Low power laser and LED irradiation effect on proliferation and differentiation of Wistar rats mesenchymal stem cells

Low power laser and LED irradiation effect on proliferation and differentiation of Wistar rats mesenchymal stem cells

Diana Mancera(a), Efrain Solarte*(a), Leonardo Fierro (b), William Criollo(b) a Quantum Optics Group, Department of Physics, Universidad del Valle, AA 25360, Cali, Colombia

b Pharmacology Group, Heath Sciences School, Universidad del Valle, AA 25360, Cali, Colombia

ABSTRACT

It has been demonstrated that appropriately cultured and stimulated mesenchymal cells, can give rise to cells of all tissues of the body. We evaluate the cell proliferation and differentiation induced by low power light irradiation in cell cultures of mesenchymal cells, isolated and previously characterized, from Wistar rats. Roche® XTT and LDH tests were used to assess proliferation and cytotoxicity. Cellular differentiation was determined by optical microscopy and using specific fluorescent markers. We report laser cellular proliferation enhancement by 532 and 473 nm, and the best cell culture response by a dose of 2 Jcm-2. Although a three day irradiation protocol the cultures grown and no cytotoxicity was detected. Cellular differentiation occurred, and the production of cardiomyocytes was promoted by the cell proliferation stimulated by low power laser irradiation.

Keywords: Low Power Laser Irradiation, Cell Differentiation, Mesenchymal Stem Cells, Cardiomyocytes

1. INTRODUCTION Cell therapies comprise a set of treatment strategies to handle dreaded diseases and in the last decades have achieved important clinical advances, as well as exploring and defining new concepts, developing new technologies and developing new models and therapies for human diseases treatment. Although no all trials have got the hoped results, cell therapy is consider one the most promising medical advance in this century to treat diseases such as Alzheimer's, Parkinson's, diabetes, stroke, and traumatic neurological injuries. One important issue is to study and produce cellular lines capable to be used in order to produce different other cells, and in this field is a main task to find and define the procedures to produce this cellular differentiation. It has been demonstrated that appropriately cultured and stimulated mesenchymal cells, can give rise to cells of all tissues of the body. Although laser irradiation has been used in different forms of therapy as the Low Level Laser Therapy, for healing and pain relief treatments, and these applications are more or less known and extended1, 2 other possibilities of laser biomedical applications as cell culture stimulation and cell proliferation modulation are only in the recent times explored 3, 4, 5, 6. In particular, cell proliferation modulation via laser irradiation has been studied in the last years and these studies have demonstrated the role of grow factors in the activation of the proliferation, nevertheless it was only very recently7 that the fluorescent imaging techniques allowed the following of the cellular activity to study and discuss the mechanisms associated with the cellular physiology changes induced and stimulated by the low power laser irradiation. Moreover8, cell proliferation is enhanced, and this enhancement is important to produce cell differentiation from stem cells

In our country, as the National Department for Statistics (DANE) and the Pan-American Health Organization found9, cardiovascular disease is the most important death cause, over violent death or cancer victims. When a person survives a heart attack, it is found that some of the myocardium tissue has suffered permanent injury, because its regeneration capacity is limited. Recent studies suggest cell therapy as an alternative for other promising for such problems, and it has been shown that adequately cultivated and stimulated mesenchymal cells (mMSC's), may give rise to cells of all tissues of the body.

8th Iberoamerican Optics Meeting and 11th Latin American Meeting on Optics, Lasers, and Applications, edited by Manuel Filipe P. C. Martins Costa, Proc. of SPIE Vol. 8785, 8785DY

© 2013 SPIE · CCC code: 0277-786X/13/$18 · doi: 10.1117/12.2021766

Proc. of SPIE Vol. 8785 8785DY-1

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We performed this work to evaluate the cell proliferation and differentiation induced by low power laser irradiation in cell cultures of mesenchymal cells, isolated and previously characterized, from Wistar rats. Roche® XTT and LDH tests were used to assess proliferation and cytotoxicity and microscopic observation has been done continuously to follow the growth and the differentiation processes.

