DMSO and non DMSO clonogenic assays from thawed cord blood
P Solves, L Larrea, MA Soler, V
Mirabet, R Moraga, EM Franco
Valencia Cord Blood Bank, Processing and Cryopreservation Service,
Valencia Transfusion Center, Spain
Correspondence: Pilar Solves, Valencia Transfusion Centre, Avda
del Cid 65-A, 46014 Valencia, Spain. Phone: 00 34 96 386 81 42.
Fax: 00 34 96 350 24 69. E-mail: solves_pil@gva.es
Clonogenic assays are currently used to determine in vitro
engraftment potential of bone marrow, mobilized peripheral
progenitor cells and recently umbilical cord blood.1
The main problem of this test is the lack of standarization as
showed by inter-laboratory, intra-laboratory and inter-personal
variations.2,3 Commercial kits availability have
decreased the variation although it persists.4
Quantification of clonogenic assays is performed in our laboratory
after cryopreservation and post-thawing with dimethyl sulfoxide
(DMSO). We compared post-thawing umbilical cord blood clonogenic
assays with and without DMSO.
Cord blood units were collected, depleted volume and cryopreserved
following the method described by Rubinstein et al. For each unit,
three satellite cryovials were also cryopreserved. Each cryotube
containing frozen cells was placed in a 37ºC water bath and
thawed with a gentle agitation. Samples for DMSO assays were drawn
from the cryovial and cultured directly in Petri dishes. The rest
of cells were washed with albumin and dextran and resuspended in
washing solution to remove dimethyl sulfoxide, as described
previously.5 Samples from washed cells were drawn for
non-DMSO cultures. Clonogenic assays were performed using a
commercially prepared complete methylcellulose medium (Methocult
GF H4434), supporting growth of CFU-GM, BFU-E and CFU-GEMM.
Cultures were plated at a range of 1-4x104 cells in
duplicate 35 mm diameter Petri dishes and incubated for 14 days at
37ºC with 5% CO2 in a humidified atmosphere. Colonies defined
as aggregates of more than 40 cells were counted under an inverted
microscope, at the end of incubation period. Colonies were
quantified by the same technician to avoid inter-personal
variations. Cord blood units total colony forming units were also
calculated. Statistical Package for social science (SPSS) v.s 8.0
for Windows was used to perform the statistical analysis. The
non-parametric tests Mann-Whitney U and Wilcoxon were used to
compare both groups. A p value of less than 0.05 was considered
significant (Table
1).
Thirty-eight samples of cryopreserved cord blood units (CB) were
cultured. CB total nucleated and CD34+ cells content mean and
range were 7.08x108 (18.66) and 2.59x104
(10.73). Cultured volume median and range were 5.5 (12) and 34.27
(84) for DMSO and non-DMSO cultures respectively (p<0.05). DMSO
final concentration median in culture medium was 0.06% (range:
0.13). Wilcoxon test showed statistically differences for all
variables except for GEMM x104 (p:0.071). Results are
shown on the attached table.
There is no a general agreement about the optimal clonogenic
cultures methodology. Many factors like kind of medium
(commercial, house-in) and the subjectivity of observer can modify
the results.2-4 Because of this variability, clinical
significance of this test is not clear and clinicians usually
don´t take into account these results for decisions. On the
other hand, post-thawing clonogenic assays can serve as quality
control of freezing in cryopreservated cord blood units and cells
can be cultured with DMSO or after release it by washing. In this
sense, culture samples with DMSO simplify the methodology and
limit the intra-laboratory variability, by avoiding cells washing.
Feasibility of DMSO cultures was confirmed by Broxmeyer et al who
showed that the amount of DMSO added to the 1 mL culture dishes
were diluted below the concentration that would interfere with the
growth of the progenitor cells.6
We conclude that post-thawing DMSO cord blood clonogenic cultures
is an easy method useful as quality control of umbilical cord
blood cryopreservation. This methodology could also be used for
progenitor cells from bone marrow or peripheral blood.
