f zu{ } 18 í } 1 2014 01 01 Çf Chinese Journal of Tissue Engineering Research January 1, 2014 Vol.18, No.1 ¹ÎÎSD ÙÎε«Œ i É ¾({î Þ{ œ Þ ñ {î 116023) s Íqg Ÿjg Ðt«Éy y q u žo q ² â Ùq² k Œ ~x» q Œ ä} f Å Îo Œq«i i d Œ ŒŸÅ g¹ Œý Î Œ ±Î ýéäq ü¹ ²ŸÍ g² z u{ o ³~ «±Î ØŒg qy Œ ºÀ Œ ÎÎ Œ Îε«Œ SD Ù Éy» «Éä gè ÎΠ«Œ Ù Sprague-Dawley Œ»q «Ù ² ry ÊÈr (2011225013) Èüz NEP1-40 NT-3 ÊqÎε«ŒŠ ü n ŒÎ nq Íu{ ƒ Éy»ÇmÀg Î Œ µ q ÙÎε«Œ º g y Œnp Œz q Ð rq ~Ž ³ q SD ÙÎε«Œƒ Éy» ºmg g n¹îî SD ÙöŒ Î ŒÎÎÎ Œ²Ÿƒ Éy» «y ²Ÿ Œ ä ~ x ŒmÀ ög Í ŒmÀ ŒŸÅ g nƒ Ï ŒÉÌý Î ±Î Œ ýéä ¹ÎΫy Éy»qÎε«ŒmÀ r Î ŒmÀ À Ê ŒmÀ S mà ˆƒ Âg 29 h Œ ² ~ 10 xývº ± q Œ } 3 xîî⵫œqÿå g CD44 CD29 CD90  Ÿ² CD45 CD34 CD11b Ê Â Ÿ² } 3 xîî⵫œéì Î ±Î Œ Ï ý w w von-kossa uä n Œ O Â Í ¹ÎΫy y~žù Åqƒ Éy» Œ ±ÎqÎε«Œ ͹ } 3 xîî⵫œmg dº ý ÉäŒ ²ü gý Íqy Œ 1986 ñ õ { î Þ{ x Á n Ôd ³ Ì {î Þ{ œ Þ ñ {î 116023 doi:10.3969/j.issn.2095-4344. 2014.01.009 [http://www.crter.org] f É ú:r394.2 h u:a ú:2095-4344 (2014)01-00051-06 ÈÍ 2013-10-13 i É ¾. ¹ÎÎ SD ÙÎε«Œ[J].f zu{ 2014 18(1):51-56. Culturing bone marrow mesenchymal stem cells from Sprague-Dawley rats using whole bone marrow adherence method Gong Yu, Wang Hong-fei, Xia Hai-jun (Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China) Abstract BACKGROUND: Tissue and cell implantation entails high-quality seed cells. In order to satisfy this requirement, it is crucial to produce adequate well-conditioned, high-purity and strong proliferation ability bone marrow-derived mesenchymal stem cells. OBJECTIVE: To establish a simple, rapid and effective in vitro isolation and culture method of bone marrowderived mesenchymal stem cells, and to define the biological features of bone marrow mesenchymal stem cells. METHODS: Rat bone marrow mesenchymal stem cells were isolated from the bilateral tibial and femoral bones by the method of whole bone marrow, then purified and passaged by attachment method. The morphology and features of bone marrow mesenchymal stem cells were observed, the growth curve was drawn and the cell surface antigen was detected by flow cytometry. The bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells isolated by the whole bone marrow adherence method grew vigorously and were highly purified. The cultured cells were spindle-shaped. The growth curve was S-shaped and the population doubling time was 29 hours. The