2. MATERIALS AND METHODS 2.1 Culture of mesenchymal stem cells from rat bone marrow (mMSC’s)

We use cells that remained stored in Liquid Nitrogen at -190 ° C, at our cell culture Laboratory (Section of Physiology, Faculty of Health Sciences, Universidad del Valle). The cells were previously characterized, and immediately seeded in a culture bottle T-75 with Knockout-DMEM medium, and kept at 37 °C and 5% CO2 atmosphere. The culture medium was renewed every 2 or 3 days in vitro. Cultured cells were observed continuously for controlling the level of confluence. Once 80% confluence was reached, cells were detached with 2 ml of Trypsin / EDTA 0.25% and incubating for 5 min at 37 °C. Trypsin was neutralized by adding an equal volume of complete medium (DMEM Knockout) and detached cells were counted Tripam blue.

For the irradiation procedure cells were seeded in 96-well microplates, at a concentration of 5000 cells per well, with 200μL growth medium for mMSC's. The medium was kept at least 48 hours until a 80% confluence was achieved. Then, the growth medium was changed by a differentiation medium for cardiomyocytes (Millipore) for three days. After this, the irradiation with two different laser types began.

Figure 1. A 96 well microplate with cultured cells, prepared for laser irradiation.

2.2 Laser Irradiation

We used as radiation sources a green laser DPSSL-Nd:YAG laser @532 nm, and a blue DPSSL @ 473 nm. The optical setup was minimal to avoid contamination, and the distances were chosen so that the laser spot area coincides with those of a culture well (0.32 cm2). The maximum available power was 33 mW for the green laser, and 63 mW for the blue one. The irradiation times were selected to give doses of 1, 2 and 3 Jcm-2 applied every day to selected cell-wells. The exposure times were 10 s, 19 s and 29 s for the green laser; and 5 s, 10 s and 15 s for the blue one. The total cell irradiation process was completed in 3 days.



2.3 Cytotoxicity assays

To study the possible cytotoxicity effects, the LDH Kit ® ROCHE (cytotoxic) was used. The reaction solution LDH kit was prepared according to the following procedure:

a) Dilute catalyst powder (ROCHE) in 1 ml of distilled and sterilized water.

b) Add 290 μl of this solution to 13.05 ml of dilute solution (dye solution ROCHE). For every 116 samples.

c) Add 100 μl of the LDH solution to each well of the culture plate.

The controls were established as following: a) Background Control: 150 μl compound growth medium without cells and LDH 50l. This control provides information about the LDH activity contained in the analysis medium, b) Low Control: 150 μl Composite cell growth medium and LDH 50l. This provides information about the activity of LDH released by normal cells untreated; and c) High Control: Composed of 150 μl cell growth medium that was in contact with 50 μl of Triton X-100 (cytotoxic agent) plus 100 μl of LDH. The High control provides information about the activity of maximum LDH release from the cells. The resume of the preparation procedure is shown in the Table 1

Table 1. Preparation of the LDH Cytotoxicity Assay Summary

Low Control ⇓

High Control ⇑ Background Samples

Medium+FCS 10% 150 μL 150 μL 150 μL 150 μL

LDH 50 μL 50 μL 50 μL 50 μL

Triton - + - -

Cells + + - +

The whole assay procedure, as it was carry out on the culture plates, is indicate in the following Table 2

Table 2. Cell cultures distribution on the plates with and without radiation, for cytotoxicity determination

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H



Gray lines show the positions with mesenchymal stem cells differentiated to cardiomyocytes. At columns 1B, 1D, 1F, the first dose was delivered using the blue and green Lasers on separate plates. On columns 5B, 5D, 5F, the second light dose from the blue and green Lasers, was delivery on separate plates. On columns 9B, 9D, 9F, the third dose from blue and green lasers was delivery on separate plates. Positions 1 to 6 H, were used for the Low Control; from 7 to 12H for the High Control, and from 10 to 12G were used for the background control. Two (2) different plates were used, one for each laser beam.

