Efek dimethyl sulfoxide (DMSO) terhadap Karakteristik Sel Punca Limbal (SPL) Tikus
Abstract
Abstact : The number of donor limbal stem cells (LSCs) is limited despite their large demand for management of LSC deficiency-related corneal opacity, requiring propagation of these cells in vitro. Production of LSCs can be done through isolation and culture of limbal tissue in vitro and cryopreservation of LSCs is utilized to maintain the availability of LSCs. Upon cryopreservation, cryoprotectants are required to protect cells from thermal injury. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant for cryopreservation but the effect of its use on LSCs are still seldomly reported. This study aimed to determine the effect of the use of DMSO in cryopreservation of LSC. The study was conducted at the Stem Cell Laboratory, Center for Biomedical and Basic Technology of Health, NIHRD Ministry of Health. This research was performed by culturing and observation of  LSCs characteristic after cryopreservation. Meanwhile, level of LSC proliferation was determined by calculating population doubling (PD) and population doubling time (PDT) besides analyzing their gene expression using markers such as CD90 (Thy1) and Krt12. The results showed that PD and PDT in LSC control and post-cryopreservation without DMSO and cryopreservation using DMSO accordingly are 1.33 and 143.03, 150.65 and 1.15, and 1.31 and 155. Meanwhile, CD90 (Thy1) gene expression and Krt12 expression in the cryopreserved group with and without DMSO compared with their respective controls are 2.7 and 4.51, 2.55 and 1:44, respectively. In this study, DMSO for the cryopreservation did not affect at the LSC characteristics of rat.Key word : limbus stem cell, LSC, cryopreservation, dimethyl sulfoxide, DMSO Abstrak : Jumlah donor sel punca limbal (SPL) sangat terbatas padahal kebutuhannya cukup besar untuk penatalaksanaan kekeruhan kornea akibat defisiensi sel tersebut sehingga SPL perlu diperbanyak secara in vitro. Produksi SPL secara in vitro dapat dilakukan dengan melakukan isolasi dan kultur dari jaringan limbal, dan metode simpan beku atau kriopreservasi SPL digunakan untuk menjaga ketersediaan SPL. Pada saat kriopreservasi, dibutuhkan krioprotektan yang dapat melindungi sel dari kerusakan termal saat kriopreservasi. Dimethyl sulfoxide (DMSO) merupakan salah satu krioprotektan yang umum digunakan untuk kriopreservasi namun efek penggunaan pada SPL masih sangat jarang dilaporkan. Penelitian ini bertujuan untuk mengetahui efek penggunaan DMSO pada kriopreservasi SPL. Penelitian dilakukan di Laboratorium Stem Cell Pusat Biomedis dan Teknologi Dasar Kesehatan Badan Litbangkes. Penelitian ini dilakukan dengan melakukan kultur SPL tikus menggunakan metode eksplan secara in vitro pada cawan petri. Pengamatan terhadap karakteristik SPL pasca kriopreservasi dilakukan dengan pengamatan terhadap morfologi SPL secara mikroskopis dan mengetahui tingkat proliferasi SPL dengan menghitung population doubling (PD) dan population doubling time (PDT) serta menganalisis ekspresi gen CD90 (Thy1) dan Krt12 sebagai marker SPL. Hasil penelitian menunjukkan bahwa PD dan PDT pada SPL kontrol dan pasca kriopreservasi tanpa DMSO dan kriopreservasi dengan DMSO secara berturut-turut adalah 1.33 dan 143.03, 1.15 dan 150.65 serta 1.31 dan 155. Sedangkan tingkat ekspresi gen CD90 (Thy1) dan Krt12 SPL pada penggunaan dan tanpa DMSO dibandingkan dengan kontrol masing-masing adalah 2,7 dan 4,51 serat 2,55 dan 1,44 kali. Pada penelitian ini, DMSO tidak  mengubah  karakteristik SPL tikus. Kata kunci: sel punca limbal, SPL, kriopreservasi, dimethyl sulfoxide, DMSOReferences
DAFTAR PUSTAKA
Burman S and Sangwan V, H 2008, ‘Cultivated limbal stem cell transplantation for ocular surface reconstruction’, Clin Opthal, vol. 2, no. 3,hh. 489-502.
