Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

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Transfusion and Apheresis Science

journal homepage: www.elsevier.com/locate/transci

Thawing methods do not affect cell viability of CD45+ and CD34+ cells,
but long-term cryopreservation of umbilical cord blood units generally
decreases cell viability
Jisela Dimas-González, Adán Nieto-Linares, Miriam Millán-Rocha, José Luis Salazar-Bailón,
⁎
Bardo Abraham Lorenzo-Moreno, Julieta Rojo-Medina
Centro Nacional de la Transfusión Sanguínea, Av. Othón de Mendizábal 195, Zacatenco, Gustavo A. Madero, 07360, Mexico City, Mexico.

A R T I C LE I N FO A B S T R A C T

Keywords: Introduction: Umbilical cord blood units (UCBUs) are collected and cryopreserved in biobanks for a future
Umbilical cord blood transplant. Hematopoietic stem cells and hematopoietic progenitor cells (HSC/HPC) present in UCB can be
Cryopreservation damaged due to factors such as the cryopreservation process, the thawing process, and prolonged storage time.
vCD34+ Methods: UCBUs (n = 27) were obtained from the Biobank of the National Center of Blood Transfusion (NCBT)
vCD45+
from Mexico. They contained three attached segments of UCBU, including 1.0–2.3 × 106 CD34+ cells prior to
Colony-formed-unit
Segment
cryopreservation and were stored during the period from 2003 to 2015. Each UCB segment was thawed with
three different methods and CD34 cells, CD45 cells, and 7-AAD were identified by flow cytometry. Furthermore,
we carried out CFU assays, and trypan blue staining.
Results: Viable CD45+ (vCD45+) cells, vCD34+ cells, CFU, and percentage of E-Clone were not statistically
significant among three different thawing methods. The number of vCD45+ and vCD34+ cells diminished in
the three thawing methods compared with the same cells prior to their cryopreservation (p < 0.0001). vCD45+
and vCD34+ cells diminished in the ≥10-year cryopreservation group (p < 0.001). In addition, percentage of
recovery of vCD45+ and vCD34+ cells diminished in this same group (p = 0.013 and p < 0.0001, respec-
tively).
Conclusion: The thawing methods did not affect either cell viability (vCD45+ and vCD34+ cells) or plur-
ipotency (CFU, percentage of E-Clone) in attached segments of UCBUs. The ≥10-year cryopreservation time in
attached segments of UCBUs alters the number of vCD45+ and vCD34+ cells; however, it does not affect their
pluripotency.

1. Introduction different isotonic solutions, thawing-diluting and eliminating the

Umbilical Cord Blood (UCB) is a source of HSC/HPC, which are
utilized as a treatment alternative in patients with oncologic and/or
non-oncologic diseases [1]. HSC/HPC are multipotent, possess the ca-
pacity of self-renewal, asymmetric cell division, and have high potential
for proliferation and long telomeres [2,3]. For several years, cryopre-
servation has been used as a common process in UCB biobanks for
maintaining the HSCs/HPCs of UCBUs. When it is necessary, UCBUs are
thawed prior to their transplant [4]. Unfortunately, the processes of
cryopreservation and thawing are factors that diminish cell viability,
pluripotency, and HSC/HPC function [4,5]. There are different methods
for thawing UCBUs, which consist of thawing-washing-diluting with
washing step, and only thawing; these methods tend to be controversial
[4,6–10]. Another factor that influences the stability of cryopreserved
UCBUs is storage period. Cryopreserved UCBUs for 5, 10, 15, and > 20
years have been evaluated; in these UCBUs, the recovery percentage of
total nucleated cells (TNC) (83- > 90%), HSC/HPC, or CD34+
(> 80–100%) and CFU (35.68–73.3%) varied according to the cryo-
preservation time; however, UCBUs with the longest cryopreservation
period diminish the percentage of recovery [11–14]. The (NCBT) in
Mexico inaugurated its UCB Biobank in 2003; therefore, we proposed to
evaluate cryopreservation period and different thawing methods on cell
viability and percentage of recovery of CD45+ and CD34+ cells.

