This power point speaks about the fundamentals of cell culturing

1. BIOMATERIALS LAB #3
Cell culture and cell-(bio)material
Interactions
Monday, February 27th and Tuesday, February 28th
BME 245/445 – Lab 3

2. PRE-LAB
 Sign-in
 Quiz (Benoit. 2005)
BME 245/445 – Lab 3

3. LINEAR MOLECULES
 PEGMA
 Acr-PEG-RGDSG
BME 245/445 – Lab 3
Modified from (Lin, C.C., Pharmaceutical Research. 26(3):
631-643. 2009)

4. 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

5. SECTION 2
Cell Culture (TA demo and then hands on)
 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

6. 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

7.  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
Lab Safety

8. STERILE TECHNIQUE
 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

9. IMAGEJ
1. Open ImageJ
2. File  Open  select image file
3. Area: Polygon option
4. Diameter: Line option
5. Analyze  Set Measurements (select area)
6. Analyze  Measure (area/length)
BME 245/445 – Lab 3

10. IMAGEJ
BME 245/445 – Lab 3
Draw line of known length
Set global scale
Future measurements are calibrated
Can add scale bars

11. IMAGEJ
Polygon Selection (for Area)
Line Selection (for Diameter)
BME 245/445 – Lab 3

12. 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

13. BME 245/445 – Lab 3

14. 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