IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 An Experimental Study o f Collection ,Separation , Enumeration and Cryopreservation of Umbilical Cord Blood Stem Cells B. H. Mutlak, I. M. Mnati Departement of Biology,College of Education I bn Al-Haitham, Unive rsity of Baghdad Abstract Umbilical cord blood (UCB) contains hematop oietic and mesenchymal st em cells(HSCs,M SCs) t hat have proven useful clinically to reconst itut e the hematop oietic sy st em in children and some adults . Fifteen cord blood samples were collected from placenta of newly delivered women in Al- Kadhemia hosp ital in Baghdad for normal vaginal delivery . M ono nucleated cells (M NCs) were isolated by using density gradient centrifugation and the M NCs count and viability were determinated by try p an blue.M NCs were cry op reserved using the cryop rotectant solution of 10% concentration of dimethy l sulfoxid (DM SO)using slow cooling and rap id thaw. The aim of the p resent st udy is to collect, sep arate and enumerate the st em cells in cord blood(CB) and st udy the effect of cryop reservation on stem cells count and viability . The results of the present st udy showed that t he average cord blood volume collected was 81.66 ml. M ononuclear cell count ranged from 5.6-14.6 x10 6 .Viability count of mononuclear cells was 93.9%.Aft er three months of cryop reservation , the viability count on revival was 73.9%.This st udy represents an attemp t to isolate the st em cells from UCB. The results of the p resent st udy confirmed that UCB p rovides a great source of st em cells for using in medical app lications. Introduction Transp lantation of UCB was successfully p erformed for the first time in 1989[1], the p otential of UCB as a source of HSCs and M SCs for transp lantation rapidly became an area of intense clinical and scientific interest [2]. In order to effectively utilize cord blood(CB) clinically ,it must be frozen and banked .The p rotocols used for this have largely been adap ted from those originally designed for bone marrow (BM )st em cells ,and there is no consensus on op timal p rocedures for CB cells [3] . Cry op reservation p rocessing that y ields consistent recovery of functionally viable cells is crucial for the further successful use of this important cell resource [4] . To develop a successful cell cryop reservation st rategy ,the key events inherent t o freezing a biological cell must be considered and each p otential source of damage should be mitigated if at all possible.A simple way to organize these st eps includes:- A:-Determining the cry op rotectant (CPA)t y p e and concentration . B:-Determining the cooling and warming rats. C:-Preparation of cells for use including any washing p rocedures [4,5] . The closest consensus considering CB st em cells is p resented as 5-10% concentration of dimethy l sulfoxide (DM SO) using slow cooling and rap id thaw[5]. Previous st udies have examined factors that influence the viability and recovery of cells obtained from UCB after st orage .Shlebak and colleagues [6] observed that liquid st orage of CB for 9 h resulted a decline in p ost thaw recovery of colony forming unit-granulocytes monocy tes (CFU-GM ).Rogers and colleagues [7] observed that the post thaw viability of CB IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 decreased when it was st ored for 24 h of st orage p rior to cryop reservation.In contrast ,Beaujean and colleagues observed that p erip heral blood (PB)stem cells could be effectively st ored for 24 h p rior to cryop reservation without significant loss of p ost –thaw viability [8].The recent series of st udies have examined op timal liquid st orage conditions and duration for PB, BM and CB. In general ,these st udies showed that dilution the p roduct with st orage solution imp roved recovery of mononuclear cells .Furt hermore, favorable recovery was observed using st orage solutions app rop riate for human use [4 ,5]. Thus ,t he aim of the present st udy were to develop technique for collection of CB