Titanium Phosphate Glass Microspheres as Microcarriers for In Vitro Bone Cell Tissue Engineering - Biointerfaces: Where Material Meets Biology - page 117

Optical microscope (DMIRB microscope; Leica Microsystems, Germany)

NOTE: This microscope was procured together with the incubator sys-
tem, but conventional optical microscopes having a camera port and
appropriate adapters can be used.

d n 3 r 4 n g | 3

Pipette with a capacity of 10 mL (Finnpipette; Thermo Scientific, UK)
Pipette tips (10 mL; Thermo Scientific, UK)
Tissue culture flask, 25 cm 2 (TPP; Helena Biosciences, UK)
Type 1 ultra-pure water purification system capable of dispensing water
with a resistivity of 18.2 MO cm 1 (Purelab UHQ-PS; Elga Labwater, UK)
Weighing boats

5.3.3 Setting Up a Time-lapse Experiment
1. Switch on the incubator system about 2 h prior to setting up the ex-
periment and set the temperature to 37 1C. Just before the experiment,
switch on the desktop PC and load the mManager software.
2. Weigh 100 mg of glass microspheres on an analytical balance and pour
into a T-25 cell culture flask. Add 10 mL of ultra-pure water to the flask
using a pipette.

TIP: Prior to the addition of microspheres and water, the bottom inner
surface of the flask can be scraped with a brush in order to provide
surface roughness so that the microspheres remain reasonably station-
ary during the experiment. If this step is performed, the flask should
then be washed two to three times with ultrapure water in order to wash
away any debris resulting from the scraping action.

.

3. Place the flask on the microscope stage and adjust the vertical stage
position so that the microspheres are brought into focus on the soft-
ware display screen (the Live View window, which can be opened by
clicking on the Live option on the software interface). Adjust the
horizontal stage position so that the display screen shows approxi-
mately eight to ten microspheres.

TIP: It is advisable to focus on microspheres that do not touch each other
so that the image processing software will count the spheres as individual
objectsandprovidediametervaluesforeachsphere;thisisbecausespheres
touching each other may be interpreted by the software as a single object.

4. To begin the time lapse sequence, click on Multi-D Acq. On the fol-
lowing window, tick the boxes for Time Points and Save Images
(Figure 5.3). Below Time Points, list the number of images to be taken
and the interval between images. Below Save Images, write the file
pathway to be used to store the files. For Saving Format, click the op-
tion for Separate image files. Click on Acquire.