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B IOMATERIALS L AB #3 Cell culture and cell-(bio)material Interactions

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. P RE-LAB. Sign-in Quiz (Benoit. 2005). BME 245/445 – Lab 3. L INEAR M OLECULES. PEGMA Acr-PEG-RGDSG.

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B IOMATERIALS L AB #3 Cell culture and cell-(bio)material Interactions

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  1. BIOMATERIALS LAB #3Cell 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 Modified from (Lin, C.C., Pharmaceutical Research. 26(3): 631-643. 2009) BME 245/445 – Lab 3

  4. SECTION 1- Done before lab PEGMA Network Synthesis (done before lab – as previous) • 10wt% PEGMA in PBS • +/- 5 mM Acr-PEG-RGDSG • 10% photo-initiator by volume (LAP) • 0.75 mL Placed between two glass slides with spacer • Exposed to UV-lamp for 10min • Remove, punch out cylindrical samples • 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. 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

  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 • 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

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

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

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