IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Morphological and Growth Properties of Albino Rat Bone Marrow Stromal Cells In Vitro I. M.Mnati and B.H. Mutlak Deparatme nt of Biology, College of Education , Ibn Al- Haitham , Unive rsity of Baghdad Abstract The p otential use of bone marrow st romal cells as a cellular therapy for chronic diseases relies on the ability of the cell to replicate extensively in vitro.For this reason the p resent st udy invest igated the replication lifesp an and examined the growt h p rop erties of albino rats mesenchymal stem cells(M SCS)in vitro. To establish an in vitro sy st em for isolating and culturing the M SCs of albino rats and to p rovide research data for its further app lication,the bone marrow (BM )was collected from y oung male rats and sep arated by gradient centrifugation.Then, the mononuclear cells(M NCs) were retrieved from the buffy lay er and cultured in M odified Eagle , s M edium (M EM ) sup p lemented with 10%Fetal Calf Serum (FCS)and incubation at 37C o in humid air with 5%CO2 . The non-M SCs were screened out.A p assage culture was undertaken while the monolayer of adherent cells formed .The sp indle –shap ed M SCS with a single nucleus formed a monolay er after the 10-12 days of p rimary culturing,and the cells app eared in an oriented array with a swirling growt h trend. In the anap hase of p assage culture ,the cell p roliferation rate was decreased and the morphology changed into broad flat app earance.These results suggested that M SCS of the albino rats can be p assaged in vitro with the established op timized culture sy st em,in addition, the growt h curves can be divided into three p hases:the initial quiescent p hase,the logarithmic replication p hase and after logarithmic replication p hase . Introduction Bone marrow is the major source of st romal cells and adult hematop oietic stem cells (HSCS) that renew circulating blood elements;these cells can also be found in other tissues .Adult BM also contains M SCs. M SCs are thought to be multip otent cells that can rep licate as undifferentiated cells and have the p otential to differentiate to lineages of mesenchymal tissues,including :bone ,cartilage muscle, nerves and marrow st roma [1,2].Considering their advantages ,such as ease of obtaining by a simp le routine BM a sp iration ,ability to self –renew and recently reported p otential to differentiate into cardiomy ogenic cells, M SCs have been considered as one of the most p romising candidates for this p urp ose and the app arent multipotent nature of M SCs makes them excellent candidates for t issue engineerin g [3]. The M SCs were first described in 1970 by Friedenst ein et al., [4]who discovered that the cells adherent to tissue culture p lates resembled fibroblasts in vitro and formed colon ies. He and others demonstrated M SCs could differentiate into multiple p athway s t hat include cardiac myogenesis[5,6].Among the most extensively st udied of adult st em cells from BM , if plated at low densities, M SCs generate cell sin gle d erived colonies and the colonies can b e differentiated into several cell phenotyp es in culture.These characteristics have been identified from numerous sp ecies includ ing humans, rats ,mice, and monk eys [7,8 ]. Interest with BM SCs has begun since 1960 and since those several techniques have been developed to p roliferate these cells and then to differentiate them into the required cells to benefit from them in the basic and clinical researches.Recent att ention has focused on BM as a IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 source of st em cells for transp lantation into t he heart.At p resent, t he BM transp lantation are normal op eration which are used for treatment of many diseases [9].T he use of M SCs in such therap eutic st rategies relies on the ability of the cell to replicate