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