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International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

In-vitro Study of MIRB Labeled Ovine Bone Marrow Derived Mesenchymal Stem Cells by MRI Technique R. Gnanadevi1, Geetha Ramesh2, T. A. Kannan3, B. Justin William4, G. Sathyan 5and Sabiha Hayath Basha6 Department of Veterinary Anatomy, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007 INDIA 1- Ph.D Scholar; 2,6-Professor, Dept. of Veterinary Anatomy; 3,4-Professor, Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, Chennai; 5- Associate Prof., Dept. of Radio-diagnosis, Govt. Stanley Medical College, Chennai *Corresponding author: [email protected] Rec. Date:

Aug 22, 2016 21:38

Accept Date:

Sep 17, 2016 08:28

Published Online:

September 19, 2016

DOI

10.5455/ijlr.20160917082803

Abstract The study involved labeling Ovine bone marrow derived mesenchymal stem cells (oBM-MSCs) using Molday Ion Rhodamine-B (MIRB). In-vitro culture of expanded oBM-MSCs from passage 4 (P-4) to passage 6 (P-6) were used for the study. Intracellular MIRB distribution, cell viability and in-vitro tracking of the labeled cells using MRI was performed. The cultured oBM-MSCs were labeled with MIRB at the concentration of 25µg Fe/ml in DMEM. Internalized MIRB was observed only after 72 hrs of post-incubation and showed decreased intensity on subculturing. MIRB labeled cells showed positive reaction with Prussian blue staining demonstrating the iron uptake of the cells. There was no significant difference in viability of MIRB labeled oBM-MSCs when assessed by 0.4% typan blue exclusion test. The viability of the MIRB labeled oBM-MSCs ranged between 98-99 per cent. The T2 weighted images of MIRB labeled MSCs showed increased signal intensity with increasing concentrations of SPIOs. Key words: Labeling, Bone Marrow Derived Mesenchymal Stem Cells, Ovine, MRI Imaging

How to cite: R., G., Ramesh, G., A., K. T., B., J. W., G., S. & Basha, S. H. (2016) In-vitro Study of MIRB Labeled Ovine Bone Marrow Derived Mesenchymal Stem Cells by MRI Technique. International Journal of Livestock Research, 6 (9), 38-48.doi:10.5455/ijlr.20160917082803

Introduction Stem cells were described as a subset of cells, showed definitive stem cell characteristics, included adherence to tissue culture plastic, multipotency upon in-vitro expansion and self-renewal capacity over

regeneration of mesenchymal tissue such as bone. Various studies have been published demonstrating the

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bone-building capacity of mesenchymal stem cells and even their usefulness in treating critical size bone

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the long term (Zuk, 2012).Mesenchymal stem cells (MSC) are an attractive cell population for

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Vol 6 (9) Sept’16

International Journal of Livestock Research eISSN : 2277-1964

defects. Most of these studies were conducted with MSC derived from bone marrow (BMSC) (Niemeyer et al. 2010). Sheep is an ideal model for bone tissue engineering and has been proposed as an animal model for a wide range of applications in biomedical research, such as for the studies of respiratory diseases, cardiomyopathies, neurological disorders and prion diseases (Lyahyai et al., 2012). To understand the mechanisms behind a successful stem cell-based therapy, monitoring of transplanted cell's migration, homing as well as the engraftment efficiency and functional capability in-vivo has become a critical issue (Henning et al., 2009, Shen et al., 2013). Tracking methods should ideally be noninvasive, high resolution and allow tracking in three dimensions. Molday ION Rhodamine-B™ (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non-phagocytic cells. It is also visualized by both MRI and fluorescence microscopy and assessed the potential for imaging and monitoring of MSCs transplantation (Addicott et al., 2011). Hence, the present study is designed to label the BM-MSCs using MIRB and its visualization using MRI technique. Materials and Methods Preparation of Cells for Labeling Ovine Bone marrow derived mesenchymal stem cells (oBM-MSCs) were separated from femur by density centrifugation method using percoll as per Bourzac et al. (2010). These MSCs were cultured and expanded using Dulbecco’s Modified Eagle’s Medium with fetal bovine serum and antibiotic-antimycotic solution as reported by Polisetti et al. (2010). The oBM-MSCs monolayer was typsinised and sub cultured once it attained 70 per cent confluency until passage 6 (P-6). Plates of oBM-MSCs, from passage 4 to passage 6 with 70 per-cent confluency were used for labelling (Castaneda et al., 2011 and Jasmin et al., 2012). The sample design was given in the Table 1. Table1: Sample Design S. No 1. 2. 3.