2.4 Cell proliferation assays (XTT® ROCHE Kit)

The XTT reactive solution was prepared using the following procedure:

Discard the culture medium solution from the wells of the culture plate. Mix 0.15 ml of the electrolyte solution (Roche) with 7.5 ml of labeling solution. Add 50 ml of the XTT solution to each well of the culture plate. For the proliferation assay a normal, laser free, control set was established. For all the cultures 200 µl of cardiomyocytes differentiation medium was used and 50µl of XTT solution was added to each well. The experimental conditions were stable and controlled to be: 37°C, 5 % CO2, and HR= 95%. Under this situation the maximum growth and a normal metabolic activity are warrantied. The Table 3 summarized the data for preparing the proliferation assays.

Table 3. Preparation of the Proliferation Assay Summary

Control Samples

Medium 200 μL 200 μL

XTT 50 μL 50 μL

FCS 10 % 10 %

Cells + +

The whole proliferation assay procedure, as it was carry out on the culture plates, is indicate in the following Table 4

Table 4. Cell cultures distribution on the plates with and without radiation, for XTT proliferation determination

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H



Gray lines show the positions with mesenchymal stem cells differentiated to cardiomyocytes. At columns 1B, 1D, 1F, the first dose was delivered using the blue and the green Lasers on separate plates. On columns 5B, 5D, 5F, the second light dose from the blue and the green Lasers was delivery on separate plates. On columns 9B, 9D, 9F, the third dose was applied from both lasers on separate plates. Positions 3H, 7H, 11H were used for proliferation control for the blue and green laser irradiated cells. Two (2) different plates were used, one for each laser beam.

3. EXPERIMENTAL RESULTS

3.1 Cytotoxicity assays

Cytotoxicity results are summarized Table 5, and in the Figure 2, as is shown the cultures do not exhibit cytotoxicity, all the results are smaller than +15% .

Table 5. Cytotoxicity percent in mesenchymal stem cell irradiated with low power lasers

Irradiation Dose [Jcm-2]

Green Laser Blue Laser

1 -8,6 -8,6 2 -1,5 -15,4 3 3,4 2,4

Figure 2. Cytotoxicity percent for mesenchymal stem cell irradiated with 532nm and 473nm DPSSL.

3.2 Cell Proliferation assays

For 3 days the cells were subjected to doses of 1, 2 and 3 J cm-2. Proliferation assays were performed on the 5, 6, 7 and 8 days. After120 hours from the last irradiation, there are not significant changes in cell proliferation observable at the different applied doses. But, after 144 hours, the cultures irradiated with the 2 Jcm-2 dose, exhibit the higher proliferation rate for both laser for types, as shown in Figure 3 for the cultures irradiated with the blue (A) and the green laser (B).



2,3

2,2

Cl..,, 1 _ 8

-- CONTROL-0-1 Jcm -2

-2 Jcm -2A 3 Jcm -2

1,7- / /.01,6 -

1,5 -

1,4 - -

110 120 130 140 150 160 170 180 190 200

Post irradiation Time [h]

2,6

2,4 -

2,2 -

2,0 -

1,8 -

1,6 -

1,4 -

-- CONTROL-0 -1 J /cm2-0-2 J /cm2

3 J /cm2

' ' ' 1 ' ' ' ' ' '110 120 130 140 150 160 170 180 190 200

Post irradiation Time [h]

2,3

2,2

2,1

C 2,07,

d 1,9EO , n

- Post Irratiation time [hours]- +120 h- -0-144 h_ -A-168h_ f 192 h

L 1,6o_

Ú 1'71,6

1,5

1,4 a0,0

_o/ _

i , i , i

0,5 1,0 1,5

i i

2,0 2,5 3,0

Irradiation Dose [J cm-2 ]

2,5 -

2,4 -

2,3 -

2,2 -

Ç 2,1 -:Pi 2,0 -a>w' 1 9 -

Post Irradiation time [hours]í 120h-0-144h-A- 168 h

h

L

C)AO

O

'0 -

0,0 0,5 1,0 1,5 2,0 2,5 3,0

Irradiation Dose [J cm-2 ]

Moreover, the cell proliferation assays show that all the irradiated samples grow following an expected behavior, but it is remarkable that the proliferation rate of those cultures irradiated with the 2 Jcm-2 remains the higher along all the measurement period.

