet, allow cells to stain, wash wells with dH2O until the water washed off is clear.
5. Add acetic acid and allow cells time to “lift” off the plate on shaker-- take the absorbance with a spectrophotometer.
Transfer the cells to a 96 well plate and take the absorbance again.

Slide 10: data Growth Curve

Slide 11: Data Standard Curve

Slide 12: Seeding density- too high try a lower seeding density with cell counting
Lab Technique- Create consistency within the team- Trypsin make sure that we aren’t getting clumps or something- using common
sense with strange cell counts

Slide 13: thanks to the Team!

Cell Counting Specifics
1. Prepare cells in four, 24 well plates
2. Grow cells, removing one plate after
0 hours(24 hours), 24 hours, 48
hours, and 72 hours.
3. When plate is removed wash and
trypsinize each well with .5 ml.
4. Add .5 ml of complete media to
each well, take 10 ul samples of each
well, add 10 ul of Trypan Blue to the
sample
5. Place on Hemocytometer and count
primaries under a microscope.
Crystal Violet Specifics
1. Prepare cells in four 24 well plates
2. Grow cells, removing one plate after,
0 hours(24 hours), 24 hours, 48 hours
and 72 hours
3. Fix cells to plate buy washing and the
adding .5 mL Methanol to each well,
allow cells to die, then wash the plate
5 times with dH2O and let dry
4. Add crystal violet, allow cells to stain,
wash wells with dH2O until the water
washed off is clear.
5. Add acetic acid and allow cells time to
“lift” off the plate on shaker-- take the
absorbance with a spectrophotometer.
Transfer the cells to a 96 well plate
and take the absorbance again.