Tests Intracellular MIRB distribution Viability In-vitro MRI tracking Total

No. of Samples Test 6 6 6 18

Control 6 6 6 18

Labeling of oBM-MSCs

with an interval of 24 hrs, 48 hrs and 72 hrs (Shen et al., 2013). After 72hrs of incubation, the spent media contained MIRB solution was removed by aspiration. The cells were washed twice with [email protected]

DOI 10.5455/ijlr.20160917082803

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(Addicott et al., 2011 and Ren et al., 2011) at 37°C with 5 per-cent CO2 and monitored for the integration

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The BM-MSCs at P4 were cultured in labeling solution with a concentration of 25 µg Fe per ml

International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

buffered saline (GIBCO) to remove extracellular MIRB. The MIRB labeled oBM-MSCs were used for subsequent experiments. Intracellular MIRB Analysis Loading properties of MIRB for BM-MSCs were evaluated for intracellular MIRB localization and distribution using fluorescent microscope (Olympus) (Addicott et al., 2011) and Prussian blue staining. The unlabelled and MIRB labelled oBM-MSCs (72hrs MIRB incubation) of all the three passages were fixed with biopal fixative and further subjected to prussian blue staining to demonstrate the iron uptake of the cells (Natalio et al., 2014). In-vitro MRI Detection of Iron in Phantom Design The labeled oBM-MSCs were dissociated using 0.25% of Trypsin-EDTA solution (GIBCO) as per Polisetti et al. (2010). For in-vitro MRI evaluation of MIRB labeled oBM-MSCs, phantom models were used. Phantoms were constructed as groups of 10 ml sample tubes (Borosilicate glass culture tubes) containing MIRB labeled BM-MSCs suspended in agar. 72 hrs post-incubation, the MIRB labeled oBMMSCs were centrifuged into a pellet and resuspended with melted 0.5 per-cent agarose and sandwiched between two layers of 0.5 per-cent plain agar. Each tube contained 1 × 106 MIRB labeled 0BM-MSCs. Phantom models of unlabelled oBM-MSCs also prepared in the same manner. The tubes were imaged with an eight-channel phased-array head coil on a clinical 1.5 T Siemens Symphony MRI unit (Addicott et al., 2011; Fan et al., 2013). Cell Viability Cell viability was assessed by Trypan blue exclusion test using the formula i.e unstained cells / total number of cells x 100 as per Nan et al. (2013). The viability of labeled and unlabelled cells was analyzed by chi-square test statistically as per standard protocol mentioned in Snedecor and Cochran, (1994). Results and Discussion Intracellular MIRB Distribution 1. Fluorescence of Rhodamine After 24 hrs post-incubation, these labeled cells were examined under fluorescent microscope under green filter with an excitation and emission wavelength of 555 and 565nm, respectively. No fluoresce of MIRB

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Labeled cells did not exhibit any fluorescence even 48 hrs of post-incubation (Fig 1).

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could be detected within the cytoplasm of cells of all the three passages P-4, P-5 and P-6 of BM-MSCs.

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International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

Fig 1: Fluorescence Image of oBM-MSCs Labeled with MIRB-48 hrs Post Incubation [X200] After 72 hrs of incubation, internalized MIRB was observed as fluorescent red colored intracytoplasmic inclusion in oBM-MSCs of all the three passages viz., P-4 (Fig 2), P-5 (Fig 3) and P-6 (Fig 4). Some of the MIRB labeled cells from each passage namely P-4, P-5, P-6 were used for further subculturing and observed that the intensity of fluorescence cells found to decrease on subculturing (Fig 5).