A

B

Figure 3. The figure shows the time behavior of the measured cell proliferation. It is shown that all cultures grow, but whereas the control and the 1 and the 3 Jcm-2 irradiate cultures grow in a similar way, the cultures irradiated at 2 Jcm-2 grow at a higher rate. C

D

Figure 4. The figure shows the behavior of the cell proliferation with the irradiation dose, taking zero for the control cultures. The extraordinary behavior of the cultures irradiated with the 2 Jcm-2 is clearly demonstrated in both types of laser the blue 473nm laser presented in graph C, and the green 532 nm laser shown in graph D.

The comparison between the non-effect (control, 1 and 3 Jcm-2 doses) and the grown stimulated cultures at 2 Jcm-2 is shown in figure 5.



2,4

2,2

1,6

1,4

110 120 130 140 150 160 170 180 190 200

Post Irradiation Time [h]

U

110 120 130 140 150 160 170 180 190 200

Post Irradiation Time [h]

2,15

2,10

2,05

2,00

C 1,95r.d 1,90

A 1 4G

-0- Proliferation Average

L I V l

a= 180d '

U1,75

1,70

1,65i i i i i i

0,0 0,5 1,0 1,5 2,0 2,5 3,0

Irradiation Dose [ J cm-2 ]

2,3

2,2 -

2,1 -

2,0 -

c0 1,9 -toL

Proliferation Average

/I\1,6 -

1,5

s,

0,0 0,5 1,0 1,5 2,0 2,5 3,0

Irradiation Dose [ J cm-2 ]

D

E

Figure 5. The average of non-effect results are compared with the 2 Jcm-2 cases. Both laser types, D graph shows the blue laser results and E those of the green laser, caused a remarkable effect in the cell proliferation, distinguishable 140 h after the laser irradiation procedure.

In order to exclude the possibility of fluctuations, data from an Anova test of the proliferation measurements were taken and the average proliferation values and the variances are plotted to make visible the Anova test results. Figure 6 shows the result of this comparison, and allows considering that, although the control, 1 Jcm-2 and 3 Jcm-2 cases are statistically indiscernible, the 2 Jcm-2 dose produces a very real effect on the cell cultures. Graph F show the situation for the blue laser treated samples, and the graph G for the green laser case.

F

G

Figure 6. Graphical presentation that shows the statistical distinguishability of the 2 Jcm-2 irradiation results from the other cases, as it is explained in the text.



Finally, it is valuable report that the differentiation process is achieved in the cultures as shown in the figures 7 and 8.

Figure 7. Mesenchymal stem cells culture at the beginning of the cell differentiation process

Figure 8. Cardiomyocytes developed from mMSC’s cells

According to the graphs in figures 4 and 5, 120 hours after the irradiation procedure, there is not a significant difference between the time evolution of cells that received 1, 2 and 3 Jcm-2, however from the sixth day (144 hours) onwards, the effect of the 2 Jcm-2 dose, begins to be predominated, and it is in this way for both laser types, this dose enhances the cell proliferation. There is however a small difference between the results of both lasers and it is shown to be even more effective the green laser, because it presents a greater absorbance than the blue laser.

4. CONCLUSIONS We presented evidence of low power laser irradiation (LPLI) effects on cell cultures by laser light doses of 2 Jcm-2. The effects are notable and occurred at 532 nm as well as at 473 nm. Although doses of 1 and 3J cm-2, do not exhibit a big increase in the cell proliferation, these dose allowed the culture growth with a small increment compared with the normal control. On the other side, green laser light produces a higher increment in the cell proliferation as compared with the blue laser light.

Cellular proliferation and differentiation from Mesenchymal Stem cells to cardiomyocytes occurred in both control and irradiated samples, but the enhancement of cell proliferation promoted by the laser light allows considering the possibility of a cell culture protocol to control and to stimulate the production of differentiated cells from mMSC’s. This can be considered an important step in the way to reach an effective cellular therapy, especially in the case of myocardial infarction.

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Low power laser and LED irradiation effect on proliferation and differentiation of Wistar rats mesenchymal stem cells

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