IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 An Experimental Study of Collection ,Separation , Enumeration and Cryopreservation of Umbilical Cord Blood Stem Cells B. H. Mutlak, I. M. Mnati Departement of Biology,College of Education Ibn Al-Haitham, University of Baghdad Abstract Umbilical cord blood (UCB) contains hematopoietic and mesenchymal stem cells(HSCs,M SCs) that have proven useful clinically to reconstitute the hematopoietic system in children and some adults . Fifteen cord blood samples were collected from placenta of newly delivered women in Al- Kadhemia hospital in Baghdad for normal vaginal delivery. Mono nucleated cells (MNCs) were isolated by using density gradient centrifugation and the MNCs count and viability were determinated by trypan blue.MNCs were cryopreserved using the cryoprotectant solution of 10% concentration of dimethyl sulfoxid (DM SO)using slow cooling and rapid thaw. The aim of the present study is to collect, separate and enumerate the stem cells in cord blood(CB) and study the effect of cryopreservation on stem cells count and viability. The results of the present study showed that the average cord blood volume collected was 81.66 ml. Mononuclear cell count ranged from 5.6-14.6 x10 6 .Viability count of mononuclear cells was 93.9%.After three months of cryopreservation , the viability count on revival was 73.9%.This study represents an attempt to isolate the stem cells from UCB. The results of the present study confirmed that UCB provides a great source of stem cells for using in medical applications. Introduction Transplantation of UCB was successfully performed for the first time in 1989[1], the potential of UCB as a source of HSCs and MSCs for transplantation 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 protocols used for this have largely been adapted from those originally designed for bone marrow (BM )stem cells ,and there is no consensus on op timal procedures for CB cells [3] . Cryopreservation processing that yields consistent recovery of functionally viable cells is crucial for the further successful use of this important cell resource [4] . To develop a successful cell cryopreservation strategy,the key events inherent to freezing a biological cell must be considered and each potential source of damage should be mitigated if at all possible.A simple way to organize these steps includes:- A:-Determining the cryoprotectant (CPA)type and concentration . B:-Determining the cooling and warming rats. C:-Preparation of cells for use including any washing procedures [4,5] . The closest consensus considering CB stem cells is presented as 5-10% concentration of dimethyl sulfoxide (DM SO) using slow cooling and rapid thaw[5]. Previous studies have examined factors that influence the viability and recovery of cells obtained from UCB after storage .Shlebak and colleagues [6] observed that liquid storage of CB for 9 h resulted a decline in post thaw recovery of colony forming unit-granulocytes monocytes (CFU-GM ).Rogers and colleagues [7] observed that the post thaw viability of CB IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 decreased when it was stored for 24 h of storage prior to cryopreservation.In contrast ,Beaujean and colleagues observed that peripheral blood (PB)stem cells could be effectively stored for 24 h prior to cryopreservation without significant loss of post –thaw viability [8].The recent series of studies have examined optimal liquid storage conditions and duration for PB, BM and CB. In general ,these studies showed that dilution the product with storage solution improved recovery of mononuclear cells .Furthermore, favorable recovery was observed using storage solutions appropriate for human use [4 ,5]. Thus ,the aim of the present study were to develop technique for collection of CB and to separate ,enumerate the stem cells in the cord blood and study the effect of cryopreservation on stem cells count and viability .This information would be essential in establishing processing guidelines for the processing of CB that ensures optimum recovery and reduces costs associated with the operation of CB processing centers. Material and Methods Fifteen UCB samples were obtained freshly from pregnant women undergoing full term vaginal deliveries were randomly selected at the time of admission for delivery from Al- Kadhmia hospital in Baghdad .All volunteers were asked to sign informed consent forms perior to collection of CB .Women with known history of hepatitis , diabetes mellitus ,severe hypertension ,abortions or bad obstetric history were excluded from the study. Umbilical cord blood samples were obtained from normal full term vaginal deliveries as per the standard method [9].The collections were made after delivery of the infant and ligation of the cord ,prior to the expulsion of the placenta. The UCB was collected while the placenta was still in utero .Using strict aseptic techniques the umbilical vein was cleansed with alcohol ,the umbilical vein was pierced and UCB collected in the standard anticoagulant treated blood collection bags .Since total collection was approximately( 65-100) ml. The details of the delivery and the new born were recorded ,UCB was transported immediately from maternity units and processed 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 grouping and revival studies . Cord blood was diluted 1:1 with phosphate buffer saline (PBS) then carefully over laid on Ficoll-paque at a ratio of 3:1 in 10 ml sterile conical tubes .The specimens were centrifuged on a cooling centrifuge for 20 min at 2000 rpm at 4C 0 .After density gradient centrifugation ,the resulting mononucleated cells (MNCs) were retrieved from buffy coat layer by pipeting and washing [1-2]times with PBS .Mononucleated cells counts and viability were done by using trypan blue dye exclusion test .UCB mononuclear cells were cryopreserved using the cryoprotectant solution which composed of (10% dimethyl sulfoxide (DM SO) +40% minimum essential medium (MEM)+50% fetal calf serum (FCS).The cells dispensed into sterile freezing ampoules (p lastic cryo vials)with 1ml per ampoules. The ampoules (which had been labeled with the name of the cells and the date of freezing ) were sealed and placed 