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Fig 2: Light Microscopic (left) and Fluorescence Image (right) of MIRB Labeled oBM-MSCs (P-4) showing MIRB Localized in the Cytoplasm [X200]

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International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

Fig 3: Light Microscopic (left) and Fluorescence Image (right) of MIRB Labeled oBMMSCs (P-5) Showing MIRB Localized in the Cytoplasm [X200]

Fig 4: Light Microscopic (left) and Fluorescence Image (right) of MIRB labeled oBM-MSCs (P-6) Showing MIRB Localized in the Cytoplasm [X200] 2. Prussian Blue Staining The unlabelled cells did not show any intracytoplasmic inclusion (Fig 6) whereas, the MIRB labeled cells of passages 4,5 and 6 showed positive blue reaction to Prussian blue (Fig 7). The labeled cells on subculturing, intensity of Molday ion reaction with Prussian blue stain

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was found to be reduced as the amount of intracytoplasmic MIRB got depleted.

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International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

Fig 5: Photomicrograph Showing Unlabelled BM-MSCs (P-4) [Prussian blue stainingX200]

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Fig 7: T2 Weighted MRI Image Showing MIRB Labeled BM-MSCs Pellet sandwiched between 0.5% agar

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Fig 6: Photomicrograph of MIRB Labeled BM-MSCs (P-4) [Prussian blue stainingX200]

International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

MRI Tracking MR signal in control group of cells was similar to that of water. The signal intensity in MIRB-labeled cells at passage 4 decreased with increasing concentrations of SPIOs. The T2 weighted images of MIRB labeled MSCs increased with increasing concentrations of SPIOs (Fig-8).

Fig 8: Trypan Blue Exclusion Test of MIRB Labeled BM-MSCs (P-5) x 100 Viability Assay In the present study, the viability of labeled BM-MSCs after 72hrs of MIRB incubation at P4, P5, P6 were given in Table 2. There was no significant difference in the viability of MIRB labeled BM-MSCs compared to the unlabeled BM-MSCs.. The viability of the MIRB labeled BM-MSCs ranged between 9899 per cent.

= Labeled Unlabeled

2.25 2.95

Labeled Unlabeled

3.08 2.43

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2.28 2.99

98.51 98.53

3.11 2.45

99.00 99.00

3.215957114NS 0.030 0.024

=

Per-cent of Viable Cells 98.75 98.75

0.000507241NS 0.034 0.044

= P6

Total Cells (in 106) 3.22 3.13

0.907691248NS

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P5

Non-Viable Cells (in 106) 0.040 0.039

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Table 2: Viability in MIRB labeled BM-MSCs Viable Cells (in 106) Labeled 3.18 P4 Unlabeled 3.10

DOI 10.5455/ijlr.20160917082803

International Journal of Livestock Research eISSN : 2277-1964

Vol 6 (9) Sept’16

Discussion The monolayered cells were used at 70-90 percent confluency for labeling, to track the cells in-vitro in the present work as stated by Bennewitza et al. (2012) and Blaber et al. (2013). Labeling was done by using superparamagnetic iron oxide nanoparticle (SPIO) conjugated with Rhodamine-B fluorescent dye in the present study as the iron oxide nanoparticles are concurrently demonstrated to be useful for specific invivo targeting applications, such as MRI tumor detection by way of antibody-coated particles (Renshaw et al., 1986 a) and passive targeting studies (Renshaw et al., 1986 b) and Saini et al., 1987). In the present study, when the cells reached 70 percent confluency, the cells were trypsinized and sub cultured as mentioned by Taylor and Clegg (2011) who worked on equine BM-MSCs and Ooi et al. (2013) in mice BM-MSCs. Intracellular MIRB Distribution 1. Fluorescence of Rhodamine MIRB used in the present study had uniform labeling of the cells and found to be an ideal tagging material without affecting the cell proliferation as per Shen et al. (2013). In the present study, labeling was found to be efficient at a concentration of 25µg Fe/ml in DMEM in contrast to the findings of Addicott et al. (2011), Ren et al. (2011) and Shen et al. (2013) who mentioned 20µg Fe of MIRB/ml of culture media. Similarly, intracytoplasmic localization of MIRB was observed after 72 hrs as per Shen et al. (2013) and in contrast to Addicott et al. (2011) and Ren et al. (2011) who observed localization after 24 hrs of incubation. In the present study, the intensity of the fluorescence in the labeled cells was found to decrease when the cells were subcultured. 2. Prussian Blue Staining In the present study, the unlabeled cells did not show any intracytoplasmic reaction whereas, the MIRB labelled cells of all the passages showed positive blue reaction to prussian blue staining

as per

Schmidtke-Schrezenmeier et al. (2011), Nan et al. (2013) and Shen et al. (2013). In the subsequent passaging of labelled cells, the intensity of iron reaction with Prussian blue stain was found to be reduced as the amount of intracytoplasmic MIRB got depleted. Viability Assay In the present study, the viability of the MIRB labeled BM-MSCs ranged between 98-99 per-cent as per

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Lee et al. (2009), McFadden et al. (2011), Nan et al. (2013), Shen et al. (2013) and Talaie et al. (2015).