extensively in vitro .For t his reason t he present study sy st ematically examined the growt h p rop erties of rat M SCs in vitro and we report here the isolation ,exp ansion and characterization of the multip otent rat M SCs. Materials and Methods - Isol ation of MS Cs from albino rats bone marrow This st udy comp rised 30 young male rats(Rattus rattus norv egicus albinos ) aged (180-200g) were used for the isolation of M SCs from the BM and were culturing in vitro, ,these animals were obtained from the M edical Research Unit College of M edicine in Al-Nahrain University . The primary culture of M SCs were p erformed according to Dodson [10].In brief,after rats were killed with an of diethy l ether,the femoral and tibial bones were collected for isolation of MSCs. The marrow p ellet was washed in p hosp hate buffer saline( PBS),centrifuged at 2000 rp m for 10 min and then resusp ended in M odified Eagle , s M edium(M EM ) .The cell susp ension was loaded on 60% p ercoll and then centrifuged in a cooling centrifuge for 20 min at 2000 rp m at 4C o .After centrifugation, the mononuclear cells (M NCs)was removed from the interface and resusp ended in M EM . M SCs were p lated at concentration of 1X10 6 cells/ml in 50 cm 2 p last ic tissue culture flask. -Culture and expansi on Following p lating ,M SCs were maintained in a humidified incubator at 37C o /5%CO 2 in M EM +10%FCS . The medium was changed after 24 hours t o remove nonadherent cells and twice weekly thereafter. Culture M SCs were observed under inverted microscop e to assess the level of exp ansion and to verify the morp hology at each culture medium change. To prevent the M SCs from differentiating ,each p rimary culture was rep lated for subculture. Cells were p assaged with 2 ml of 0.25% trypsin- 0.1%Ethy lene diamine tetra acetic acid (EDTA) up on reaching 70-80% confluency and exp anded until p assage 3,where upon they were analyzed. -Quantification of MS Cs growth To determine the initial density of M SCs in p rimary cultures,the adherent cells were detached with 0.25% try p sin- 0.1% EDTA and st ained with 0.2% try p an blue .Triplicate samples from each well of 4-well plast ic p lates were resusp ended in 2ml of PBS and count with a hemocy tometer under light microscope [11]. The average cell number calculated from four wells of each of three p lates (1X10 4 cells/well ; n=12) was t aken as the initial density of M SCs in p rimary cultures and the seeding density for every following subcultures .T his cells counting p rocedure was repeated every 48 h until the cells almost ceased proliferation.The growt h curves and the precise growt h p rop erties of M SCs in the p rimary cultures (P0)and the three subcultures (P1-P3) were determined .The numbers of cell doublings (NCD) were calculated with t he formula : NCD= log 10 ( Y / X ) / log 10 2 where X represents the initial seeding density of the cells and Y the end of the logarithmic replication phase in every p assage . Re sults - Rat MS Cs primary culture IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 The bone marrow isolates were removed by changing the medium at 24 h . In the first hours of culturing ,the adherent cells grew in a round shap e with few morp hological changes.Under an inverted microscope, morphological conservation was observed , the results showed that the p arental M SCs were characterized by an enlarging app earance.After three days of p rimary culture,the M SCs were observed att ach to the culture flask sp arsely and these cells began to p roliferate in culture medium(Fig.1 A). At day 7,sp indle shap e was observed in well- sp read colony –forming cells.Subsequently ,most adherent cells grew in a sp indle shap e characterist ic of fibroblasts (Fig.1 B). By day 10,the M SCs was dup licating rapidly and the cell morphology was mainly sp indle- shap ed(Fig.1C). Observation