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 C0 for 24 h and in -196C0 in liquid nitrogen tank for long –term storage [10]. After a minimum of three months of storage in liquid nitrogen , one ampoule of each sample was thawed in a 37 C0 water path for viability assessment .The resulting solutions containing the thawed cells were centrifuged in a cooling centrifuge .After removal of the supernatant ,cells were re-diluted for analysis. Results 15 samples of UCB were collected and cryopreserved. Information regarding the antenatal history ,delivery ,newborn and any complications 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. SCI VOL.23 (1) 2010 in this study was -0.78,-0.51 and 0.57 respectively ,showing thereby that these 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 paired (t)test. The (t) was 0.786 and was found to be statistically 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% prior to preservation of the sample Table (2). One ml of each sample was revived after a variable period of time but within three months of preserving the sample. The average nucleated cell count /ml of the revived sample was 0.68 x106 (i.e 68%) with a range of 42% to 99% and average viability count of 73.9% of progenitor cells Table (3). This indicated an average loss of 20% of progenitor cells Table (4) and Figure (3). Discussion Until recently ,blood that remained in the umbilical cord and placenta after delivary was routinely discarded .Human UCB is now considered a valuable source for stem cells ,this blood is known to contain both HSCs and MSCs [11] .Since the first cord blood transplant performed in 1989 [1],there has been a substantial increase in the clinical use and research investigation of UCB in haemopoietic transplantation and regenerative medicine [12]. This study was prompted by the demonstration that UCB can be used as a source of stem cell for transplantation .UCB is abundantly available and easy to collect ,and frozen ,CB is immediately available for transplantation .UCB is normally discarded ,it can be readily collected without danger to the mother or infant and technical feasibility of using UCB for transplantation has been established [13]. A number of different procedures have been proposed for UCB collections ,including open systems in which CB is collected by gravity in bottles or closed systems in which modified blood collections are used [14] .Data collected in the present study indicated that the closed system allows an average collection of 81.66 ml of CB. As our experiments have demonstrated 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 transplantation [13]. In the present study ,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 compares well with other studies . Increased volume of CB sample resulted in increased recovery of NCCs ,an important surrogate marker of HSCs and in turn of the transplant potential of the CB sample [14,15]. It appeared that cell losses did not occur in frozen samples but were due to variables in freezing and thawing procedures that were not under direct control .However ,it should be noted that it was routinely possible to obtain cell recoveries of more than 73.9% viability and cell recoveries of 68% on an average ,which though slightly similar to those in the previous studies ,were comparable [16]. References 1- Broxmeyer,H.E.;Douglas ,G.W. ;Hangoc,G.and Cooper,S .(1989) . Proc .Natl.Acad.Sci.USA,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). Cytother. 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 .Med. Hemother. 34:276-285. 6-Shlebak,A.A.;Marley,S.B.and Roberts,I.A.(1999).Bone Marrow Transplant . 23:131- 136(abstract). 7-Rogers,I.;Sutherland,D.and Holt,D. (2001).Cytother. 3:269-276. IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL.23 (1) 2010 8-Beaujean,F.;Pico,J.and Norol,F.(1996).j.Hematother. 5:681-686. 9-Rubinstein,P.;Dorila,L.and Rosenfield,R.E.(1995).Proc .Acad.Natl . Sci.USA,1995:10119-10122. 10-Al-Azawi,I.N.(2003).Experimental study on culture of mammalian embryonic stem cells .Ph.D.,thesis ,College of Science ,Al- Mustensiriya Univ.:70-72. 11-Mutlak,B.H.(2007).In vitro study of UCB-derived stem cells and their neurogenic differentiation.Ph.D.thesis,College of Education /Ibn –Al- Haitham,Baghdad Univ.:62-88. 12-Moise,K.J.(2005).Obst.Gyne. 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-Shpall,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. SCI VOL.23 (1) 2010 Table (2): Volume of cord blood collected, nucleated cell count (NCC) ,and viability count Table( 3): Volume of cord blood cryopreserved, nucleated cell count (NCC) ,and viability count Viability count % NCC\ml x106 Volume collected (ml) Sample 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 Mean Viability count % NCC/ml x106 Volume preserved Sample 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. SCI VOL.23 (1) 2010 Table (4): Comparative pre-cryo preservation and post –cryo preservation nucleated cell count and viability. Cell counts 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. SCI 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 nucleated cell 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 د،ابن الهیثم-كلیة التربیة ،قسم علوم الحیاة جامعة بغدا الخالصة ة للــدم والخالیـا الجذعیـة اللُحمیـة التـي اثبتـت فائـدتها مـن الناحیــة یحـوي دم الحبـل السـري علـى الخالیـا الجذعیـة المكونـ . السریریة ألعادة تكوین النظام المكون للدم لدى األطفال وبعض البالغین ـات اللـــواتي ولــدن طب "انموذجـــأ 15تــم جمــع ــًا فــي مستشـــفى لــدم الحبـــل الســري مـــن مشــیمة الــوالدات الحدیثـــة لألمهـ یعیـ .الكاظمیة في بغداد حیویــة واعـداد الخالیــا بأســتخدام تد، وتـم حــدعزلـت الخالیــا األحادیــة األنویـة بأســتخدام النبــذ المركـزي المتــدرج الكثــافي ا بأسـتعمال. ل األزرق صـبغة المثیـ DM) % 10محلـول الحفـظ بالتجمیـد والمكـون مـن حفظـت الخالیـ SO) وبأسـتعمال .واألذابة السریعة ، التجمید البطيء ـة أثیر الحفــظ هاوتعـداد ان هـدف الدراسـة الحالیـة هــو جمـع وفصـل الخالیـا الجذعیـ ة تـ المشـتقة مـن دم الحبــل السـري ودراسـ . بالتجمید في حیویة واعداد الخالیا الجذعیة ـا اعــداد الخالیــا األحادیــة النــواة فقــد ،مــل 66.81هــو جمــع بینـت نتــائج الدراســة الحالیــة إن معــدل حجــم الــدم الــذي امـ 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