Prevention and Detection of Mycoplasma Contamination in Cell Culture

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Fibrosis

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* Corresponding Addresses: P.O.Box: 14515- 775, Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran

Email: kazem_ parivar@yahoo.com

P.O.Box: 16635-148, Department of Regenerative Medicine and Cell Therapy, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Email: Nasser.aghdami@royaninstitute.org

5HFHLYHG6HS$FFHSWHG-DQ

Abstract

2EMHFWLYHThe aim of this study was to test the effect of intravenous injection of mesenchy-PDOVWHPFHOOV06&VRQGR[RUXELFLQ'2;LQGXFHG¿EURVLVLQWKHKHDUW:HLQYHVWLJDWHG the mechanisms that possibly mediate this effect.

0DWHULDOV DQG 0HWKRGV,QWKLVH[SHULPHQWDOVWXG\¿EURVLVLQWKHP\RFDUGLXPRIDGXOWPDOH :LVWDUUDWVZHLJKWVJZHHNVRIDJHWRWDOQ ZDVFUHDWHGE\DOX administra-tion. DOXPJNJZDVDGPLQLVWHUHGLQWUDSHULWRQHDOO\WLPHVDZHHNIRUDWRWDOGRVHRIPJ kg over a period of 2 weeks. MSCs from Wistar rats were separated and cultured in Dulbecco’s PRGL¿HGHDJOHPHGLXP'0(07KHFRQGLWLRQPHGLXP&0ZKLFKFRQWDLQHGIDFWRUVVHFUHWHGE\ 06&VZDVDOVRFROOHFWHGIURP06&VFXOWXUHGLQVHUXPIUHH'0(07ZRZHHNVDIWHUWKH¿UVWLQMHF -tion of DOX, MSCs, CM and standard medium (SM) were transplanted via intravenous injec-tion. )RXUZHHNVDIWHUWUDQVSODQWDWLRQKLVWRORJLFDO0DVVRQ¶VWULFKURPHVWDLQLQJIRU¿EURVLVGHWHFWLRQ and molecular [real-time polymerase chain reaction (RT-PCR)] analyses were conducted. In addi-tion, insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF) in the CM were meas-ured with an enzyme-linked immunosorbent assay (ELISA). For immunosuppressive treatment, cyclosporine A was given (intraperitoneally, 5 mg/kg/day) starting on the day of surgery until the end of study in all groups. Fibrosis rate and relative gene expression were compared by analysis of vari-ance (ANOVA) and post-Tukey’s test. HGF and (IGF-1 in the CM were analyzed by independent VDPSOHWWHVW3ZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQL¿FDQW

5HVXOWV2XUGDWDGHPRQVWUDWHGWKDWLQWUDYHQRXVO\WUDQVSODQWHG06&VDQG&0VLJQL¿FDQWO\UH -GXFHG¿EURVLVDQGVLJQL¿FDQWO\LQFUHDVHG%FOH[SUHVVLRQOHYHOVLQWKHP\RFDUGLXPFRPSDUHGWR WKH'2;JURXSS+RZHYHUWKHUHZDVQRVLJQL¿FDQWGLIIHUHQFHEHWZHHQ%D[H[SUHVVLRQ OHYHOVLQWKHVHJURXSV,QDGGLWLRQVHFUHWLRQRI+*)DQG,*)ZDVGHWHFWHGLQWKH&0S

&RQFOXVLRQWe conclude that intravenous transplantation of MSCs and CM can attenuate P\RFDUGLDO¿EURVLVDQGLQFUHDVH%FOH[SUHVVLRQ7KLVPD\EHPHGLDWHGE\SDUDFULQHVLJQDO -LQJIURP06&VYLDDQWL¿EURWLFDQGDQWLDSRSWRWLFIDFWRUVVXFKDV+*)DQG,*)

Keywords: Mesenchymal Stem Cell, Doxorubicin, Heart, Apoptosis, Fibrosis Cell Journal(Yakhteh), Vol 14, No 2, Summer 2012, Pages: 142-151

FK\PDOVWHPFHOOVRQGR[RUXELFLQLQGXFHG¿EURVLV&HOO-Introduction

&DUGLDF¿EURVLVLVDPDMRUFDXVHIRUWKHSURJUHV -sion of heart failure, and its prevention is a critical

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antibiotic) is a useful anti-tumor agent for treating

