Biomedical Engineering Reference
In-Depth Information
−120°C to −150°C (mechanical freezer or steam of nitrogen), or at −196°C (liquid
nitrogen); and (c) cell thawing in a water bath at 37 ± 3°C.
The cryopreservation of peripheral blood vs. bone marrow-derived SCs has to be
adapted to conditions which depend on the: (a) higher mononuclear cell count; (b)
presence of plasma proteins; and (c) absence of lipid and bone particles in cell sus-
pension [
33
]. Immediately after thawing in a water bath at 37 ± 3°C, cells are rein-
fused through a central venous catheter. Generally, patients tolerate the infusion of
unprocessed SCs well, with no side effects [
21,
32,
33,
42
]. However, the grade of
the potential reinfusion-related toxicity is associated with DMSO quantity in the
cell concentrate. Thus, the recovery of SCs cryopreserved by 5% DMSO reported
by Abrahamsen et al. is rather impressive [
19,
46
]. They showed that the use of 5%
rather than 10% DMSO results in an improved CD34
+
cell recovery (low apoptotic
and necrotic CD34
+
cell fraction) with a high potential for in vivo engraftment and
ex vivo manipulations of these cells. Similar findings were also reported by Rowley
et al. [
15
]. Finally, there are reports [
14,
26
] that DMSO concentrations lower than
5% (2.2 and 3.5% DMSO) are also sufficient for acceptable SC recovery.
Cryoprotectant agent can be removed by washing after thawing, but this proce-
dure results in substantial SC loss [
10,
22,
33
]. The integrity of residual granulo-
cytes is compromised within cryopreserved SCs and consequential DNA release
during the thawing procedure may lead to cell “clumping” with resulting additional
cell loss. To avoid this problem, a washing protocol by recombinant human deoxy-
ribonuclease (rHu-DNase) is recommended [
36
]. The addition of rHu-DNase to cell
concentrate seemingly proves to be effective in preventing “clumping” and it does
not cause decreased expression of adhesion molecules, although it is not free of
potential risks for patients. Moreover, the use of specific additives (e.g., membrane
stabilizers) could improve postthaw cell recovery and it is probably a more effective
approach than the decrease of DMSO concentration [
34
]. Our results are in agree-
ment with the above-mentioned studies.
Namely, we have found that the recovery of mature population of the pluripotent
and committed hematopoietic progenitors (CFU-Sd12 and CFU-GM) in the pres-
ence of 5% vs. 10% DMSO is superior [
11,
20
]. However, it has also been demon-
strated that the recovery of very primitive pluripotent hematopoietic stem cells
(Marrow Repopulating Ability—MRA) is better when 10% DMSO is used. These
results imply a different “cryobiological request” of MRA cells in comparison with
the nucleated cells and progenitors. Moreover, we have demonstrated that differ-
ences in cell recovery are not related to the changes in the total number of frozen/
thawed cells, regardless of the use of cryopreservation strategy [
11
] . Our clinical
studies showed that therapeutic use of the controlled-rate cryopreserved SCs in
treatment of leukemia (ALL, ANLL, CML), multiple myeloma, Hodgkin's and non-
Hodgkin's lymphoma, breast and ovarian cancer, and extragonadal non-seminal germ
cell tumor resulted with high cell recovery (91%) and rapid posttransplant
hematopoietic reconstitution—on the 11th day in average. Fig.
13.3
.
Finally, in Transplant Center of MMA—in addition to the routine “initial” cell-
mediated treatment using fresh ex vivo manipulated or unmanipulated cells—cryo-
preserved autologous SCs were applied for “repeated” therapy or “retreatment” of
patients with large myocardial infarction [
20,
40,
41,
47,
48
] . Fig.
13.4
.
