Collection, Separation, Enumeration and Cryopreservation of Umbilical Cord Blood Haematopoietic Stem Cells – An Experimental Study

Abstract

Cord blood as a source of haematopoietic stem cells (HSC) has several advantages as it is easily available, involves non-invasive collection procedure and is better tolerated across the HLA barrier. Since the first cord blood transplant in 1988, over 1000 cord blood HSC transplants have been done world wide. The present study was carried out for collection, separation, enumeration and cryopreservation of cord blood HSC. 30 samples of cord blood HSC were collected after delivery of infant prior to expulsion of placenta. The average cord blood volume collected was 101.33ml. Mononuclear cell count ranged from 7.36 to 25.6 X 10 7 ml. Viability count of mononuclear cells was 98.4%. After 6 months of cryopreservation, the viability count on revival was over 82.1%. MJAFI 2003; 59 : 298-301Key Words : Cord blood; Cryopreservation; Haematopoietic stem cell; Viability yield. Secondly, to separate and enumerate the HSC in the cord blood and study the effect of cryopreservation on stem cell count and viability. Material and Methods30 full term pregnant women undergoing full term vaginal deliveries were randomly selected at the time of admission for delivery. All volunteers were asked to sign informed consent forms prior to collection of cord blood. Women with known history of hepatitis, infectious diseases, diabetes mellitus, severe hypertension, abortions or bad obstetric history were excluded from the study. Umbilical cord blood (UCB) samples were obtained from normal full term vaginal deliveries as per the standard method [4,5]. 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 followed by betadine. The umbilical vein was pierced and UCB collected in the standard blood collection bags containing citrate phosphate dextrose adenine-1 (CPDA-1) anticoagulant (approximately 25 ml) since total collection was approximately 100-120 ml. During collection the blood bag was shaken gently, so that the anticoagulant freely mixed with UCB. The blood bag with anticoagulant was weighed before and after collection blood to find out the volume collected. The details of the delivery and the new born were recorded and documentation carried out meticulously. UCB was transported immediately from maternity units carefully in a plastic box at 4°C without delay. UCB units were stored at 4°C and processed within 24 hours. 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, sterility and tests for HIV-1 and 2, HBsAg, HCV and Syphilis. Aerobic bacterial cultures

Conditioned medium: a new alternative for cryopreservation of equine umbilical cord mesenchymal stem cells

“…The reduction in cell viability after cryopreservation (M1–M3), compared to fresh cells (P2), is possibly due to osmotic stress which occurs during the freezing and thawing process, which proved to be more pronounced in cells cryopreserved in the absence of FBS (M2). The loss of cell viability was also observed in other studies after cryopreservation of different cell (Dhot et al, ; Vrhovac et al, ; Ribeiro et al, ; Inamdar and Inamdar, ; Kim et al, ; Munévar et al, ). It is noteworthy that the loss of viability did not affect the adhesion and clonicity shown in CFU‐F assay of UCIM‐MSC cryopreserved with medium M1 and M3, since the CFU‐F efficiency did not differ ( P < 0.05) in these groups to fresh cells.…”

Therapeutic effect of human umbilical cord blood progenitor (CD34+) cells in experimentally induced renal failure in adult albino rats. A histological and immunohistochemical study

“…The renal function was impaired 24 h after glycerol administration, which was identified by the significant The samples were stored at 4°C and processed within 24 h of collection [16].…”

Following the banking cycle of umbilical cord blood in India: the disparity between pre-banking persuasion and post-banking utilization

“…However, there are reports that suggest stem cell research using UCB is being carried out at several centers. For example, a joint study by Dhot et al (2003) collected and cryopreserved. The study aimed to improve techniques for the collection of cord blood, to study the influence of cryopreservation on stem cell count, and to carry out cord blood stem cell transplantation at AHRR in New Delhi.…”

“…The study aimed to improve techniques for the collection of cord blood, to study the influence of cryopreservation on stem cell count, and to carry out cord blood stem cell transplantation at AHRR in New Delhi. For this study, pregnant women undergoing full term vaginal deliveries at these centers were randomly selected at the time of admission for delivery, with informed consent (Dhot et al: 2003;Wadhwa et al 2013). …”