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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. - PowerPoint PPT Presentation
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BIOMATERIALS LAB #3Cell 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
BME 245/445 – Lab 3
Modified from (Lin, C.C., Pharmaceutical Research. 26(3): 631-643. 2009)
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)
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
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
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
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
IMAGEJ
BME 245/445 – Lab 3
Draw line of known lengthSet global scale
Future measurements are calibratedCan add scale bars
IMAGEJ
Polygon Selection (for Area)
Line Selection (for Diameter)
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 Responsibilities0 Monday, Feb 27th Tuesday, Feb 28th Feed cells
Collect Images
1 Tuesday, Feb 28th Wednesday, Feb 29th Feed cellsCollect Images
2 Wednesday, Feb 29th Thursday, March 1st Feed cellsCollect ImagesLive/Dead
3 Thursday, March 1st Friday, March 2nd Collect Images
BME 245/445 – Lab 3