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

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

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

4. S ECTION 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. S ECTION 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. S ECTION 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. S TERILE T ECHNIQUE  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. I MAGE J 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. I MAGE J BME 245/445 – Lab 3 Draw line of known length Set global scale Future measurements are calibrated Can add scale bars

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

12. S PINDLE F ACTOR  Ratio of major axis to minor axis SF = 96.566/86.770 = 1.11SF = 118.127/47.676 = 2.48 BME 245/445 – Lab 3

14. Scheduling DayTuesday Lab Responsibilities 0Monday, Feb 27 th Tuesday, Feb 28 th Feed cells Collect Images 1Tuesday, Feb 28 th Wednesday, Feb 29 th Feed cells Collect Images 2Wednesday, Feb 29 th Thursday, March 1 st Feed cells Collect Images Live/Dead 3Thursday, March 1 st Friday, March 2 nd Collect Images BME 245/445 – Lab 3