cells still maintained a strong proliferative capacity after they were passaged for 10 generations. The surface markers such as CD44, CD29, CD90 were positive, while CD45, CD34, CD11b were negative. At the third passage, bone marrow mesenchymal stem cells were induced to differentiate along the osteogenic, chondrogenic and adipogenic lineages, Gong Yu, Studying for master s degree, Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China Corresponding author: Wang Hong-fei, M.D., Professor, Master s supervisor, Department of Orthopedics, the Second Affiliated Hospital of Dalian Medical University, Dalian 116023, Liaoning Province, China Accepted: 2013-10-13 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 51
respectively. Following induction, Alizarin red staining, alkaline phosphatase staining, von-kossa mineralized nodules staining, toluidine blue staining, and oil red O staining were all positive. This shows that the whole bone marrow adherence method is a simple and reliable method for the in vitro isolation, culture and proliferation of bone marrow mesenchymal stem cells. Moreover, they have multi-lineage differentiation capacity under different inducers. The third passage bone marrow mesenchymal stem cells have the highest biological activity and can act as the ideal seed cells for subsequent experiments. Subject headings: bone marrow; mesenchymal stem cells; rats, Sprague-Dawley; cells, cultured Funding: the Science and Technology Plan Project of Liaoning Province, No. 2011225013 Gong Y, Wang HF, Xia HJ. Culturing bone marrow mesenchymal stem cells from Sprague-Dawley rats using whole bone marrow adherence method. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(1):51-56. 0 Introduction ² u{ÿ Îε«Œ(bone marrow mesenchymal stem cells BMSCs) º p ²³ ùžƒz ƒ ± iî} g d ü Ï» ùéì Éäg Î Œ ±Î Œ Œ Œ x Œö Œ} [1-4] g²ü Œz qy Œ xö oq k Å Œ [5-6] ÎÎfÎε«Œÿ ƒ uëîî Œ q0.001%0.1% g Œz ö o µ qîî⵫œ g ¼ u{q Îε«Œ»mº Ø«g Í n«~ê ³ qsd ÙÎε«ŒÉy» ä º mà mg g Í ºý Î ±Î ŒÉ äœ g² u{îî⵫œz o ö z Í u 1 Materials and methods Œ ƒ Í Âö p2011 4 Ž2012 8 ²çy Ùg Íf 6' ÙÎε«Œƒ Éy»ö¹ Äg Ï y Ïöy LG-DMEM» fž Trypsin-EDTA pn qu βm w ¾ β j m C-2 w ¾ n Œ Trizol Ï Cell Counting kit-8 Hyclone Gibco Sigma Invitrogen Trypan Blue CD44 CD29 CD90 CD45 CD34 Santa cruz CD11b ˆr À CO 2»~ üuä u{ ²ø ä Ï É Olympus Thermo ÍÙg SPF SD