on day 10-12 revealed that the cell monolay er was forming with constant orientation and a whirling tendency , which means the cells have the p otency to be p assaged in ratio 1: 2 . - Rat MS Cs passage culture Passaged M SCs behaved similarly to those in p rimary cultures. The M SCs in subcultures could be divided into two ty p es: small sp indle- like cells and broad flattened cells.T he flattened cells seldom p roliferated and were gradually surrounded by the small –like cells that replicated faster and were more inclined to form colonies.It seemed that the sp indle-like cells gradually transformed into broad flattened cells with later p assages.P1 of the mesenchymal cells was characterized by the ability to p roliferate in culture with an att ached well-sp read morphology , reaching confluence on day 6-8.In this p assage,the cells continued to dup licate rapidly (Fig.2 A). M icroscop ic observations showed that P2 ,cells remain fast growing and reach70-80%confluence by day 6 (Fig.2 B). However the cell p roliferation rate decreased in P3 comp aried with the earlier p assages,the cell p roliferation obviously slowed down on day 6.In this case,a few ty p ical sp indle cells can be observed under microscope and more flat cells were found in the field (Fig.2 C). In this st udy , the criterion for p assaging was not iced a half of colonies reached 70-80% confluence, usually 10-12 days after seeding for p rimary culture and 6-8 day s after subculture. -Growth propertie s of MS Cs in cul ture The growt h curves of M SCs in p rimary culture and in the three p assages are shown in (Fig.3).For p rimary cultures, the cells remained quiescent during the first two day s of culture and then quickly replicated until day 10 when the average cell number reached 13.4X10 4 cells/well, the number of cells did not increase during the following five days of culture 10-14 days.T he p att erns of the growt h curves of M SCs in P1 and P2 were similar to those of p rimary cells.However, the cells showed a short ened quiescent p eriod 1-2 days before p roliferation and the cells number kept increasing for a short er p eriod 4-6 days in P1 and P2.T he increase of average cell numbers was considerably slowed in P3 and it is exp ected the cells almost ceased proliferation in later passages. To determine the changes in precise growt h p rop erties of M SCs with p assages, the growt h course of M SCs in the p rimary culture and in each successive subcultures was divided into three p hases according to their growt h curves: the initial quiescent p hase,the logarithmic replication p hase,and after logarithmic replication p hase.Rat M SCs(rM SCs) could be exp anded up to 15.35cell doublings of successive subcultures.T he results of this assay are summarized in Table 1,which show the number of cell doublings(NCD) from P0-P3,and the accumulative NCD with p assages. Discussion M esenchy mal st em cells are thought to be multipotent cells,which are p resent in adult marrow.T hey can replicate as undifferentiated cells and have the p otential to be induced to IBN AL- HAITHAM J. FOR PURE & APPL. . VOL.22 (4) 2009 differentiate exclusively into chondrocy tic or ost eocy tic lineages[8].This st udy has imp ortant limitations which are represented by the use of y oung animals as the source of BM -derived M SCs. Our data is in line with the st udy made by Tomita et al., [12] who found t hat BM of y oung and growing p igs may have contained p rogenitor cells than would be found in older animals.Primary cell cultures from y oung animals grow more rapidly than do cells obtained from older animals. However,the exp andability of M SCs in vitro varied dramatically among different sp ecies and different methodologies for isolation and p lating of the cells.It has been reported that murine M SCs were much more difficult to grow than other sp ecies[6] and