Albert R, Vereb Z, Csomos K, Moe MC, Johnsen EO, Oldstad OK, et al., H 2011, ‘Cultivation and characterization of corneal limbal ephitelial stem cell on lens capsule in animal material-free medium’, PLoS ONE, vol. 7, no. 10, hh. 1-11.
Lekhanont K, Choubtum L, Chuck RS, Sa-ngiampornpanit T, Chuckpaiwong V and Vongthongsri A. H 2009. ‘A Serum-and feeder-free technique of culturing human corneal epitheliual stem cells on amniotic membrane’, Mol Vis, 2009, vol. 15, no. 1, hh. 294-302.
Loureiro RR, Cristovam PC, Martins CM, Covre JL, Sobrinho JA, Ricardo JRS, Hazarbassanov RM, et al, H 2013, ‘ Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells’, Mol Vis, vol. 19, hh. 69-77.
Sancak İG, Ozen A, Pinarli FA, Tiryaki M, Ceylan A, Acar U and Delibasi T, H 2014, ‘Limbal stem cells in dogs and cats. Their identification, culture and differentiation into keratinocytes’, Kafkas Univ Vet Fak Derg,, vol. 6, hh. 909-914.
Meyer-Blazejewska E.A, Kruse F.E, Bitterer K, Meyer C, Hofmann-Rummelt C, Peter H, H 2010, ‘Preservation of the limbal stem cell phenotype by appropriate culture techniques’, IOVS, vol. 51, no. 2, hh. 765-774.
Yeh HJ, Yao CL, Chen HI, Cheng HC, Hwang SM, H 2008, ‘Cryopreservation of human limbal stem cell ex vivo expanded on amniotic membrane’ Cornea, vol. 27, no. 3, hh. 327-333.
El-Danasaouri I. and Selaman H. , H 2007, ‘Vitrification versus conventional cryopreservation technique’, Middle East Fertlity Society Journal, vol. 10, no. 3, hh. 205-206.
Meryman HT., H 2007, ‘Cryopreservation of living cells: principles and practice’, Transfusion ,vol. 47, hh. 935-945.
Bagis H, Akkoc T, Taskin C and Arat S, H 2009, ‘Comparison of different cryopreservation techniques: Higher survival and implantation rate of frozen-thawed mouse pronuclear embryos in the presence of beta-mercaptoethanol in post-thaw culture’, Reprod Dom Anim, Doi: 10.1111/j.1439-0531.2009.01570.x
Kito K, Kagami H, Kobayashi C, Ueda M, Terasaki H, H 2005, ’ Effect of cryopreservation on histology and viability of cultured corneal epithelial cell sheets in rabbits’, Cornea, vol. 24, no. 6, hh. 735-741.
Desai N, Xu J, Tsulata T, Lawson JS, Hafez AF, Goldfarb J and Falcone T, H 2010, ‘Vitrification of mouse embryo-derived ICM cells: a tool for presering embryonic stem cell potential?’, J Assist reprod Genet , DOI 10.1007/s10815-010-9500-x.
Djuwantono T, Wirakusumah F F, Achmad T H, Sandra F, Halim D, Faried A., H 2011, ‘Comparison of cryopreservation methods: slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells, BMC Research Notes, vol. 11, no. 4, hh. 371.
Aye M, Giorgio C D, Mo M D, Botta A, Perrin J, Courbiere B., H 2010. ‘Assessment of genotoxicity of three cryoprotectants used for human oocyte vitrification: dimethyl sulfoxide, ethylene glycol and propylene glycol’ Food Chem Toxicol, vol. 48, hh. 1905-12.