Ù6ù Ú ÄÄ Â ƒ«80100 g n ²çy Ùg Íf Ùg«ü ú SCXK(²)2008-0002 Í Îε«ŒqñxÉy» 10% ü Ø Ù È ŒŽ Ù¹ pƒzé g75% j f 30 min p Âú ºöŒ Î ŒÎ PBSÀ ÓôÎe Î n10 ml LG-DMEM» ö À ÎÎ Ä Œ}² ýq Œ x1 000 r/miny 5 min Ø LG-DMEM» (ÿö ) Žy ~ µ Œ âé x 1 10 9 L 1 q Œ yp25 cm 2» m ˆp37 ƒzé g5%co 2 Ê»~f» ŒmÀ À 24 hý¹ 2 5 d ÉÌé 7 d¹ Îε«Œq~x»»mf ŒÉ 80%90% üê n1.5 ml Trypsin-EDTA y Œ ŒµÉy Ø 5 mlqÿž» ä y Œ 1 000 r/miny 5 min Ø ÿƒzé g10% fž qlg-dmem» q Œ x1 2 ²Ÿ~x Îε«Œq ñx }8x Œ n ˆr À ºmÀ ög dr mà q Œpÿ10%pn quqž fá 2 ý š Í rö Œ p37 ƒzé g5%co 2»~f» Îε«ŒmÀ q Í }1 3x Œ Œ äý x5 10 4 f/ yp96 Ø 10 μl CCK-8 37 Ê ƒzé g5%co 2»~ 2 hý n Š o yp450 nm À ý }1 2 3 4 5 6 7 ü  rü Í Œ Îε«ŒŸÅ g¹ üž90% úmàg }3xÎε«Œ Œ q ä y Œ Œ g1 10 9 L 1 É ŽüPE~f Ø ê à ƒcd44 CD29 CD90 CD45 CD34 CD11b ~ ü  d ( d f Ø{ ƒ) 40 min PBS 52 P.O. Box 10002, Shenyang 110180
A B C D 1 Îε«Œq ( 100) Figure 1 Morphology of bone marrow mesenchymal stem cells ( 100) f A gñx» 3 d q SD ÙÎε«Œ² g Ê Ê Ê B gñx» 7 d q SD Ù Îε«Œ x Œg µé Œ Ê p Ÿ µé gmà C g} 3 xîî⵫œmà±ñ x Œ x Œgg D g} 8 xîî⵫œg x q Œgg À Œ Œ Œy É Îε«Œ ý Éä mà q}3 xîî⵫œ x5 10 7 L 1 yp24 Œ «mà üž70%80% ýü féìø Î Œ Ï(ÿ βm w ¾ m C-2w ¾) ±Î Œ Ï(ÿ ±ämà β m C) Œ Œ Ï(ÿ Œ ˆ±) ü x kqîî⵫œ» g d Î Œ Ï 11 dý 40 g/l Šn Œ Ÿ w w von-kossauä ±Î Œ Ï 21 dý 40 g/l Šn Œ uœéfý Ÿn Œ Œ Œ Ï 19 dý 40 g/l Šn Œ Ÿ O g Îε«Œq ömà Îε«ŒŸÅ gq¹ Îε«Œý Î ±Î Œ ÉäŒ q¹ 2 Results 2.1 Åp h Ë yp»mq ÎÎ Œ p» f Œ y24 hý «³²» ôã «Œ 48 hý«œ² g Ê Ê Ê( 1A) 7 dý «Œ x Œg µé Œ Ê p Ÿ µé gmà( 1B) }3xÎε«ŒmÀ±ñx Œ x Œgg( 1C) Œ~xŽ}8x Œ ä g x q Œgg( 1D) 2 Œy } 3 x SD ÙÎε«ŒŸÅ gqÿ² Figure 2 Surface antigens of passage 3 bone marrow mesenchymal stem cells from Sprague-Dawley rats detected by flow cytometry ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 53
} 1 x } 3 x A B C 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 1 2 3 4 5 6 7 8»  d) D E F 3 } 3 x SD ÙÎε«ŒqmÀ xö ý Éä¹ Figure 3 Growth curves and multi-differentiation of passage 3 bone marrow mesenchymal stem cells (1) f A g} 1 3 x SD ÙÎε«ŒqmÀ } 1 3 xîî⵫œmà S ŒmÀ ï }mà (12 d) mà (35 d) mà ú (68 d) (2) f B g} 3 x SD ÙÎε«Œ Î 11 d ý ŒÂù ¾«z Œ f q Œ ü ô Œ ¾ ( 200) (3) f C g} 3 x SD ÙÎε«Œ Î 11 d ýw w ù Œ «Â Ÿ²( 200) (4) f D g} 3 x SD ÙÎε«Œ Î 11 d ý vonkossa uä ù ŒŠÄ Ø gé  Ÿ ²( 200) (5) f E g} 3 x SD ÙÎε«Œ ±Î 21 d ýn Œ q±î Œ ŸÅ n Œ ù Œ«( 200) (6) f Fg} 3xSD ÙÎε«Œ Œ 19 dý O qœ ŒŒ«z Œ Ø rp ü( 200) 2.2 Åp Â Ç Œy }3xÎε«ŒŸÅ gqÿ² x