IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.22 (4) 2009 Morphological and Growth Properties of Albino Rat Bone Marrow Stromal Cells In Vitro I. M.Mnati and B.H. Mutlak Deparatment of Biology, College of Education , Ibn Al- Haitham , University of Baghdad Abstract The potential use of bone marrow stromal cells as a cellular therapy for chronic diseases relies on the ability of the cell to replicate extensively in vitro.For this reason the present study investigated the replication lifespan and examined the growth properties of albino rats mesenchymal stem cells(M SCS)in vitro. To establish an in vitro system for isolating and culturing the MSCs of albino rats and to provide research data for its further application,the bone marrow (BM)was collected from young male rats and separated by gradient centrifugation.Then, the mononuclear cells(M NCs) were retrieved from the buffy layer and cultured in Modified Eagle , s Medium (MEM) supplemented with 10%Fetal Calf Serum (FCS)and incubation at 37C o in humid air with 5%CO2 . The non-MSCs were screened out.A passage culture was undertaken while the monolayer of adherent cells formed .The sp indle –shaped MSCS with a single nucleus formed a monolayer after the 10-12 days of primary culturing,and the cells appeared in an oriented array with a swirling growth trend. In the anaphase of passage culture ,the cell proliferation rate was decreased and the morphology changed into broad flat appearance.These results suggested that MSCS of the albino rats can be passaged in vitro with the established optimized culture system,in addition, the growth curves can be divided into three phases:the initial quiescent phase,the logarithmic replication phase and after logarithmic replication phase . Introduction Bone marrow is the major source of stromal cells and adult hematopoietic stem cells (HSCS) that renew circulating blood elements;these cells can also be found in other tissues .Adult BM also contains MSCs. MSCs are thought to be multipotent cells that can replicate as undifferentiated cells and have the potential to differentiate to lineages of mesenchymal tissues,including :bone ,cartilage muscle, nerves and marrow stroma [1,2].Considering their advantages ,such as ease of obtaining by a simple routine BM a spiration ,ability to self –renew and recently reported potential to differentiate into cardiomyogenic cells, MSCs have been considered as one of the most promising candidates for this purpose and the apparent multipotent nature of MSCs makes them excellent candidates for tissue engineering [3]. The MSCs were first described in 1970 by Friedenstein et al., [4]who discovered that the cells adherent to tissue culture plates resembled fibroblasts in vitro and formed colonies. He and others demonstrated MSCs could differentiate into multiple pathways that include cardiac myogenesis[5,6].Among the most extensively studied of adult stem cells from BM, if plated at low densities, MSCs generate cell single derived colonies and the colonies can be differentiated into several cell phenotypes in culture.These characteristics have been identified from numerous species including humans, rats ,mice, and monkeys [7,8 ]. Interest with BMSCs has begun since 1960 and since those several techniques have been developed to proliferate these cells and then to differentiate them into the required cells to benefit from them in the basic and clinical researches.Recent attention has focused on BM as a IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.22 (4) 2009 source of stem cells for transplantation into the heart.At present, the BM transplantation are normal operation which are used for treatment of many diseases [9].The use of MSCs in such therapeutic strategies relies on the ability of the cell to replicate extensively in vitro .For this reason the present study systematically examined the growth properties of rat M SCs in vitro and we report here the isolation ,expansion and characterization of the multipotent rat MSCs. Materials and Methods - Isolation of MSCs from albino rats bone marrow This study comprised 30 young male rats(Rattus rattus norvegicus albinos ) aged (180-200g) were used for the isolation of MSCs from the BM and were culturing in vitro, ,these animals were obtained from the Medical Research Unit College of Medicine in Al-Nahrain University . The primary culture of MSCs were performed 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 pellet was washed in phosphate buffer saline( PBS),centrifuged at 2000 rpm for 10 min and then resuspended in Modified Eagle ,s M edium(MEM) .The cell suspension was loaded on 60% percoll and then centrifuged in a cooling centrifuge for 20 min at 2000 rpm at 4C o .After centrifugation, the mononuclear cells (M NCs)was removed from the interface and resuspended in MEM. MSCs were plated at concentration of 1X10 6 cells/ml in 50 cm2 plastic tissue culture flask. -Culture and expansion Following plating ,MSCs were maintained in a humidified incubator at 37C o /5%CO2 in MEM +10%FCS . The medium was changed after 24 hours to remove nonadherent cells and twice weekly thereafter. Culture MSCs were observed under inverted microscope to assess the level of expansion and to verify the morphology at each culture medium change. To prevent the MSCs from differentiating ,each primary culture was replated for subculture. Cells were passaged with 2 ml of 0.25% trypsin- 0.1%Ethylene diamine tetra acetic acid (EDTA) upon reaching 70-80% confluency and expanded until passage 3,where upon they were analyzed. -Quantification of MSCs growth To determine the initial density of MSCs in primary cultures,the adherent cells were detached with 0.25% trypsin- 0.1% EDTA and stained with 0.2% trypan blue .Triplicate samples from each well of 4-well plastic plates were resuspended in 2ml of PBS and count with a hemocytometer under light microscope [11]. The average cell number calculated from four wells of each of three plates (1X10 4 cells/well ; n=12) was taken as the initial density of MSCs in primary cultures and the seeding density for every following subcultures .This cells counting procedure was repeated every 48 h until the cells almost ceased proliferation.The growth curves and the precise growth properties of MSCs in the primary cultures (P0)and the three subcultures (P1-P3) were determined .The numbers of cell doublings (NCD) were calculated with the 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 passage . Results - Rat MSCs primary culture IBN AL- HAITHAM J. FOR PURE & APPL. SCI. 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 shape with few morphological changes.Under an inverted microscope, morphological conservation was observed , the results showed that the parental MSCs were characterized by an enlarging appearance.After three days of primary culture,the MSCs were observed attach to the culture flask sparsely and these cells began to proliferate in culture medium(Fig.1 A). At day 7,sp indle shape was observed in well- spread colony –forming cells.Subsequently ,most adherent cells grew in a sp indle shape characteristic of fibroblasts (Fig.1 B). By day 10,the MSCs was duplicating rapidly and the cell morphology was mainly sp indle- shaped(Fig.1C). Observation on day 10-12 revealed that the cell monolayer was forming with constant orientation and a whirling tendency , which means the cells have the potency to be passaged in ratio 1: 2 . - Rat MSCs passage culture Passaged MSCs behaved similarly to those in primary cultures. The MSCs in subcultures could be divided into two types: small sp indle- like cells and broad flattened cells.The flattened cells seldom proliferated and were gradually surrounded by the small –like cells that replicated faster and were more inclined to form colonies.It seemed that the spindle-like cells gradually transformed into broad flattened cells with later passages.P1 of the mesenchymal cells was characterized by the ability to proliferate in culture with an attached well-spread morphology, reaching confluence on day 6-8.In this passage,the cells continued to duplicate rapidly (Fig.2 A). Microscopic observations showed that P2 ,cells remain fast growing and reach70-80%confluence by day 6 (Fig.2 B). However the cell proliferation rate decreased in P3 comparied with the earlier passages,the cell proliferation obviously slowed down on day 6.In this case,a few typical sp indle cells can be observed under microscope and more flat cells were found in the field (Fig.2 C). In this study, the criterion for passaging was noticed a half of colonies reached 70-80% confluence, usually 10-12 days after seeding for primary culture and 6-8 days after subculture. -Growth properties of MSCs in culture The growth curves of MSCs in primary culture and in the three passages are shown in (Fig.3).For primary cultures, the cells remained quiescent during the first two days 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.The patterns of the growth curves of MSCs in P1 and P2 were similar to those of primary cells.However, the cells showed a shortened quiescent period 1-2 days before proliferation and the cells number kept increasing for a shorter period 4-6 days in P1 and P2.The increase of average cell numbers was considerably slowed in P3 and it is expected the cells almost ceased proliferation in later passages. To determine the changes in precise growth properties of MSCs with passages, the growth course of MSCs in the primary culture and in each successive subcultures was divided into three phases according to their growth curves: the initial quiescent phase,the logarithmic replication phase,and after logarithmic replication phase.Rat MSCs(rM SCs) could be expanded up to 15.35cell doublings of successive subcultures.The 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 passages. Discussion Mesenchymal stem cells are thought to be multipotent cells,which are present in adult marrow.They can replicate as undifferentiated cells and have the potential to be induced to IBN AL- HAITHAM J. FOR PURE & APPL. . VOL.22 (4) 2009 differentiate exclusively into chondrocytic or osteocytic lineages[8].This study has important limitations which are represented by the use of young animals as the source of BM-derived MSCs. Our data is in line with the study made by Tomita et al., [12] who found that BM of young and growing pigs may have contained progenitor cells than would be found in older animals.Primary cell cultures from young animals grow more rapidly than do cells obtained from older animals. However,the expandability of MSCs in vitro varied dramatically among different species and different methodologies for isolation and plating of the cells.It has been reported that murine MSCs were much more difficult to grow than other species[6] and this was probably due to the higher sensitivity of the murine MSCs to the initial seeding density[13].Here,the present study