OPTIMIZATION OF THE CONDITIONS IN ISOLATION OF NUCLEATED CELLS FROM UMBILICAL CORD BLOOD USING HYDROXYETHYL STARCH

Implication of Umblical cord blood (UCB) as a safe and rich source of stem
cell has opened a new window toward treatment of different disease in regenerative medicine. Many
various methods have been exploited to purify the UCB in order to increase the percent of nucleated
cells (NCs) and decrease the amount of red blood cells (RBCs) which is required for better outcome of
transplantation. Hydroxyl ethyl starch (HES) is a sedimentation factor used for RBCs sedimentation
and raised some paradox competence in different studies. Thus, in the present study we are aimed to
determine the correlation among HES concentration, centrifugation force, temperature, tube diameter
and time on the isolation efficiency of NCs and RBCs sedimentation.
Materials and methods: UCB was collected from the woman who was undergone normal vaginal
delivery. The efficiency of isolation was investigated through enumeration and viability of NCs and
RBCs in each step on taken UCB beyound the different concentrations of HES (0.5%, 1.1%, 1.8%,
2.5% and 3%), various relative centrifugation forces (RCFs) of 50g, 100g and 200g for 1, 5 and 10
minutes, diverse 22° C, 10°C and 4°C temperatures and different . In addition UCB sample was
incubated in 22 ° for 30, 60, 90, 120, 150 and 180 minutes to test the effect of time on RBCs
sedimentation. The height to diameter ratio effect of test tube also was considered.
Results: The mean percents of isolated NCs were 66.8±4.1%, 73.3±3.2%, 75.1±3.6%, 76.9±2.9 % and
69.9±4.3% for the concentrations of 0.5 %, 1.1%, 1.8%, 2.5% and 3% respectively. The most NCs
were isolated by employing RCF of 50g and temperature of 22 °. Incubation in 30, 60, 90 minutes led
to isolation of 57±2.6%, 63±3.7% and 64±3.1% of NCs. Using of tubes with the diameters of 25 mm
and 15 mm resulted in 58±6% and 76±3.8 % isolation.
Discussion and conclusion: The number of NCs and RBCs in UBC palys pivotal role in
transplantation success. The maximum NCs isolation and RBCs sedimentation percent were 76% and
90% respectively under the following condition: RCF of 50g, centrifugation for 10 minutes, and
incubation for 60 minutes (p<0.05) and HES 2.5%. Further studies are required to assess the effect of
other factors on isolation rate of NCs and RBCs sedimentation.