The cryopreservation protocol optimal for progenitor recovery is not optimal for preservation of marrow repopulating ability

Abstract

Summary:The efficiency of five different cryopreservation protocols (our original controlled-rate and noncontrolled-rate protocols) was evaluated on the basis of the recovery after thawing of very primitive pluripotent hemopoietic stem cells (MRA CFU-GM ), pluripotent progenitors (CFUSd12) and committed granulocyte-monocyte progenitors (CFU-GM) in mouse bone marrow. Although the nucleated cell recovery and viability determined immediately after the thawing and washing of the cells were found to be similar, whether controlled-rate or noncontrolled-rate cryopreservation protocols were used, the recovery of MRA CFU-GM , CFU-Sd12 and CFU-GM varied depending on the type of protocol and the cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better MRA CFU-GM , CFU-Sd12 and CFU-GM recovery from frozen samples. The most efficient was the controlled-rate protocol of cryopreservation designed to compensate for the release of fusion heat, which enabled a better survival of CFU-Sd12 and CFU-GM when combined with a lower (5%) DMSO concentration. On the contrary, a satisfactory survival rate of very primitive stem cells (MRA CFU-GM ) was achieved only when 10% DMSO was included with a fivestep protocol of cryopreservation. These results point to adequately used controlled-rate freezing as essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. At the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of very primitive stem cells, but not, however, for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in primitive MRA cells, which is not the case for less primitive progenitors.

Viabilidade celular da fração mononuclear da medula óssea e fração vascular estromal do tecido adiposo de equinos após o processo de congelamento e descongelamento

“…Esta injúria pode resultar da extensiva desidratação celular e/ou devido à formação intracelular de cristais de gelo. Além disso, um alto grau de destruição celular está relacionado à liberação de calor de fusão observada quando o período de transição entre as fases líquida e sólida se prolonga excessivamente (Balint et al 1999).…”

“…Para que o processo de criopreservação tenha sucesso, é necessário o uso de um agente crioprotetor que diminua o gradiente osmótico e a diferença de pressão de vapor entre os compartimentos intra e extracelular (Balint et al 1999). As soluções crioprotetoras são classificadas de acordo com a sua capacidade de atravessar a membrana plasmática.…”

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days

“…These results are comparable with recovery data reported after cryopreservation of PBSC (70-90% for nucleated cells, 33-80% for committed progenitors. 12,[30][31][32][33][34][35] In the experiments described in Table 4, three washes were performed before the addition of medium. As a consequence, recovery data were variable.…”

Cryopreservation of Hematopoietic and Non-Hematopoietic Stem Cells – A Review for the Clinician

“…However, several investigators examined the effects of lower DMSO concentrations on recovered cell counts, viability and colony forming capacity. DMSO concentration as low as 2% were used (Bakken, O Bruserud, & J F Abrahamsen, 2003;Balint et al, 1999;Galmés et al, 1999;Halle et al, 2001;Syme et al, 2004;Zeisberger et al, 2010). Overall, 5% DMSO concentrations delivered comparable results to the standard 10% concentration, whilst DMSO concentrations of less or equal to 2% revealed inferior cell integrity, at least in some reports (Zeisberger et al, 2010).…”

“…Nuclear cell count, viability and several functional assays (MRA, CFU-S and CFU-GM) were assessed after thawing and washing. Although comparable cell counts and viability assays were achieved, superior functional assays (particularly CFU-S and CFU-GM) were achieved with the controlled rate freezing procedure (Balint et al, 1999). In a clinical study from Japan, two different freezing protocols were compared in the cryopreservation of peripheral blood stem cells (PBSC) (Y Takaue et al, 1994).…”