Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

Summary

An original experimental setup for heating cells in a culture dish using 1.94 µm continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

Abstract

An original method to heat cultured cells using a 1.94 µm continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 µm is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.

To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.

The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

Introduction

Understanding temperature-dependent cell biological responses is of great importance to successful hyperthermia treatments. Retinal laser photocoagulation with a thermal laser, used in ophthalmology, is one of the most established laser treatments in medicine. Visible light, mostly from green to yellow wavelengths, is used in retinal laser treatment. The light is highly absorbed by the melanin in retinal pigment epithelial (RPE) cells, which form the outermost cell monolayer of the retina. There has been recent interest among physicians and researchers in very mild thermal irradiation (sub-visible photocoagulation) as a new therapeutic strategy for different kinds of ....

Protocol

1. RPE Cell Culture

1. Isolation of RPE cells from porcine eyes
   1. Obtain freshly enucleated porcine eyes from the local slaughterhouse. Keep them cool (4 °C) and in a dark environment.
   2. Remove extracellular tissues with scissors and soak the eyes in an antiseptic solution for 5 min.
   3. Place the eyes in sterilized phosphate-buffered saline without calcium and magnesium (PBS (-)) until use.
   4. Using a scalpel, penetrate the sclera at about 5 mm posterior to the corneal limbus. Resect the whole anterior part of the eye by cutting with scissors all way through, parallel to the corneal limbus.
   5. Remove....

Results

Temperature distribution after different power settings

All temperature developments for each single irradiation were monitored in the temperature calibration. From this data, the maximal temperature at the measured point was obtained and defined as T_max (°C). As shown in Figure 3A, the program was executed at the time point when the culture dish was placed on the heating plate. After the 14.......

Discussion

In discussing temperature-related biological cellular responses, not only the temperature, but also the time duration of the increased temperature, is of importance, since most biochemical processes are time-dependent. Particularly in the field of laser-induced hyperthermia in ophthalmology, due to the short time range-from milliseconds to seconds-it is difficult to investigate cellular thermal effects with precise temperature control. Therefore, a laser irradiation setup suitable for the cell culture model and with an o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Dulbecco’s Modified Eagle’s Medium - high glucose	Sigma-Aldrich	D5796-500ML	Add (2)-(4) before use. Warm in 37 °C water bath before use.
Antibiotic Antimycotic Solution (100 ×)	Sigma-Aldrich	A5955-100ML	Containing 10,000 units penicillin, 10 mg streptomycin and 25 μg Amphotericin B in 1mL. Add 5.5 mL in 500 mL medium bottle (1) before use.
Sodium pyruvate (100 mM)	Sigma-Aldrich	S8636-100ML	Add 5.5 mL in 500 mL medium bottle (1) before use (final concentration: 1 mM)
Porcine serum	Sigma-Aldrich	12736C-500ML	Add 50 mL in 500 mL medium bottole (1) before use (final: 10%)
Phosphate Buffered Saline (PBS)	Sigma-Aldrich	D8537-500ML
Trypsin from porcine pancreas	Sigma-Aldrich	T4799-25G
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	ED-100G
Human VEGF Quantikine ELISA Kit	R&D System	DVE00
Oxiselect Total Glutathione Assay Kit	Cell Biolabs, Inc	STA-312
Apoptotic/Necrotic/Healthy Cells Detection Kit	PromoKine	PK-CA707-30018
Name	Company	Catalog Number	Comments
Equipments
Thulium laser	Starmedtec GmbH	Prototype	0-20 W
365 mm core diameter fiber	LASER COMPONENTS Germany	CF01493-52
Thermocouple	Omega Engineering Inc	HYP-0- 33-1-T-G-60-SMPW-M
Heating plate	MEDAX
Microplate reader (spectrofluorometer)	Molecular Device	Spectramax M4
cell homogenizer	QIAGEN	TissueLyser LT
Fluorescence microscope	Nikon	ECLIPSE Ti
mathematical software program	The Mathworks. Inc	MATLAB Release 2015b
system-design platform	National Instrument	Labview	Laboratory Virtual Instrument Engineering Workbench