Œ Ÿ²CD44 CD29 CD90  iéìg 99.69% 83.99% 95.05% CD45 CD34 CD11b/c Ê Â Ÿ² ÉÌg0.28% 0.62% 4.25%( 2) 2.3 Åp j }1 3xÎε«ŒmÀ S ( 3A) Îε«ŒmÀ²z Çj }mà (12 d) (35 d) Ãi² m À ú (68 d) Á 2 ý Œ Œ 95%x v q Œ vmà ² xç ŒmÀ³ }3 ²ÍÈ í»m ¼qÎε«ŒÄ ²Ÿ~x ² É Çq~x  r Žq 2.4 Åp Œ ÆgÎ«Ç Î Ï»11 dý ŒŒ«¼µ È Œ Ä mà ŒÂù ¾«z Œ f q Œ ü ô Œ ¾ ( 3B) w w ù Œ «Â Ÿ²( 3C) vonkossauä ù ŒŠÄ Ø gé  Ÿ ²( 3D) Îε«Œ ±Î Œ Ï» 21 dý q±î Œ ŸÅ q n Œ ù Œ«( 3E) ÎΠµ«œ Œ Ï»19 dý qœ ŒŒ«z Œ Ø rp ü ŒnÀ g ² O x ŒqŒ ¼ Œ«( 3F) 3 Discussion ²vg } oœî q f Œz Ÿ g q o i [7] rð Œ x Œ ² x Œö Æ Œ µ Œ ¹ Œz oö z y Œq [8-9] u º ƒ» Ä}ÁÈ d² Í kqí [10] Îε «Œ Îμq y Œ Ѓ Œ ²ù g z»éägx Œ ºŽ ²³ ~Ž o ö ƒ ± 54 P.O. Box 10002, Shenyang 110180
iî pg n}g g Œz ±k qy Œ [11] Îε«Œ Œz oxö z}u{è g qu{ [12-15] rðîî⵫œƒ Éy» ä ö Œ¹ ie q  [16] x ~ê ³ qéy» ä ö Œ¹ q g Îε«ŒŸÅ Œ È rð nqéy ¹ÎΫy y owj Œyɲ [17-21] ü Ž º~ ºf¹ÎΫy ~Ž Œ n [22] Îε«Œ ~Ž qéy ä npîî⵫œ ig ŒŸÅ g Žˆ µéu{ Í Îε«Œ¹ ü¹ â «ŒmÀ ö Œ ŒŸÅ g¹ Œ ý Éä Œ ³² ² ƒ Éy» ŒgÎε «Œ Í n¹îî«y ƒ Éy»SD ÙΠε«Œ Œy ŒŸÅÉ Ÿ² Éy Ÿ ²n«y ~ê ³ qj ~± Äy ùx Œ q g y»ý}2 ù 굃 Œ«³ ž Œög «q Œ Œ qvf v» f ñx Œ y»24 hë ý ùx 굃 Œ«² d ³ zš Œ mà ý ñx Œ«m À815 d²í ŒË üý ³ž Œö r Œ ôã ý ù²ÿîî⵫œ~x» ³² Œ~xÃô «ö «Œ ù Î Œ Í qîî⵫œ«œ Œ ŒƒÊ º ±Îqmg ²ü gy Œ vsd Ù}1 3xqmÀ r SD ÙÎΠµ«œqmà ïo }² mà ú }1xÍ}3x ŒmÀ Œ pñx Œ ¹ÎΫy ²npÎε«ŒqÉ y» ~Îε«Œ ig ŒŸ Å g º»²zfùŸ²Ç y ŒCDŸ gù ÿgîî⵫œ [23-24] u{ µ«Œ i³ž ŒŸÅ â CD34 CD45öCD11b SH2 SH3 CD29 CD44 CD71 CD90}µ«ŒŸÅ  Ÿ² [25] Í n Œy ~xž}3xsd ÙÎε«ŒqCD44 CD29 CD90 CD45 CD34öCD11b Ÿ»q ŒŸ²Çµ«Œqg CD44 CD29 CD90}  Ÿ² CD45 CD34 CD11b Ê Â Ÿ² Woodbury} [26] Hofstetter} [27] ƒ u{ Îε«Œº ý ÉäŒ Í ± q}3xîî⵫œ³² Î ±Î Œ Ï» Í º ýéäœ Œ«70% üý Ø Î Ï» 3 dý Œƒz u }6 Œ Œ«¼ ŒÈ }11 Œ«¼µ È Œ Ä mà ŒÂù ¾«z Œ f q Œ² ü ô Œ ¾ w w ù Œ «Â Ÿ² vonkossauä ù ŒŠÄ Ø gé  Ÿ² Œ» ±Î Ï» Œv«Âpy ~ µq Œ ² Œk ùx Í Œk² ŸÅ 21 dý Œk uœéfý²ÿn Œ ù Œ«Œ«100% üý Ø Œ Ï Œ 96 hý Œ¼ Œ Ç j 19 dýœ Ø rp ü ŒnÀ g ² O x Œ«Œ Ï» g ý ÉÌ Ø Î Œ ±Î Œ Œ Œ ² Í ƒ Éy»qSD ÙÎÎ Œg ±ÎqÎε«Œ źw³ž Œ o ~ê ±ÎqÎε«Œ Éy» ¹ q ³² ŒŸ² qg ŸÅ ñ Ÿ²³ž Œg ñ ºº ý Î Œ Œ ±Î Œ Éäq Œ}g o ÍÉ y»q ÙÎÎ ŒgÎ qîî⵫œ Å ºw³ž Œ Œ~xŽ}3x ù Î mg ÎqÎε«Œ g o v NT-3 ± SD ÙÎε«Œq Íu{ o uxö o µ qy Œ Ž {î Þ{ œ Ì ÀÁ ì c«} «n d «h  c c Ϋc c ÚÒ«d ÌrÀ{ À À í q d k «n t 2009 wethical issues in animal experimentationx üì íd }ß Ý ó Êöi c Ý } Ý Ú y Ý d u k À í À d ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 55
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