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damage might increase in reactive oxygen spe-cies, which in turn causes oxidative harm to car-diac mitochondria, eventually leading to

apopto-VLVDQGP\RFDUGLDO¿EURVLV)RUPHUVWXGLHV

have demonstrated that cell transplantation could

DWWHQXDWH FDUGLDF ¿EURVLV RI WKH UHJLRQDOO\ LP -paired myocardium, such as those prevailing after

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shown promising results with mesenchymal stem

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related to their differentiation into reparative or

re-SODFHPHQWFHOOW\SHV6HYHUDOUHFHQWVWXG -ies have demonstrated that other mechanisms, in-cluding the regeneration of cardiomyocytes, may contribute to improved heart function (17). Recent reports have suggested that some of these repara-tive effects are related to paracrine factors secreted

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potentially repair damaged heart tissue (17). In this research, we investigate whether intravenous

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predict that paracrine signaling also participates in

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Materials and Methods

Animals

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were obtained from the Central Animal

Laborato-U\3DVWHXU,QVWLWXWH,UDQ$QLPDOVZHUHNHSWLQWKH &HQWUDO$QLPDO+RXVHRIWKH0D]DQGDUDQ8QLYHU

-VLW\RI0HGLFDO6FLHQFHXQGHUDKRXUOLJKW KRXUGDUNUHJLPHQDWÛ&IHGZLWKFRPPHU

-cial standard rodent chow, and allowed free access to water. All animal experimental protocols were

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-WHHRI0D]DQGDUDQ8QLYHUVLW\RI0HGLFDO6FLHQFH 0D]DQGDUDQ,UDQ$IWHUDWZRZHHNDFFOLPDWL]D -tion period, animals were randomly assigned

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,QGXFWLRQRIGR[RUXELFLQ'2;LQGXFHG¿EURVLV model

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Histological assessment of myocardial damage

)RXU ZHHNV DIWHU WKH ¿QDO LQMHFWLRQ RI '2; KHDUWV Q IURP HDFK JURXS ZHUH UDSLGO\ UH -moved and ventricles dissected from the atria. The

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sections were randomly obtained from three levels

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Olysai software) and the area of the collagenous fraction was calculated as the sum of all areas that contained connective tissue divided by the total area of the image.

RNA isolation and reverse-transcription

To evaluate apoptotic gene expression in the

my-RFDUGLXPIURPHDFKH[SHULPHQWDOJURXSQ SHU JURXSPJRIYHQWULFXODUWLVVXHZHUHGLVUXSWHG DQGKRPRJHQL]HGLQ,$]ROO\VLVUHDJHQW4LDJHQ

Europe). Total RNA was isolated using an RNeasy

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a Quantitect Reverse Transcription Kit (Qiagen,

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-IHFWLYHO\UHPRYHFRQWDPLQDWLQJJHQRPLF'1$$IWHU JHQRPLF'1$HOLPLQDWLRQ51$VDPSOHVZHUHSUH -pared for reverse transcription by using a master mix

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Quantiscript RT buffer, and RT primer mix. The

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were described earlier (Table 1).

Real-time polymerase chain reaction (RT-PCR) analysis

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gel electrophoresis and by melting curve analysis at

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in duplicate and the mean value of each duplicate was used for all further calculations. Reverse transcriptase minus samples and no template controls were run

to-gether with test samples. The level of expression of

HDFKJHQHZDVQRUPDOL]HGE\WKDWRIȕDFWLQUHIHU -ence gene). The relative expression ratios were calcu-lated by the 2±¨¨&7_{/LYDNPHWKRG2XWSXWGDWDZHUH}

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Mesenchymal stem cells (MSCs) isolation

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Germany). The cultures were then incubated in an

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Four days after plating, non-adherent cells were removed and adherent cells were propagated for four passages prior to transplantation.

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Flow cytometric analysis was used to determine the

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antigens. For this purpose, cells at the fourth passage

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Table 1 : Primer sequences, expected fragment sizes and annealing temperatures of apoptotic and housekeeping genes

Annealing Size (bp)

Primer sequences (5'-3') Name

Û&

F: GGATGACTTCTCTCGTCGCTACCGT R: CGAGTGAGGATGTGCATGAA

Bcl2

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F: CCAGGACGCATCCACCAAGAAGC R: TGCCACACGGAAGAAGACCTCTCG

Bax

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F: GAACCCTAAGGCCAACCGTG R: AGGCATACAGGGACAACACAGC

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marrow were differentiated into osteogenic and adi-pogenic cell lineages. For osteogenic differentiation,

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(22). For adipogenic differentiation, cells were ex-posed to adipogenic induction medium that consisted