this was p robably due to the higher sensitivity of the murine M SCs to the initial seeding density [13].Here,the p resent st udy showed that at seeding density of 1X10 4 cells/well,rat M SCs could be exp anded to 10.4 cell doublings after 17 days of culture(P0-P1)and up to a maximum 15.35 cell doublings in 30 days,t his resembles t he rep orted in vitro lifesp an of human M SCs[14]. M ultip le factors,such as medium ,serum selection ,seeding ratio and micro-environment (p H and temp erature)can affect the cell y ield and p assage.In our exp erience, M SCs can be isolated by their adherence p rop erties to the p last ic surfaces of culture flasks,disp lay ing fibroblast-like growt h characterist ics and increased p roliferation[15].After two days of culture,M SCs app eared sp arsely and began to p roliferate in culture medium, and in the end of the second week they formed the monolayer of adherent cells .These results corresp ond to other st udies results [11,16 ,17] who have arrived at regarding forming the monolayer of p rimary culture app roximately in the second week of culturing. In our p ilot exp eriments,it was found that if the cells were grown over two weeks before subcultures,t hey quickly lost the ability to p roliferate within 1-2 p assages. It is known t hat p roliferation of cultured cells is usually p romoted when cells begin to release their own cy tokines and inhibited by cell-to-cell contacts up on confluency .Although a more frequent subculture can delay the senescence of M SCs,t hey gradually lost the ability to p roliferate with the increasing of cell doublings,and this was accomp anied by a gradual morphological conversion of the small sp indle-like M SCs into the broad flattened cells,t ogether with the evidence from several other laboratories [11,14]. Because the amount of M SCs is too low (0.001-0.01%) in adult BM (1),the best way to obtain sufficient M SCs is in exp anding in culture.The growt h kinetics of these cell p op ulations revealed rapid adherence through to P2,but the cell proliferation rate decreased in P3 compared with the earlier p assages, which was similar to[11,17]. According to these results, it ap p eared that the nature of rM SCs have several p rop erties as follows:- 1. M SCs are more sensitive to p lating density . 2. They exp and more rap idly after low-density p lating. 3. They require frequent culture medium chan ging. 4. They form confluent cultures after low-density p lating. From the results of this st udy ,it was concluded that,t he BM -derived M SCs of alb ino rats can be isolated,proliferated and maintained them in active state for several weeks and examin ed the growt h p rop erties of M SCs in vitro. Re ferences 1-M inguell,J.;Er ices,A.andCon get,P.(2001).Exp .Biol.M ed.,226:507-520. 2-Pittenger, M .F. and M artin, B.J. (2004). Circ. Res., 95:9. 3-M akino,S.;Fukuda, K;Pan, and Ogawa,S.(1999).J. Cl in. Invest ., 103(5): 697-705. 4-Friedenst ein,A.J.; Deriglasova, U.F. and Kulagina, N.N. (1974). Exp . Hematol., 2: 83-92 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 5-Piersma, A.H.; Ploemach er, R.E. and Brockbank, K.G.M . (1983). J. Haematol., 54(2): 285- 290. 6- Clark, B. and Keating, A. (1995). Ann. N. Y. Acad. Sci.,770 : 70-78. 7-Hai-jing, S.H.I. ;Zheng, Z.H.U.; Long-ding, L.I.U. and Yun-zhang,H.U. (2007).Zool.Res., 28(2):213-216. 8- Prockop , D.J. (1997). Sci., 276: 71-74. 9-Nilsson, S.K.; Simmons, P.J. and Bertoncello, I. (2006). Exp . Hematol., 34: 123-129. 10-Dodson, S.A. (2000). Cell suspension comp arisons of bone p roduction in bone marrow st em cells of y oung and adult aged rats as measured in vitro by bone colony assay s .Thesis.www.term p aperp ower.com./ stem-cell-thesis. 11-Liu,Y. ;Son g,J.; Liu,W.;Wan,Y.;Chen,X.andHu,C.(2003).Cardiovasc. Res.., 58: 460-468. 12- Tomita,S.;.; Weisel, R.D.; Jia, Z.Q.; Tumiati, L.C.; Allid ina ,Y. ; and Li,R.K. (2002). J. Thorac. Cardiovasc. Sur g., 123:1132-40. 