⁎
Corresponding author. Tel.: +52 55 63922250.
E-mail addresses: jisela.dimas@salud.gob.mx (J. Dimas-González), anieto2711@gmail.com (A. Nieto-Linares), mirimillan@hotmail.com (M. Millán-Rocha),
jose.salazar@salud.gob.mx (J.L. Salazar-Bailón), bardo.lm@hotmail.com (B.A. Lorenzo-Moreno), julieta.rojo@salud.gob.mx (J. Rojo-Medina).

https://doi.org/10.1016/j.transci.2019.03.008
Received 17 September 2018; Received in revised form 23 January 2019; Accepted 11 March 2019
1473-0502/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Jisela Dimas-González, et al., Transfusion and Apheresis Science, https://doi.org/10.1016/j.transci.2019.03.008
J. Dimas-González, et al. Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

Table 1 representative attached segments of UCBU and ≤2.3 × 106 CD34+

Characteristic of umbilical cord blood units. cells prior to cryopreservation. Inclusion criteria were as follows: signed
Characteristic UCB Units ≥10 years Cryo < 10 years Cryo P value letter of informed consent; negative family history of hereditary and
(N = 13) (N = 14) transmissible diseases; ≥34 weeks of gestation, and absence of con-
genital diseases. Quality control criteria included: > 80 ml in vo-
Cryopreservation time 10–15 9-3
lume; > 8 × 108 leukocytes; negative microbiological cultures; and not
(years)
Maternal age (years) 29.2 ± 5.43 28.4 ± 5.63 0.6229
reactive to HIV, HCV, HBV, Treponema pallidum, or Trypanosome cruzi.
Gestations 1.92 1.93 0.7582
Births 0.69 1.36 0.1207 2.2. Thawing methods
Cesareans 1.15 0.5 0.0608
Abortions 0.23 0.07 0.3259
Gestation weeks 38.92 ± 1.32 38.94 ± 0.78 0.8941 For each attached segment of UCB, we utilized a specific thawing
Sex (%) 46.15 57.14 0.7064 method: Method 1, segment was quickly thawed in the hands; Method
Female 53.85 42.86 2, segment was quickly thawed in the hands and diluted at a 1:1 ratio
Male
with isotonic solution (v/v Dextran 40 [Laboratorios PiSA, S.A de C.V.,
Birth weight (kg) 3.28 ± 0.41 3.13 ± 0.27 0.3434
Initial hematic biometry 11.02 ± 1.86 11.15 ± 2.65 0.9332
Guadalajara, Mexico] and human albumin at 20% [Grifols, Barcelona,
(x106) Spain]) [4,15]; and Method 3, segment was thawed in a water bath at
Final hematic biometry 30.04 ± 4.68 39.4 ± 11.57 0.0168 37 °C [9,10]. Then each segment was utilized immediately for flow
(x106) cytometry, CFU assays, and trypan blue staining to observe cell mor-
% recuperation 64.47 ± 14.82 72.58 ± 14.46 0.1408
phology.
Initial white blood cell 10.97 ± 2.95 11.15 ± 2.75 0.6498
count (x108)
Final white blood cell count 6.70 ± 0.89 8.08 ± 2.45 0.1976 2.3. Flow cytometry
(x108)
ABO blood group (%) 15.38 21.43 0.999
A 84.62 78.57
Flow cytometry analysis was carried out using BD Stem Cell
O Enumeration Kit (BD Biosciencies, San Jose, CA, USA), which allows for
Time from collection to 20 ± 5.93 28.42 ± 15.55 0.0776 quantifying the number of CD34+ cells, CD45+ cells, and cell viability
processing (h) through 7-AminoActinomycin D (7-AAD). Flow cytometry assay was
carried out in the BD FASCalibur Flow Cytometer and results were
analysed with BD CellQuest Pro software.
2. Materials and methods

2.1. UCBUs criteria 2.4. CFU assay

UCBUs (n = 27) selected from the NCBT Biobank were collected The assays were carried out in MethoCult H4434 classic or H4034
during the period from 2003 to 2015; they contained three optimum medium (StemCell Technologies, Inc., Vancouver, Canada).
Briefly, 50,000 vCD45+ cells of UCB were seeded in 35-mm Petri

Fig. 1. Representative examples of flow cytometric analysis UCB segments of three thawing methods. A) Represent UCB segments frozen > 10 years, B) Represent
cells frozen < 10 years. Viable cells are showed in R2.