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Preparation of MSC condition medium (CM)

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(Gibco, Germany). The medium from 10 _{cells was}

collected and centrifuged at 700 g for 10 minutes and

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Animal groups and transplantation of MSCs, MSCs-CM, and CM

'2;WUHDWHG DQLPDOV Q ZHUH UDQGRPO\ VXE -divided into four groups: i.group 1 which received

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and administered via the tail vein. Following injec-tions for immunosuppressive treatment, cyclosporine

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starting the day of surgery and continuing until the

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Evaluation of cell therapy

Histological assessment of myocardial damage after MSC and MSC-CM injections

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-MHFWLRQV KHDUWV Q IURP HDFK JURXS ZHUH UDS -idly removed and ventricles dissected from the atria. Transverse sections were randomly obtained from three levels (basal, middle, and apical) and

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mentioned above was performed.

RNA isolation and RT-PCR analysis

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from each group) were evaluated as mentioned above.

Enzyme-linked immunosorbent assay (ELISA): Analysis of MSC-CM

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from 10_{FHOOVDQGFHQWULIXJHGDWJDWÛ&IRU}

minutes and supernatants were stored at -20ÛC. Then, 100 µL of the supernatants were assayed for IGF-1

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Statistical analysis

Statistical analysis was performed using the

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Results

Model induction results

Pathological studies +LVWRORJLFDOFKDQJHVRIWKHP\RFDUGLXPLQYHVWL -JDWHGE\0DVVRQ¶VWULFKURPHVWDLQLQJUHYHDOHGWKDWWKH '2;JURXSVKRZHG¿EURVLVLQWKHFDUGLDFPXVFOHDQG P\RFDUGLDOLQWHUVWLWLXP)LJ'LVSURSRUWLRQDWHDF -FXPXODWLRQRI¿EURVLVZDVPRUHFRQFHQWUDWHGQHDUWKH

epicardium than the endocardium. The area of

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Cardiac gene expressions in the ventricles

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Table 2: Mean and SD of statistical analysis

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group; bar=20 µm, A. Control group; B. DOX group, (p<0.01).

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Fig 4: Fold differences of expression of relative apoptotic genes in DOX and control groups.

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Cell therapy results

Characteristics of MSCs

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the total cells counted. In contrast the majority of cells

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population. In addition, the results of oil red staining

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Pathological studies

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Fig 5: A. Adherent, spindle-shaped MSCs. B. Adipogenic induction, oil red staining. MSCs developed some lipid droplets. C. Osteogenic induction, alizarin red staining. Mineralized matrix formed in the MSCs. D. Flow cytometric analysis. Most

cultured MSCs expressed CD90. In contrast, the majority of MSCs were negative for CD34.

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compared to the DOX group; (bar=20 µm). A. DOX group; B. MSC group; C. MSC-CM group; D. SM group (p<0.01).

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Cardiac gene expressions in the ventricles

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had the relative expression levels of apoptotic genes

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Assay of HGF and IGF-1 for evaluating parac-rine function of MSCs

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and IGF-1 (pg/ml) are shown in Fig 10.

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Discussion

In this article we provided evidence that

trans-SODQWDWLRQ RI 06&V DWWHQXDWHG FDUGLDF ¿EURVLV DVVRFLDWHG ZLWK '2; LQFUHDVHG %FO H[SUHVVLRQ LQWKHP\RFDUGLXPWUHDWHGZLWK'2;DQGH[HUWHG DQWL¿EURWLFDQGDQWLDSRSWRWLFHIIHFWVRQWKHP\R -cardium, probably by acting in a paracrine manner.

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expression levels compared to the control group.

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The results of this study also showed that intravenous

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of acute myocarditis (27). This study revealed that

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of apoptosis (29); these proteins control the unity of

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protein family consists of both anti-apoptotic

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inhibits cytochrome c release into the cytosol and prevents apoptosis), and pro-apoptotic members that

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release of cytochrome c by promoting mitochondrial

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In the next step, we aimed to investigate

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Administration of IGF-1 after myocardial infarc-tion improves cardiac funcinfarc-tion through the increase

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apoptotic properties of IGF-1 have been demon-strated in various models of myocardial ischemia

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reoxygenation-induced apoptosis, prevented the re-lease of cytochrome C from the mitochondria, and

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with a mitochondria-mediated apoptotic pathway in

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According to our observations in this study and

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and IGF-1 may participate in the anti-apoptotic and

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Conclusion

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Acknowledgments

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