and to sep arate ,enumerate the st em cells in the cord blood and st udy the effect of cryop reservation on st em cells count and viability .This information would be essential in establishing p rocessing guidelines for the p rocessing of CB that ensures op timum recovery and reduces costs associated with the operation of CB processing centers. Material and Methods Fifteen UCB samples were obtained freshly from p regnant women undergoing full term vaginal deliveries were randomly selected at the time of admission for delivery from Al- Kadhmia hosp ital in Baghdad .All volunteers were asked to sign informed consent forms p erior to collection of CB .Women with known hist ory of hepatitis , diabetes mellitus ,severe hy p ertension ,abortions or bad obst etric hist ory were excluded from the study . Umbilical cord blood samples were obtained from normal full term vaginal deliveries as p er the st andard method [9].The collections were made after delivery of the infant and ligation of the cord ,p rior to the exp ulsion of the p lacenta. The UCB was collected while the placenta was still in utero .Using st rict aseptic techniques the umbilical vein was cleansed with alcohol ,the umbilical vein was p ierced and UCB collected in the st andard anticoagulant treated blood collection bags .Since total collection was app roximately( 65-100) ml. The details of the delivery and the new born were recorded ,UCB was t ransp orted immediately from maternity units and p rocessed within 6 h .Laminar flow cabinet was cleansed with 70% ethanol and volume of UCB was measured .A volume of 5 ml of UCB was kept in aliquots for routine testing of blood group , Rh group ing and revival st udies . Cord blood was diluted 1:1 with p hosp hate buffer saline (PBS) then carefully over laid on Ficoll-paque at a ratio of 3:1 in 10 ml st erile conical tubes .The sp ecimens were centrifuged on a cooling centrifuge for 20 min at 2000 rp m at 4C 0 .Aft er density gradient centrifugation ,the resulting mononucleated cells (M NCs) were retrieved from buffy coat lay er by p ipeting and washing [1-2]times with PBS .M ononucleated cells counts and viability were done by using try p an blue dy e exclusion test .UCB mononuclear cells were cryop reserved using the cryop rotectant solution which comp osed of (10% dimethy l sulfoxide (DM SO) +40% minimum essential medium (M EM )+50% fetal calf serum (FCS).The cells disp ensed into st erile freezing amp oules (p last ic cryo vials)with 1ml p er ampoules. The ampoules (which had been labeled with the name of the cells and the date of freezing ) were sealed and p laced in +4C 0 for 12 h then -4C 0 for 12 h . The frozen vials were transferred into -20 C 0 for 24h then -70 C 0 for 24 h and in -196C 0 in liquid nitrogen tank for long –term st orage [10]. Aft er a minimum of three months of st orage in liquid nitrogen , one ampoule of each sample was thawed in a 37 C 0 water p ath for viability assessment .The resulting solutions containing the thawed cells were centrifuged in a cooling centrifuge .Aft er removal of the sup ernatant ,cells were re-diluted for analy sis. Re sults 15 samples of UCB were collected and cryop reserved. Information regarding the antenatal history ,delivery ,newborn and any comp lications were noted in Table (1). The average volume of CB collected was 81.66 ml with arrange of 65-100 ml Table( 2). The volume of CB collected was analyzed for correlation with the haemoglobin of mother ,birth weight of child ,sex of the child Table (1) and Figure( 1).The coefficient of correlation IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 in this study was -0.78,-0.51 and 0.57 resp ectively ,showing thereby that t hese parameters do not effect the volume of CB collected .The effect of volume of CB collected on nucleated cell count Figure (2) was analyzed using single tailed p aired (t)test. The (t) was 0.786 and was found to be statist ically significant; p<0.10. The average nucleated cell count /ml of CB was 9.88x10 6 and with a range of 5.6-14.6 x10 6 