Effects of cryoprotectants on freezing, Culture behaviour and apoptosis of buffalo umbilical cord matrix cells
Abstract
Umbilical cord represents the link between mother and fetus during pregnancy. Postnatally umbilical cord is a discarded organ and the collections of umbilical cord cells do not require an invasive procedure with ethical concerns. It is composed of a special embryonic mucous connective tissue called Whartson’s jelly and its cells have the properties of stem cells. Umbilical cord matrix cells are cryopreserved to study their future applications. Cryopreservation is a method to protect cells from freezing injury. Cells are vulnerable to cryopreservation induced apoptosis due to artificial cleavage of apoptotic protein during freezing and thawing. A family of cysteine proteases present in most cells is responsible for apoptosis. In the present study confluent cultures from buffalo umbilical cord matix cells were frozen at three different passages (P3, P6, P9) in three different combinations of cryoprotectants viz. DMEM culture medium containing 20% FBS and 10% DMSO, 1.5 M ethylene glycol and 0.2 M sucrose in PBS and 4% DMSO, 6% trehalose and 90% FBS. Pre and post freezing cell viability (%) was assessed using trypan blue dye exclusion method. The cells in (FBS-DMSO) and (EG-Sucrose) had more post thaw viability than (DMSO- trehalose –FBS) cryoprotectant combination. In post thaw culture behavior studies cells cryopreserved in (FBS-DMSO) started adhering to the surface of flask within 4 hrs and could proliferate well with similar morphology as that of continuous culture. Most of the cells cryopreserved in (EG-Sucrose) could not adhere to the surface of flask even after 24 hrs. The cells cryopreserved in (DMSO- trehalose –FBS) could adhere to surface of flask after 10-12 hrs and could grow well without any marked change in morphology. The RT–PCR analysis of antiapoptotic gene, Bcl-2, and proapoptotic gene, Bax with house keeping gene (β actin) of pre-freezing and post thawed cells showed that both the genes were expressed from cells of each group at P3, P6 and P9 before freezing and after thawing. In conclusion, the umbilical cord matrix cells have similar post thaw viability in (FBS-DMSO) and (EG-Sucrose) cryoprotectanat combinations but post thaw culture behavior is better in (FBS-DMSO). The expression of Bcl-2 and Bax genes in pre and post thawed cells indicate that quantitative expression studies are required to ascertain their level of expression.