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Vol 6 (9) Sept’16

Magnetic Resonance Imaging (MRI) In the present study, it was observed that on the whole that the internalization of the iron nanoparticle did not affect cell growth and its viability. However, there has been an evidence to suggest that endocytic lifecycles can be affected due to nanoparticles residing in endosomes (Arbab et al., 2005). Summary Internalized MIRB was observed only after 72 hrs of incubation of the cells. The MIRB labeled cells showed decreased intensity when subcultured. Prussian blue staining was done to demonstrate the iron uptake of the cells. MIRB labeled cells showed positive reaction whereas; the unlabelled cells did not show any positive reaction. There was no significant difference in the viability of MIRB labeled BMMSCs and ADMSCs compared to the unlabelled BM-MSCs and ADMSCs, respectively. The signal intensity in MIRB-labeled cells at passage 4 decreased with increasing concentrations of SPIOs. It is concluded that SPIONs could be used to label BM-MSCs at various passages and they emit signals sufficient to pick up at MRI T2 weighted images. Acknowledgement The author acknowledges the Professor and Head for permitting to utilize the facilities available at the Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, Chennai-7 and Govt. Stanley Medical College for permitting to utilize the MRI facilities.

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1. Addicott B, Willman M, Rodriguez J, Padgett K, Han D, Berman D, Hare JM and Kenyon NS. 2011. Mesenchymal stem cell labeling and in vitro MR characterization at 1.5T of new SPIO contrast agent: Molday ION Rhodamine-B™ Contrast Media. Mol Imaging. 6: 7-18. 2. Arbab AS, Wilson LB, Ashari P, Jordan EK, Lewis BK and Frank JA. 2005. A model of lysosomal metabolism of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles: implications for cellular magnetic resonance imaging. NMR Biomed. 18(6): 383–389. 3. Bennewitza MF, Tanga KS, Markakisb EA and Shapiro EM, 2012. Specific chemotaxis of magnetically labeled mesenchymal stem cells: Implications for MRI of glioma. Mol Imaging Biol. 14(6): 676–687. 4. Blaber SP, Hill CJ, Webster RA, Say JM, Brown LJ, Wang SC, Vesey G and Herbert BR. 2013. Effect of Labeling with Iron Oxide Particles or Nanodiamonds on the Functionality of AdiposeDerived Mesenchymal Stem Cells. PLOS ONE. 8 (1): e52997. 5. Bourzac C, Smith LC, Vincent P, Beauchamp G, Lavoie JP and Laverty S. 2010. Isolation of equine bone marrow derived mesenchymal stem cells, a comparison between three protocols. Equine Vet J. 42(6): 519-27. 6. Castaneda RT, Khurana A, Khan R and Daldrup-Link HE. 2011, Labeling stem cells with ferumoxytol, an FDA-approved iron oxide nanoparticle. J. Vis. Exp. 10: 3791-3482. 7. Fan J, Tan Y, Jie L, Wu X, Yu R and Zhang M. 2013, Biological activity and magnetic resonance imaging of superparamagnetic iron oxide nanoparticles-labeled adipose-derived stem cells. Stem Cell Res Ther. 4: 44.

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25. Talaie T, Pratt SJP, Vanegas C, Xu S, Henn RF, Yarowsky P and Lovering RM. 2015. Site-specific targeting of platelet-rich plasma via superparamagnetic nanoparticles. Orthop J Sports Med. 3(1): doi: 10.1177/2325967114566185. 26. Taylor SE and Clegg PD. 2011. Collection and Propagation Methods for Mesenchymal Stromal Cells. Vet Clin Equine. 27(2011): 263–274. 27. Zuk P. 2012. Adipose-Derived Stem Cells in Tissue Regeneration: A Review. ISRN Stem Cells. 2013: 1- 35.

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