and an average viability count of 93.9% p rior to p reservation of the sample Table (2). One ml of each samp le was revived after a variable p eriod of time but within three months of p reserving the sample. The average nucleated cell count /ml of the revived sample was 0.68 x10 6 (i.e 68%) with a range of 42% to 99% and average viability count of 73.9% of p rogenitor cells Table (3). This indicated an average loss of 20% of p rogenitor cells Table (4) and Figure (3). Discussion Unt il recently ,blood that remained in the umbilical cord and p lacenta after delivary was routinely discarded .Human UCB is now considered a valuable source for st em cells ,this blood is known to contain both HSCs and M SCs [11] .Since the first cord blood transp lant p erformed in 1989 [1],there has been a substantial increase in the clinical use and research invest igation of UCB in haemopoietic transp lantation and regenerative medicine [12]. This st udy was p rompted by the demonstration that UCB can be used as a source of st em cell for transp lantation .UCB is abundantly available and easy to collect ,and frozen ,CB is immediately available for transp lantation .UCB is normally discarded ,it can be readily collected without danger to the mother or infant and technical feasibility of using UCB for transp lantation has been established [13]. A number of different p rocedures have been p rop osed for UCB collections ,including op en sy st ems in which CB is collected by gravity in bottles or closed sy st ems in which modified blood collections are used [14] .Data collected in the p resent study indicated that the closed sy st em allows an average collection of 81.66 ml of CB. As our exp eriments have demonst rated that collection of increased volume of CB results in increased number of recovered cells ,the ability to routinely harvest large amounts of CB should result in more cells available for use in transp lantation [13]. In the p resent st udy ,the haemoglobin of the mother ,birth weight of child ,six of children did not affect the volume of CB sample collected or the NCCs of the sample which comp ares well with other st udies . Increased volume of CB sample resulted in increased recovery of NCCs ,an imp ortant surrogate marker of HSCs and in turn of the transp lant p otential of the CB sample [14,15]. It app eared that cell losses did not occur in frozen samples but were due to variables in freezing and thawing p rocedures that were not under direct control .However ,it should be noted that it was rout inely p ossible to obt ain cell recoveries of more than 73.9% viability and cell recoveries of 68% on an average ,which though slightly similar to those in the p revious st udies ,were comp arable [16]. Re ferences 1- Broxmeyer,H.E.;Douglas ,G.W. ;Hangoc,G.and Cooper,S .(1989) . Proc .Natl.Acad.Sci.U SA,86:3828-3832. 2-Hows,J.M.(2001).J.Clin.Pathol.,54:428-434. 3-Hubel,A.;Carlquist ,D.;Clay ,M .and Cullough,J.(2003). Cy tother. 5 (5) :370-376. 4-Berz,D.;Cormack,M .and Peter ,J.(2007).Am.J.Hematol.,110:645-650. 5-Woods,E.J.;Pollok,K.E.;Byers,M .and Perry ,B.C.(2007). Transfus .M ed. Hemother. 34:276-285. 6-Shleb ak,A.A.;M arley,S.B.and Roberts,I.A.(1999).Bone M arrow Transp lant . 23:131- 136(abstract). 7-Rogers,I.;Sutherland,D.and Holt,D. (2001).Cy tother. 3:269-276. IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 8-Beaujean,F.;Pico,J.and Norol,F.(1996).j.Hematother. 5:681-686. 9-Rubinstein,P.;Dorila,L.and Rosenf ield,R.E.(1995).Proc .Acad.Natl . Sci.U SA,1995:10119-10122. 10-Al-Az awi,I.N.(2003).Exp erimental st udy on culture of mammalian embry onic st em cells .Ph.D.,thesis ,College of Science ,Al- M ust ensiriy a Univ.:70-72. 11-M utlak,B.H.(2007).In vitro study of UCB-derived st em cells and their neurogenic differentiation.Ph.D.thesis,College of Education /Ibn –Al- Haitham,Baghd ad Univ.:62-88. 12-M oise,K.J.(2005).Obst.Gy ne. 106(6):1393-1407. 13-Dhot ,C.P.;Sirohi,M .D.and Swamy,B.G.(2003).MJAFI,59:298-301. 14-Cairo,M .S.and Wagner,J.E.(1997).Blood,90(12):4665-4675. 