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Stem Cell Culture in Bioreactor

Hosseini Zand, Hasti | 2012

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 43783 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Abdekhodaie, Mohammad Jafar; Ebrahimi, Marziye; Yaghmaei, Soheyla
  7. Abstract:
  8. Static culture systems, such as well-plates, T-Flasks and gas-permeable blood bags are restricted by their limited number of hematopoietic stem cells (HSCs) available. Hence, stirred culture systems are alternative options due to their appropriate culture conditions. Ex-vivo expansion of HSCs in suspension bioreactors has been successfully developed in recent years. The purpose of this study is comparing HSCs expansion in bioreactor with reciprocating impeller and static culture, investigation the effect of rotational speed changes in suspension culture on HSCs expansion and comparing the expansion potential of static and suspension cultures with rotational movement.
    Expansion of umbilical cord blood mononuclear cells (MNCs) was carried out in a 200 ml Lambda bioreactor at 0.1 Hz reciprocational speed, a 50 ml spinner-flask at 20, 40 and 60 rpm rotational speeds and a 20 ml glass petri dish in serum-containing IMDM medium supplemented with 10% Fetal Bovine Serum (FBS), 10% Cord Blood Serum(CBS) and relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 2.13 ng/ml GM-CSF and 7.47 ng/ml FLt-3) for 14 days. Lambda bioreactor operating conditions were temperature of 37° C, pH of 7.2 and dissolved Oxygen concentration of 5.4 mg/l. the two other culture systems were incubated in a humidified incubator (37°C, 5% CO2 pressure and 95% humidity). Initial cell density in all systems was 106 cell/ml. Sampling was done on days 0, 3, 7 and 14 in order to evaluate cell expansion, viability percent, potential of colony forming, expression of surface markers and glucose consumption and lactic acid production rates. For sampling, half of the medium was removed, after centrifugation and collecting sufficient number of cells, rest of them with fresh medium were returned to culture. Medium exchange was performed on alternative days if necessary. The expansion results of Lambda bioreactor were so weak. The low cell expansion rate of Lambda bioreactor came from the specific moving style of impeller which resulted in cell damages. At the end of the culture, the total cell number in suspension culture at the rotational rate of 40 rpm and static culture were 5.32×106±0.0588 and 2.43×106±0.205, respectively. Total colony-forming units in suspension culture at the rotational rate of 40 rpm and static culture were 59.67±9.84 and 19.22±3.54, respectively. It is demonstrated that spinner-flask at 40 rpm rotational rate can provide HSCs with more appropriate and homogenous environment for expansion than static culture and spinner-flask at other rotational rates. In addition, appropriate result of cell expansion in Spinner-Flask was gained in the conditions with low doses of cytokines, which was a crucial point of this study
  9. Keywords:
  10. Shear Stress ; Reproduction ; Umbilical Cord Blood Hematopoietic Stem ; Suspension Bioreactor ; Static Culture

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