showed that at seeding density of 1X10 4 cells/well,rat M SCs could be expanded to 10.4 cell doublings after 17 days of culture(P0-P1)and up to a maximum 15.35 cell doublings in 30 days,this resembles the reported in vitro lifespan of human MSCs[14]. Multiple factors,such as medium ,serum selection ,seeding ratio and micro-environment (pH and temperature)can affect the cell yield and passage.In our experience, MSCs can be isolated by their adherence properties to the plastic surfaces of culture flasks,displaying fibroblast-like growth characteristics and increased proliferation[15].After two days of culture,MSCs appeared sparsely and began to proliferate in culture medium, and in the end of the second week they formed the monolayer of adherent cells .These results correspond to other studies results [11,16 ,17] who have arrived at regarding forming the monolayer of primary culture approximately in the second week of culturing. In our pilot experiments,it was found that if the cells were grown over two weeks before subcultures,they quickly lost the ability to proliferate within 1-2 passages. It is known that p roliferation of cultured cells is usually promoted when cells begin to release their own cytokines and inhibited by cell-to-cell contacts upon confluency.Although a more frequent subculture can delay the senescence of MSCs,they gradually lost the ability to proliferate with the increasing of cell doublings,and this was accompanied by a gradual morphological conversion of the small spindle-like MSCs into the broad flattened cells,together with the evidence from several other laboratories [11,14]. Because the amount of MSCs is too low (0.001-0.01%) in adult BM(1),the best way to obtain sufficient MSCs is in expanding in culture.The growth kinetics of these cell populations revealed rapid adherence through to P2,but the cell proliferation rate decreased in P3 compared with the earlier passages, which was similar to[11,17]. According to these results, it appeared that the nature of rMSCs have several properties as follows:- 1. MSCs are more sensitive to p lating density . 2. They expand more rapidly after low-density plating. 3. They require frequent culture medium changing. 4. They form confluent cultures after low-density plating. From the results of this study,it was concluded that,the BM-derived MSCs of albino rats can be isolated,proliferated and maintained them in active state for several weeks and examined the growth properties of MSCs in vitro. References 1-Minguell,J.;Erices,A.andConget,P.(2001).Exp.Biol.Med.,226:507-520. 2-Pittenger, M.F. and Martin, B.J. (2004). Circ. Res., 95:9. 3-Makino,S.;Fukuda, K;Pan, and Ogawa,S.(1999).J. Clin. Invest., 103(5): 697-705. 4-Friedenstein,A.J.; Deriglasova, U.F. and Kulagina, N.N. (1974). Exp. Hematol., 2: 83-92 IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.22 (4) 2009 5-Piersma, A.H.; Ploemacher, 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 comparisons of bone production in bone marrow stem cells of young and adult aged rats as measured in vitro by bone colony assays .Thesis.www.term paperpower.com./ stem-cell-thesis. 11-Liu,Y.;Song,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.; Allidina ,Y. ; and Li,R.K. (2002). J. Thorac. Cardiovasc. Surg., 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-Friedenstein,A.J.;Gorskaja,U.andKalugina,N.N.(1976).Exp.Hematol., 4: 267-274. 16- Xu, W.; Qian, H.; Zhu, W.; Sun, X.; Hu, J.; Zhou, H. and Chen, Y. (2004). Exp. Biol. Med., 229: 623-631. 17- Mnati, I. M. (2007).In vivo and in vitro studies of adult bone marrow stem cells and its role in induced myocardial infarction in albino rats. Ph .D. Thesis,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) Logarithmic replication phase (y) Intitial quiescent phase(x) Passage number 6.7 6.7 13.4X104 1X104 P0 10.4 3.7 7.4 X104 1X104 P1 13.65 3.25 6.5 X10 4 1X10 4 P2 15.35 1.7 3.4 X104 1X104 P3 IBN AL- HAITHAM J. FOR PURE & APPL. SCI. 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(arrows)(X63).(B): The cells at seven days in culture,the MSCs were appeared a spindle-like shape(arrows)(X160). (C): The cells after ten days of culture,the cells formed a monolayer of adherent cells (X63) A B C IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.22 (4) 2009 Fig.(2): Morphology of Rat MSCs passage culture.(A):The cells from P1 of culturing, the flattened cells (thick arrows) surrounded by number of spindle cells(thin arrows). (X100.8).(B):The cells from P2 of culturing,the cells showed that continued to duplicate rapidly (X160).(C): The cells from P3 of culturing,the cells proliferation rate was decreased and the morphology changed into flat appearance (arrows)(X160) A B C IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.22 (4) 2009 Fig .(3): Growth curves for MSCs in primary culture (P0) and three successive subcultures (P1,P2,P3 ). 2009) 4 (22مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد 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 مـضاف MEM،وبعد عملیة النبذ المتدرج الكثافة سحبت الخالیا االحادیـة النـواة مـن الطبقـة الـضبابیة وزرعـت فـي وسـط زرعـي م37مصل جنین العجل وحضنت المزارع بدرجة حراریة % 10 هالی .غاز ثاني اوكسید الكربون %5 وفي جو رطب مع 0 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 ه بأتجـاه نمــو دورانـي"ا یومـ12-10بعـد مــرور ـا علـى شــكل اشـعة موجــ وفــي االطــوار . مــن الـزرع االبتــدائي، وقـد ظهــرت الخالیـ 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