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J. Dimas-González, et al.
Fig. 2. Flow cytometric analysis of UCB segments by thawing methods. A)
vCD45+ cells (x108) from UCB segments, B) vCD34+ cells (x106) from UCB
segments, C) CD34+ cell viability from UCB segments. Results are given as
mean ± SD. *** p < 0.0001.
dishes with MethoCult medium in duplicate and were incubated under
standard conditions at 37 °C, 5% CO2, and ≥95% humidity for 14 days.
Then, red, white or mixed colonies were identified and quantified in an
inverted microscope with 10X objective. In order to obtain total CFU x
106, we first calculated total CFU/μl (average total CFU/volume of
seeded UCB [50,000 vCD45+ cells]); later, the value obtained was
multiplied by the number of microliters of each UCBU. Finally, the
result obtained was raised to 106. The percentage of E-Clone (Estimated
Clonogenic) was obtained: total CFU x 106 / (CD34+ cells x 106)
(100%) [16,17].
2.5. Statistical analysis
Statistical analysis was performed with GraphPad Prism 7 software
(GraphPad Software, CA, USA). Comparisons of UCBUs, thawing
methods, and time of cryopreservation (Cryo, ≥10 years and < 10
years) were analysed using the U-Mann-Whitney nonparametric test;
results are presented as mean with Standard Deviation (SD). Values of
p < 0.05 were statistically significant.
3
Transfusion and Apheresis Science xxx (xxxx) xxx–xxx
3. Results
3.1. Characteristics of UCBUs
The characteristics of the UCBUs included in this study are de-
scribed in Table 1. Only the final hematic biometry variable was sta-
tistically significant (30.04 × 106 ± 4.68, 39.4 × 106 ± 11.57; p =
0.0168).
3.2. Analysis of number of vCD45+ and vCD34+ cells in UCB segments by
thawing methods
To determine the number of viable CD45+ and CD34+ cells in UCB
segments after different thawing methods we carried out flow cyto-
metry. Fig. 1 shows flow cytometry histograms of thawed UCB seg-
ments with the different thawing methods and different years of cryo-
preservation.
The average number of vCD45+ cells (Fig. 2A) and vCD34+ cells
(Fig. 2B) diminished significantly in thawed UCB segments with the
different methods compared with the number of cells quantified before
cryopreservation of the UCBUs (p < 0.0001); vCD45+ and vCD34+
cells did not present significant differences among the three thawing
methods. However, average CD34+ cell viability was not statistically
significant among the thawing methods (Fig. 2C).
The cell morphology of UCB segments was observed by trypan blue
and confirmed that mean cell viability percentage was similar with cell
viability by flow cytometer (data not shown).
3.3. Analysis of CFU in cells of UCB segments by thawing method
Representative images of the Burst-Forming Unit-Erythroid (BFU-E),
CFU-Granulocyte-Macrophage (GM), and CFU-Granulocyte-
Erythrocyte-Monocyte-Megakaryocyte (GEMM) of CFU assays are
shown in Fig. 3A. Mean CFU (Fig. 3B) and percentage of Estimated
Clonogenic (E-Clone) (Fig. 3C) were not statistically significant in any
of three thawing methods. The mean of percentage of E-Clone was as
follows: Method 1 (27.14% ± 17.12), Method 2 (27.05% ± 15.46), and
Method 3 (27.57% ± 15.11).
3.4. Analysis of vCD45+ cells, vCD34+ cells and CFU by cryopreservation
time
Mean vCD45+ cells (Fig. 4A) and vCD34+ cells (Fig. 4B) sig-
nificantly diminished in UCB segments cryopreserved ≥10 years
(p < 0.0001). The percentage of viability of CD34+ cells, CFU, and
percentage of E-Clone were not statistically significant (Figs. 4C-4E);
however, mean percentage of E-Clone of the ≥10 years group was
30.67% ± 16.95, and < 10 years group was 25.47% ± 13.93 (Fig. 4E).
The percentage of recovery of vCD45+ cells was 37.97% in the
≥10-years group and 55.99% in the < 10 years group of cryopre-
servation (p < 0.0001). The percentage of recovery of vCD34+ cells
was 55.23% and 67.24% according to the duration of cryopreservation
(p = 0.013) (Table 2).
4. Discussion
In this study, three different thawing methods were evaluated in
attached segments of the UCBUs. The methods did not affect the
number of vCD45+ cells, vCD34+ cells, CFU, and percentage of E-
clone. On the other hand, cryopreservation time affected vCD45+ and
vCD34+ cells in attached segments of the UCBUs cryopreserved ≥10
years; however, CFU and percentage of E-Clone did not decrease.
It is important to mention that attached segments are representative
of the bag and are useful tool for quality control and confirmation of
cell viability in the UCBUs [18–20]; we used attached segments as
quality control of the UCBUs in our biobank.
J. Dimas-González, et al. Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

Fig. 3. CFU assays of UCB segments by thawing methods. A) Representative examples of colonies grown from UCB segment, B) Total CFU (x106) from UCB segments
C) E-Clone from UCB segments. Results are showed as mean ± SD.