13- Javazon, E.H.; Colter, D.C.; Schwarz, E.J. and Prockop , D.J. (2001). Stem Cells, 19 (3): 219-225. 14-Di Girolamo, C.M .; Stokes, D.; Colter, D.; Phinney, D.G.; Class, R. and Prockop , D.J. (1999). Br. J. Haematol., 107(2): 275-281. 15-Friedenst ein,A.J.;Gorskaja,U.andKalugina,N.N.(1976).Exp .Hematol., 4: 267-274. 16- Xu, W.; Qi an, H.; Zhu, W.; Sun, X.; Hu, J.; Zhou, H. and Ch en, Y. (2004). Exp . Biol. M ed., 229: 623-631. 17- M nati, I. M . (2007).In vivo and in vitro studies of adult bone marrow st em cells and its role in induced myocardial infarction in albino rats. Ph .D. T hesis,College of Education/Ibn- Al- Haitham, Baghdad University : 105-111p. Table (1): Growth properties of MSCs in primary and passaged cultures Accumulative NCD Numbers of cell doubling(NCD) Logarithm ic replication phase (y) Intitial quiesc ent phase(x) Passage number 6.7 6.7 13.4X10 4 1X10 4 P0 10.4 3.7 7.4 X10 4 1X10 4 P1 13.65 3.25 6.5 X10 4 1X10 4 P2 15.35 1.7 3.4 X10 4 1X10 4 P3 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Fig.(1): Morphology of Rat MSCs primary culture.(A):The cells after three days in culture,the MSCs began to proliferate in culture medium(arrow s)(X63).(B): The cells at seven days in culture,the MSCs were appeared a spindle-like shape(arrow s)(X160). (C): The c ells after ten days of culture,the c ells formed a monolayer of adherent cells (X63) A B C IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Fig.(2): Morphology of Rat MSCs passage culture.(A):The cells from P1 of culturing, the flattened cells (thick arrow s) surrounded by number of spindle cells(thin arrows). (X100.8).(B):The cells from P2 of culturing,the c ells showed that continued to duplicate rapidly (X160).(C): The cells from P3 of culturing,the cells proliferation rate w as decreased and the morphology changed into flat appearance (arrow s)(X160) A B C IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Fig .(3): Growth curves for MSCs in primary culture (P0) and three successive subcultures (P1,P2,P3 ). (22مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد 4 (2009 الخصائص المظهریة والنمو لخالیا سدى نقي العظم للجرذان البیض خارج حيالجسم ال انتظار محمد مناتي وبیداء حسین مطلك ابن الهیثم ،جامعة بغداد- قسم علوم الحیاة ،كلیة التربیة الخالصة ان االمكانیات المحتملة الستخدام خالیا سدى نقي العظم عالج خلـوي لالمـراض المزمنـة یعتمـد علـى قـدرة تكـرار الخالیـا الــسبب فــان الدراسـة الحالیــة بحثــت تكرارالخالیـا لمــدى الحیــاة وفحـصت خــصائص النمــو بـصورة كبیــرة خـارج الجــسم الحــي ،ولهـذا .للخالیا الجذعیة اللحمیة للجرذان البیض خارج الجسم الحي ولغرض انشاء نظـام لعـزل وزراعـة الخالیـا الجذعیـة اللحمیـة للجـرذان البـیض خـارج الجـسم الحـي ولتقیـیم البیانـات البحثیـة الالحـق، فقـد جمـع نقـي العظـم مـن ذكـور الجـرذان الیافعــة وفـصل بأسـتخدام النبـذ المتـدرج الكثافـة لطـرد معظـم خالیـا الــدم لتكاثرهـا M،وبعد عملیة النبذ المتدرج الكثافة سحبت الخالیا االحادیـة النـواة مـن الطبقـة الـضبابیة وزرعـت فـي وسـط زرعـي EM مـضاف م37مصل جنین العجل وحضنت المزارع بدرجة حراریة % 10 هالی 0 .غاز ثاني اوكسید الكربون %5 وفي جو رطب مع لحمیة باالستبدال المتكرر للوسط الزرعي ،وتم القیـام بـالزرع الثـانوي فـي الوقـت الـذي تكونـت ال عزلت الخالیا الجذعیة غیر لخالیـا الجذعیـة اللحمیـة المغزلیـة الـشكل االحادیـة النـواة طبقـة احادیـة مـن الخالیـا كونـت ا.فیه طبقة احادیة مـن الخالیـا الملتـصقة وفــي االطــوار . مــن الـزرع االبتــدائي، وقـد ظهــرت الخالیــا علـى شــكل اشـعة موجــه بأتجـاه نمــو دورانـي"ا یومـ12-10بعـد مــرور لمظهـري الـى مظهـر مـسطح عـریض ،وهـذه النتـائج النهائیة للزرع الثانوي فان معدل تكاثر الخالیا بدأ باالنخفـاض وتغیـر الـشكل ا تقتـرح بــان الخالیـا الجذعیــة اللحمیـة یمكــن تكاثرهــا خـارج الجــسم الحـي بتكــوین نظـام زرعــي بــشكل امثـل ،یــضاف الـى ذلــك فــان . غارتمي وطورالتكرار اللوغارتمي وطورالتكرارما بعد اللو،طور السكون االبتدائي:ثالثة اطوار على منحنى النمو یمكن تقسیمه