BIOMATERIALS LAB #3
Cell culture and cell-(bio)material
Interactions
Monday, February 27th and Tuesday, February 28th
BME 245/445 – Lab 3
PRE-LAB
 Sign-in
 Quiz (Benoit. 2005)
BME 245/445 – Lab 3
LINEAR MOLECULES
 PEGMA
 Acr-PEG-RGDSG
Modified from (Lin, C.C., Pharmaceutical Research. 26(3):
631-643. 2009)
BME 245/445 – Lab 3
SECTION 1- Done before lab
PEGMA Network Synthesis (done before lab – as previous)
1. 10wt% PEGMA in PBS
1. +/- 5 mM Acr-PEG-RGDSG
2. 10% photo-initiator by volume (LAP)
3. 0.75 mL Placed between two glass slides with spacer
4. Exposed to UV-lamp for 10min
5. Remove, punch out cylindrical samples
6. Place in PBS
BME 245/445 – Lab 3
SECTION 2
Cell Culture (TA demo and then hands on)
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Sterile Techniques
Feeding cells
Counting cells
Seeding cells (TC plastic / PEG / PEG+RGDSG)
Monitor cell-material interactions over time… (ImageJ)
 # of adherent cells
 Average adhesion area
 Average spindle factor
BME 245/445 – Lab 3
SECTION 3
Time-course data collection
 Days 0, 1, 2, 3
 # adherent cells, adhesion area, spindle factor (ImageJ)
 Day 2
 Live/Dead imaging
BME 245/445 – Lab 3
Lab Safety
 BSL 1/2
 Human cell lines in culture room  BSL 2
 We’re using animal cells (Mouse embryonic
fibroblasts)  BSL 1
 Hoods
 Filter out biologic particulates
 Gloves, glasses, caution
 No food/drink/chapstick/ect
STERILE TECHNIQUE
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Gloves!
Spray everything down with EtOH before entering hood
Do NOT reach over the top of open containers
Do NOT leave containers open unless in use
Caps always placed right-side up
Change pipette tips after every use
Clean up after done
 Soap for spills
 EtOH to sterilize
BME 245/445 – Lab 3
IMAGEJ
1.
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6.
Open ImageJ
File  Open  select image file
Area: Polygon option
Diameter: Line option
Analyze  Set Measurements (select area)
Analyze  Measure (area/length)
BME 245/445 – Lab 3
IMAGEJ
Future measurements are calibrated
Can add scale bars
Draw line of known length
Set global scale
BME 245/445 – Lab 3
IMAGEJ
Line Selection (for Diameter)
Polygon Selection (for Area)
BME 245/445 – Lab 3
SPINDLE FACTOR
 Ratio of major axis to minor axis
SF = 96.566/86.770 = 1.11
SF = 118.127/47.676 = 2.48
BME 245/445 – Lab 3
BME 245/445 – Lab 3
Scheduling
Day
Tuesday Lab
Tuesday Lab
Responsibilities
0
Monday, Feb 27th
Tuesday, Feb 28th
Feed cells
Collect Images
1
Tuesday, Feb 28th
Wednesday, Feb 29th
Feed cells
Collect Images
2
Wednesday, Feb 29th
Thursday, March 1st
Feed cells
Collect Images
Live/Dead
3
Thursday, March 1st
Friday, March 2nd
Collect Images
BME 245/445 – Lab 3