Transcript Effects of THz exposure on Human Primary Keratinocyte

Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Clothier, R.H., and Bourne, N.
School of Biomedical Sciences, Faculty of Medicine and Health
Sciences, University
of Nottingham, Nottingham. NG7 2UH, UK.
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Primary human keratinocytes driven, in vitro, to differentiate via
activation of transglutaminases, results in transglutaminase
regulated cross linking of specific amino acids with cornified
envelope formation monitored via the incorporation of
fluorescein cadaverine, as a substitute for L-Lysine (Gray et al.,
1999).
This differentiation endpoint was co-assayed with the
keratinocytes reductive capacity for converting resazurin to
resorufin, as a measure of cell activity/viability (Andrew et al.,
1997; Rasmussen 1999; O’Brien et al., 2002; Gray and Clothier
2001).
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Materials and Methods
Primary Human Keratinocytes isolated from human skin, donated with patient consent. The
primary keratinocytes were passaged once, then the cells were seeded (passage 2) from a
5x104/ml suspension, at 100µl per well into a 96 well plate.
The plates were placed into a thermal box at room temperature (approx. 22°C) and transported
to the THz source in HBSS.
THz Exposure
This took place at TeraView Ltd, Cambridge, or The Institute of Microwaves and Photonics,
University of Leeds. The keratinocytes were exposed to the THz through the base of the 96 well
plate in clusters of 4 wells per exposure time. These both used a Ti:Sapphire laser impacting on
an electro-optic photoconverter to generate THz power. The Leeds system had a total pulse
duration is 20-30ps, (90% delivered within two ps). The average output power for this
(unamplified) system is approximately 1W within the frequency range 0.2 – 3.0THz. The
repetition rate of the THZ pulse is approximately 80MHz .
At Teraview by optical excitation of a gallium arsenide wide aperture antenna. A large DC-bias
applied across the device which was excited using a Ti:Sapphire laser emitting 250 fs pulses
centered at a wavelength of 800 nm, with a 250kHz repetition rate. Frequency range of 0.1 THz
to 2.7 THz, average power of approximately 1 mW. The THz-radiation was focused paraobla
mirror on to the sample with a spot size of 130 µm to 3.7 mm. The THz spot was raster scanned
over sample for the duration of the exposure.
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Materials and Methods Cont.
Prior to the exposure to the THz, a resazurin (Sigma, Poole, Dorset) assay was performed.
Cells were exposed for periods of 10, 20 or 30 minutes, giving a total exposure of 0.15, 0.3 or
0.45mJ/cm2 or 0.15, 0.3 or 0.45J/cm2 per 4 wells.
Two sets of 4 control wells were exposed to the same conditions but not the THz beam. The
plates were returned to the thermal box and transported back (2 hours). A resazurin assay was
then performed and again at 3, 6 and 8 days. Following the initial post exposure resazurin assay
the keratinocytes were returned to Greens Medium (Gray et al., 1999) containing 20µM/ml
fluorescein cadaverine (Sigma; Gray et al., 1999; Gray & Clothier., 2001) to stimulate and
quantify differentiation.
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Methods
Grow to confluence
Isolate Human
Keratinocytes
Seed to 96 well plates,
5000 per well
Perform Resazurin assay
Return at 23 C & transport to
Nottingham (2 hrs)
Resazurin assay
THz Exposure for 10,
20 or 30 mins
Place in HBSS at 23 C & transport
to THz source (2 hrs)
Resazurin assay at 3 days then
into Green’s Medium with 20µm
Fluorescein Cadaverine
8 days post exposure Resazurin
and FC assayed Fix cells.
6 days post exposure Resazurin
and FC assayed (return to Greens
Medium with 20µm FC)
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Typical coblestone appearance of keratinocytes.
Fluorescein Cadaverine assays differentiated
cells green
Resazurin assay with cells converting it
to resorufin being red. Confocal image.
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Figure 1b. Effects of 1-3THz radiation upon
human primary keratinocyte activity (Leeds)
Figure 1a. Effects of 1-3THz radiation upon
human primary keratinocyte activity (Cambs)
200
200
180
Percent of control cell activity
Percent of control cell activity
180
160
140
120
100
80
60
40
20
160
140
120
100
80
60
40
20
0
0
post
3days
6days
8days
post
Time after exposure
non-exposed
0.15J/cm2
0.30J/cm2
0.45J/cm2
3days
6days
8days
Time after exposure
non-exposed
0.15mJ/cm2
0.30mJ/cm2
0.45mJ/cm2
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Figure 2. Effects of 1-3THz radiation upon human
primary keratinocyte differentiation via FC uptake.
Percent of control Fluorescein Cadaverine
uptake
140
120
100
80
60
40
20
0
6days
8days
Time after exposure
non-exposed
0.15J/cm2
0.30J/cm2
0.45J/cm2
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
Figure 3. Effects of 1-3THz radiation upon human
primary keratinocyte differentation.
Percent of control Fluorescein Cadaverine
incorporation
140
120
100
80
60
40
20
0
6days
8days
Time after exposure
non-exposed
0.15J/cm2
0.30J/cm2
0.45J/cm2
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
•The THz from the two sources gave comparable results with no signs of induced
stress to the cells, over the subsequent culture period.
•From previous results and the database on FC incorporation rates, it has been
shown that there is a rise in FC uptake in normal keratinocytes from <20pg per well
to 111 ± 38pg per well on 3 and 9 days respectively.
•The results presented, with an average of 184 ± 85pg per well were in line with
these results.
•The differentiation capacity was as expected for all three donors, exposed to the
higher THz levels up to 0.45J/cm2. This was also true for the donors exposed to the
lower energy levels.
•Thus the THz caused not detectable change in the activity or differentiation
capacity of confluent primary human keratinocytes from the basal and stem cell
population of the stratum basale of the epidermis.
•Dividing cells will need to be examined.
Effects of THz exposure on Human Primary Keratinocyte
Differentiation and Viability.
We acknowledge the funding under the FW5 programme THz-Bridge (QOL-20004.2.2), and the assistance of the FRAME Research Laboratory staff, THz generation
under the control of Emma Pickwell, Cambridge University or Vincent Wallace and
Philip Taday, Teraview, Cambridge Science Park, Cambridge, or Dr. M.A.Naftaly and
Prof. N.N.Zinovev, Teravision, ( Institute of Microwaves & Photonics), School of
Electronics & electrical Engineering, The University of Leeds, Leeds LS2 9JT (funded
under the EPSRC and by the EU as part of the Teravision programme (IST-1999-10154).