Fig. 4. Flow cytometric and clonogenic assays analysis of UCB segments by long term cryopreservation. A) vCD45+ cells (x108) from UCB segments, B) vCD34+
cells (x106) from UCB segments, C) CD34+ cell viability from UCB segments, D) Total CFU (x106) from UCB segments E) E-Clone from UCB segments. Results are
given as mean ± SD. *** p < 0.0001.

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J. Dimas-González, et al.
Table 2
Percent recovery of cryopreserved UCB cells after ≥10 and < 10 years.
Characteristic Recovery (%) ± SD Range (%) P value
CD45+ cells (x108)
≥10 years 37.97 ± 13.24 13.42–62.47 < 0.0001
< 10 years 55.99 ± 18.59 27.45–100
CD34+ cells (x106)
≥10 years 55.23 ± 22.23 43–100 0.013
< 10 years 67.24 ± 20.39 46.45–100
Cryopreservation and thawing processes are factors that affect cell
viability and pluripotency of UCBUs; these parameters are evaluated as
quality control for a successful transplant [4,5]. Different methods have
been developed to thaw UCBUs; one of these consists of thawing-
washing-diluting UCB in an equal volume of isotonic solution (Dextran
40/Bovine Serum Albumin [BSA]) [6,7,21]. Some methods eliminate
the washing step because TNC, CD34+ cells, and CFU recovery di-
minishes; thus, the thawing-dilution method was described with dif-
ferent isotonic solutions [4,15], and others consist of only thawing
UCBUs at 37 °C [9,10].
Thawing methods can be controversial; however, we did not ob-
serve changes in the number of vCD45+ cells, vCD34+ cells, CFU, and
percentage of E-Clone among the three methods utilized, similar to that
observed in other studies [21].
vCD45+ and vCD34+ cells diminished post-thawing in the three
methods utilized compared with pre-cryopreservation values. It should
be noted that cryopreservation affects cell viability; however, this di-
minution is not influenced by any thawing method of UCB cells post-
thawing [6,7].
Another factor that affects cell viability and pluripotency of cryo-
preserved UCBUs is long storage time. Broxmeyer et al. evaluated dif-
ferent periods of cryopreservation time of UCBUs (2–5, > 10, 15, and
23.5 years) and identified a recovery range of TNC (83- > 90%), HSC/
HPC (80–100%), and CFU (35.68–91%); however, UCBUs with the
longest cryopreservation time presented low recovery percentages
[11–14]. Similarly, we observed that a longer cryopreservation period
(10–15 years) decreases vCD45+ and vCD34+ cells; however, it does
not affect pluripotency (CFU, % of E-Clone).
The criteria for transplant of post-thawing UCB segments should be:
viable TNC (> 50%), vCD34+ cells (> 50%), CFU growth, and E-clone
(> 10%) [22]. In our study population, UCBUs with < 10 years of
cryopreservation meet the criteria for transplantation (vCD45 +
55.99%, vCD34+v 67.25%, CFU growth, and a percentage of E-Clone
of 25.47%). In the ≥10-year group, some UCBUs did not meet all cri-
teria. For example, some samples had an average percentage of
vCD45+ cells of 37.97%. However, the average of vCD34+ cells was
55.23% and the percentage of E-clone was 30.67%, which are in ac-
cordance with the established range. Furthermore, in this group HSC/
HPC do not lose their pluripotency. In addition, it has been mentioned
that TNC can diminish due to death of granulocytes, which are more
sensitive to cryopreservation and thawing processes [6,7].
In conclusion, the three thawing methods utilized in this study do
not affect vCD45+ cells, vCD34+ cells, CFU, and percentage of E-
Clone in attached segments of the UCBUs. However, longer cryopre-
servation time (10–15 years) of UCBUs generally decreases cell viability
(vCD45+ cells, vCD34+ cells) in attached segments. In contrast, HSC/
HPC conserve pluripotency because they induce formation of CFU.
Funding
This work was supported by Centro Nacional de la Transfusion
Sanguínea.
Transfusion and Apheresis Science xxx (xxxx) xxx–xxx
Authorship contributions
JDG contributed to design of study, acquisition of data, analysis and
interpretation of data, statistical analysis and writing the manuscript.
ANL and MMR contributed to analysis and interpretation of data.
JLSB and BHLM contributed to conception and design of study.
JRM contributed to conception and design of study, discussion of
results, writing and revising the manuscript.
Conflict of interest
All authors declare that they have no conflict of interest.
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