Bakhach J., H 2009, ‘The cryopreservation of composite tissues: principles and recent advancement on cryopreservation of different type of tissues’, Organogenesis,vol. 5, no.3, hh. 119-126.
Violante GD, Zerrouk N, Richard I, Provot G, Chaumeil JC and Arnaud P., H 2002, ‘Evaluation of the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures’, Biol Pharm Bull,, vol. 25, no. 12, hh. 1600-3.
Krishnan S, Iyer GK and Krishnakumar S., H 2010, ‘Culture and characterisation of limbal epithelial cells and oral mucosal cells’, Indian J Med Res,vol.13, no.1, hh. 422-8.
Li G, Zhu Y, Xie H, Chen S, Tseng S., H 2012, ‘ ‘Mesenchymal stem cells derived from human limbal niche cells’, Cornea, vo. 12, no. 53, hh. 5686-97.
Polisetty N, Fatima A, Madhira SL, Sangwan VS and Vemuganti GK., H 2008, ‘Mesenchymal stem cell from limbal stroma of human eye’, Mol Visvol. 14, hh. 431-42.
Jain J K and Paulson RJ. H 2006, ‘Oocytes cryopreservation’, Fertility and Sterility, vol.86, hh. 1037-46.
Naaldijk Y, Staude M, Fedorova V and Stolzing A., H 2012, ‘Effect of different freezing rate during cryopreservation of rat mesenchymal stem cell using combination of hydroxyethyl strach and dimethylsulfoxide’, BMC Biotechnology, vol. 12, no.49. DOI:10.1186/1472-6750-12-49.
Ock S and Rho G., H 2011, ‘Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cell (pMSC)’, Cell Transplantation, vol. 20, hh. 231-9.
Burman S and Sangwan V, H 2008, ‘Cultivated limbal stem cell transplantation for ocular surface reconstruction’, Clin Opthal, vol. 2, no. 3,hh. 489-502.
Albert R, Vereb Z, Csomos K, Moe MC, Johnsen EO, Oldstad OK, et al., H 2011, ‘Cultivation and characterization of corneal limbal ephitelial stem cell on lens capsule in animal material-free medium’, PLoS ONE, vol. 7, no. 10, hh. 1-11.
Lekhanont K, Choubtum L, Chuck RS, Sa-ngiampornpanit T, Chuckpaiwong V and Vongthongsri A. H 2009. ‘A Serum-and feeder-free technique of culturing human corneal epitheliual stem cells on amniotic membrane’, Mol Vis, 2009, vol. 15, no. 1, hh. 294-302.
Loureiro RR, Cristovam PC, Martins CM, Covre JL, Sobrinho JA, Ricardo JRS, Hazarbassanov RM, et al, H 2013, ‘ Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells’, Mol Vis, vol. 19, hh. 69-77.
Sancak İG, Ozen A, Pinarli FA, Tiryaki M, Ceylan A, Acar U and Delibasi T, H 2014, ‘Limbal stem cells in dogs and cats. Their identification, culture and differentiation into keratinocytes’, Kafkas Univ Vet Fak Derg,, vol. 6, hh. 909-914.
Meyer-Blazejewska E.A, Kruse F.E, Bitterer K, Meyer C, Hofmann-Rummelt C, Peter H, H 2010, ‘Preservation of the limbal stem cell phenotype by appropriate culture techniques’, IOVS, vol. 51, no. 2, hh. 765-774.
Yeh HJ, Yao CL, Chen HI, Cheng HC, Hwang SM, H 2008, ‘Cryopreservation of human limbal stem cell ex vivo expanded on amniotic membrane’ Cornea, vol. 27, no. 3, hh. 327-333.
El-Danasaouri I. and Selaman H. , H 2007, ‘Vitrification versus conventional cryopreservation technique’, Middle East Fertlity Society Journal, vol. 10, no. 3, hh. 205-206.