15- Rubinstein,P.(1994).Blood Cells,20:587-600. 16-Shp all,E.;Lima,M .;Jones,R.and Champlin,R.(2007).Blood 110(8):3064-3066. Table( 1):- Age of mother, blood group, haemoglobin , weight of child and sex of child. Weight Kg Child M/ F Hb g/dl Blood group Age years Sample No. 3.5 M 10 A + 18 1 2.6 M 9.5 AB + 27 2 3.1 F 10 O + 22 3 2.25 M 12.5 O + 19 4 2.8 F 10.5 B + 21 5 2.2 M 11.5 A + 25 6 3.2 M 10 AB + 20 7 3 M 12.5 O + 19 8 2.4 F 8 A+ 23 9 2.8 M 12 A + 22 10 2.75 M 11.5 B + 25 11 2.9 M 10.5 B + 26 12 3.2 M 12 B + 18 13 2.7 F 12.5 A + 21 14 2.3 F 9 O + 23 15 IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 Table (2): Volume of cord blood coll ected, nucleated cell count (NCC) ,and viability count Table( 3): Volume of cord blood cryo preserved, nucle ated cell count (NCC) ,and viability count Viability count % NCC\ml x10 6 Volume collected (ml) S ample No. 94 10.1 80 1 91 8.2 65 2 91 7.4 85 3 95 12.8 90 4 90 8 80 5 95 8.7 70 6 96 10.4 95 7 94 8.8 80 8 96 13.75 90 9 93 14.6 95 10 94 13.8 100 11 93 8.6 75 12 95 5.6 65 13 96 9 75 14 95 8.5 80 15 93.9 9.88 81.66 M ean Vi ability count % NCC /ml x1 0 6 Volume prese rve d S am ple No. 85 0.99 59 1 82 0.91 44 2 77 0.60 37 3 65 0.57 44 4 72 0.58 60 5 52 0.45 36 6 78 0.54 37 7 67 0.42 40 8 85 0.9 42 9 80 o.8 55 10 78 0.74 53 11 80 0.73 58 12 69 0.4 9 25 13 59 0.69 40 14 79 0.80 50 15 73 .9 0.68 45 .3 Mean IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 Table (4): Comparative pre-cryo preservation and post –cryo preservation nucleate d cell count and viability. Cell c ounts before and after preservation Post-cryopreservation Pre-cryopreservation 0.68x10 6 (range 0.42 to 0.99 x10 6 9.88x10 6 (range 5.6 to 14.6x 10 6 Nucleated cell count/ml 73.9% 93.9% Viability 0 10 20 30 40 50 60 70 80 90 100 123456789101112131415 Wieght Hb Volume of cord blood Fig.( 1): Histogram depiciting the haemoglobin ,weight of the child and volume of the cord blood collected for the 15 samples IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 0 2 4 6 8 10 12 14 16 020406080100120 Volume of cord blood (ml) N u c le a te d c e ll c o u n t* 1 0 ^ 6 Fig.( 2): Scatter diagram depiciting the nucleated cell count against the volume of cord blood 0 10 20 30 40 50 60 70 80 90 100 Viability NCC post-cryopreservationpre-cryopreservation Fig.( 3): Histogram depiciting the comparative pre-cryo preservation and post-cryo-preservation nucle ated ce ll count and viability 2010) 1( 23لتطبیقیة المجلدمجلة ابن الھیثم للعلوم الصرفة وا للخالیا الجذعیة المشتقة وتعداد والحفظ بالتجمید فصلو دراسة تجریبیة لجمع من دم الحبل السري بیداء حسین مطلك وانتظار محمد مناتي جامعة بغداد،ابن الهیثم-كلیة التربیة ،قسم علوم الحیاة الخالصة السـري علـى الخالیـا الجذعیـة المكونـة للــدم والخالیـا الجذعیـة اللُحمیـة التـي اثبتـت فائـدتها مـن الناحیــة یحـوي دم الحبـل . السریریة ألعادة تكوین النظام المكون للدم لدى األطفال وبعض البالغین ــًا فــي مستشـــفى لــدم الحبـــل الســري مـــن مشــیمة الــوالدات الحدیثـــة لألمهــات اللـــواتي ولــدن طب "انموذجـــأ 15تــم جمــع یعیـ .الكاظمیة في بغداد حیویــة واعـداد الخالیــا بأســتخدام تد، وتـم حــدعزلـت الخالیــا األحادیــة األنویـة بأســتخدام النبــذ المركـزي المتــدرج الكثــافي DM) % 10محلـول الحفـظ بالتجمیـد والمكـون مـن حفظـت الخالیـا بأسـتعمال. ل األزرق صـبغة المثیـ SO) وبأسـتعمال .واألذابة السریعة ، التجمید البطيء المشـتقة مـن دم الحبــل السـري ودراسـة تـأثیر الحفــظ هاوتعـداد ان هـدف الدراسـة الحالیـة هــو جمـع وفصـل الخالیـا الجذعیــة . بالتجمید في حیویة واعداد الخالیا الجذعیة امــا اعــداد الخالیــا األحادیــة النــواة فقــد ،مــل 66.81هــو جمــع بینـت نتــائج الدراســة الحالیــة إن معــدل حجــم الــدم الــذي 14.6x -5.6 تراوحـت بـین 6 وبعـد مـرور ثالثـة اشـهر مـن %.93.9فـي حـین كانـت حیویـة الخالیـا األحادیـة النـواة هـي ، 10 %.73.9الحفظ بالتجمید فأن حیویة الخالیا التي تم اعادة انعاشها هي الجذعیــة مـن دم الحبــل الســري واكـدت نتــائج الدراسـة الحالیــة ان دم الحبــل تعـد الدراســة الحالیـة محاولــًة لفصــل الخالیـا فـي كثیـر مـن األسـتخدامات ذعیـة التـي یمكـن حفظهـا واسـتعمالالتالسري یعد مصدراً سهل المنال للحصول على الخالیا الج .الطبیة