Malaysian Journal of Medicine and Health Sciences Vol. 11 (2) June 2015 The Eficiency of Cell Sorting and Harvesting Methods for In vitro Expansion of Human Umbilical Cord Blood derived CD34 + Hematopoietic Stem Cells Mohadese Hashem Borojerdi, 1,2 Maryam Maqbool, 1,2 Zuraidah Yusoff, 1,2 Sharmili Vidyadaran, 1 Ling King Hwa, 3 Elizabeth George 4 & Rajesh Ramasamy*, 1,2 1 Immunology Laboratory, Department of Pathology 2 Stem Cell Research Laboratory, Genetic & Regenerative Medicine Research Center 3 Medical Genetics Laboratory, Genetic & Regenerative Medicine Research Center 4 Hematology Unit, Department of Pathology Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia ABSTRACT Introduction: During the last three decades hematopoietic stem cell transplantation (HSCT) has become a well-established treatment for many hematologic malignancies. The most important limitation for HSC transplantation is the low number of hematopoietic stem cells (HSC) that can lead to delayed engraftment or graft failures. Numerous attempts have been made to improve in vitro HSC expansion via optimization of various methods such as isolation techniques, supplementing with growth factors, utilizing stromal cells as feeder layer and other culture conditions. Objective: This project is aimed to decipher the eficiency of an isolation technique and retrieval of culture expanded HSC from feeder layer using two different harvesting methods. Materials and Methods: Hematopoietic stem cells from human umbilical cord blood were isolated via MACS mediated CD34 + double sorting. Then, the cells were cultured onto MSC feeder layer for 3 and 5 days. Culture expanded cells were harvested using two different harvesting method namely cell aspiration and trypsinization methods. Hematopoietic stem cell expansion index were calculated based on harvesting methods for each time point. Results: The numbers of HSC isolated from human umbilical cord blood were 1.64 x 10 6 and 1.20 x10 6 cells at single and double sortings respectively. Although the number of sorted cells diminished at the second sorting yet the yield of CD34 + purity has increased from 43.73% at single sorting to 81.40% at double sorting. Employing the trypsinization method, the HSC harvested from feeder layer showed a signiicant increase in expansion index (EI) as compared to the cell aspiration harvesting method (p≤ 0.05). However, the purity of CD34 + HSC was found higher when the cells were harvested using aspiration method (82.43%) as compared to the trypsinization method (74.13%). Conclusion: A pure population of CD34 + HSC can be retrieved when the cells were double sorted using MACS and expanded in culture after being harvested using cell aspiration method. Key words: Hematopoietic stem cells, Mesenchymal stem cells, Trypsin, Expansion index, CD34 INTRODUCTION Haematopoietic stem cell (HSC) transplantation has become an inevitable therapy for many blood related disorders. Among all stem cell-based therapies, hematopoietic stem cell transplantation is the only one with a well-established clinical regimen and remains an effective approach for patients with certain haematological diseases. 1 Basically, HSC transplantation involves the intravenous infusion of autologous or allogeneic stem cells collected from bone marrow, mobilized peripheral blood stem cells or umbilical cord blood to the patients. 2 Although bone marrow serves as an ideal source for HSC yet several factors such as very painful invasive procedure, post infections risk, limitation of donor availability and the age of patients often circumvent its wide applications. 3 However, the successful transplantation of umbilical cord blood (UCB) in 1988 has open the gateway to UCB for being considered as an attractive alternative source of HSC in the treatment of haematological malignancies (leukaemia & lymphoma) and non-malignant blood diseases (thalassaemia & sickle cell disease). 4,5 Although UCB transplantation has become a big breakthrough in the ield of cellular therapy, yet the number of HSC harvested for cryopreservation or therapy still remains an issue. A substantial number of umbilical cord blood that has been collected by the national and private cord blood banks are discarded due to low number of mononuclear *Corresponding author: Rajesh Ramasamy rajesh@upm.edu.my Malaysian Journal of Medicine and Health Sciences (ISSN 1675-8544); Vol. 11 (2) June 2015: 21-28