Meryman HT., H 2007, ‘Cryopreservation of living cells: principles and practice’, Transfusion ,vol. 47, hh. 935-945.
Bagis H, Akkoc T, Taskin C and Arat S, H 2009, ‘Comparison of different cryopreservation techniques: Higher survival and implantation rate of frozen-thawed mouse pronuclear embryos in the presence of beta-mercaptoethanol in post-thaw culture’, Reprod Dom Anim, Doi: 10.1111/j.1439-0531.2009.01570.x
Kito K, Kagami H, Kobayashi C, Ueda M, Terasaki H, H 2005, ’ Effect of cryopreservation on histology and viability of cultured corneal epithelial cell sheets in rabbits’, Cornea, vol. 24, no. 6, hh. 735-741.
Desai N, Xu J, Tsulata T, Lawson JS, Hafez AF, Goldfarb J and Falcone T, H 2010, ‘Vitrification of mouse embryo-derived ICM cells: a tool for presering embryonic stem cell potential?’, J Assist reprod Genet , DOI 10.1007/s10815-010-9500-x.
Djuwantono T, Wirakusumah F F, Achmad T H, Sandra F, Halim D, Faried A., H 2011, ‘Comparison of cryopreservation methods: slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells, BMC Research Notes, vol. 11, no. 4, hh. 371.
Aye M, Giorgio C D, Mo M D, Botta A, Perrin J, Courbiere B., H 2010. ‘Assessment of genotoxicity of three cryoprotectants used for human oocyte vitrification: dimethyl sulfoxide, ethylene glycol and propylene glycol’ Food Chem Toxicol, vol. 48, hh. 1905-12.
Bakhach J., H 2009, ‘The cryopreservation of composite tissues: principles and recent advancement on cryopreservation of different type of tissues’, Organogenesis,vol. 5, no.3, hh. 119-126.
Violante GD, Zerrouk N, Richard I, Provot G, Chaumeil JC and Arnaud P., H 2002, ‘Evaluation of the cytotoxicity effect of dimethyl sulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures’, Biol Pharm Bull,, vol. 25, no. 12, hh. 1600-3.
Krishnan S, Iyer GK and Krishnakumar S., H 2010, ‘Culture and characterisation of limbal epithelial cells and oral mucosal cells’, Indian J Med Res,vol.13, no.1, hh. 422-8.
Li G, Zhu Y, Xie H, Chen S, Tseng S., H 2012, ‘ ‘Mesenchymal stem cells derived from human limbal niche cells’, Cornea, vo. 12, no. 53, hh. 5686-97.
Polisetty N, Fatima A, Madhira SL, Sangwan VS and Vemuganti GK., H 2008, ‘Mesenchymal stem cell from limbal stroma of human eye’, Mol Visvol. 14, hh. 431-42.
Jain J K and Paulson RJ. H 2006, ‘Oocytes cryopreservation’, Fertility and Sterility, vol.86, hh. 1037-46.
Naaldijk Y, Staude M, Fedorova V and Stolzing A., H 2012, ‘Effect of different freezing rate during cryopreservation of rat mesenchymal stem cell using combination of hydroxyethyl strach and dimethylsulfoxide’, BMC Biotechnology, vol. 12, no.49. DOI:10.1186/1472-6750-12-49.
Ock S and Rho G., H 2011, ‘Effect of dimethyl sulfoxide (DMSO) on cryopreservation of porcine mesenchymal stem cell (pMSC)’, Cell Transplantation, vol. 20, hh. 231-9.
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Published
2017-11-22
How to Cite
Rinendyaputri, R., Dany, F. and Uly Alfi Nikmah (2017) “Efek dimethyl sulfoxide (DMSO) terhadap Karakteristik Sel Punca Limbal (SPL) Tikus”, Indonesian Journal on Medical Science, 5(1). Available at: http://ejournal.poltekkesbhaktimulia.ac.id/index.php/ijms/article/view/124 (Accessed: 20 April 2025).
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