BIOMATERIALS LAB 3 Cell culture and cellbiomaterial Interactions

BIOMATERIALS LAB #3 Cell culture and cell-(bio)material Interactions Monday, February 27 th and Tuesday, February 28 th 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. 10 wt% PEGMA in PBS 1. +/- 5 m. M Acr-PEG-RGDSG 2. 10% photo-initiator by volume (LAP) 3. 0. 75 m. L Placed between two glass slides with spacer 4. Exposed to UV-lamp for 10 min 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) § § § Sterile Techniques Feeding cells Counting cells Seeding cells (TC plastic / PEG+RGDSG) Monitor cell-material interactions over time… (Image. J) § # 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 (Image. J) § 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 § § § § Gloves! Spray everything down with Et. OH 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 § Et. OH to sterilize BME 245/445 – Lab 3

IMAGEJ 1. 2. 3. 4. 5. 6. Open Image. J 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 Responsibilities 0 Monday, Feb 27 th Tuesday, Feb 28 th Feed cells Collect Images 1 Tuesday, Feb 28 th Wednesday, Feb 29 th Feed cells Collect Images 2 Wednesday, Feb 29 th Thursday, March 1 st Feed cells Collect Images Live/Dead 3 Thursday, March 1 st Collect Images Friday, March 2 nd BME 245/445 – Lab 3