Beruflich Dokumente
Kultur Dokumente
1, 2010
ORIGINAL ARTICLE
Christina Basford , Nicolas Forraz , Saba Habibollah , Kendal Hanger , Colin McGuckin
1
Newcastle Centre for Cord Blood, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, NE1 3BZ,
United Kingdom, 2CTI-LYON, Cell Therapy Research Institute, Parc Technologique de Lyon-St Priest, Cdre 1,
97 Alle Alexandre Borodine, 69800 SAINT PRIEST LYON, France
Background and Objectives: Well over 1 million Umbilical Cord Blood units (UCB) have been stored globally in the
last 10 years. Already, over 20,000 transplants been performed using UCB for haematopoietic reconstitution alone,
now this potential is joined by regenerative medicine. However, more needs to be known about processing of this
stem cell source for it to reach full potential.
Methods and Results: In this study we evaluated five separation methods: plasma depletion, density gradient,
Hetastarch, a novel method known as PrepaCyte-CB and an automated centrifugal machine. Sepax gives the highest
recovery of nucleated cells, an average of 78.8% (SD21.36). When looking at CD34 haematopoietic stem cells
PrepaCyte-CB provided the greatest recovery at 74.47% (SD8.89). For volume reduction density gradient was the
most effective leaving 0.03106 RBC/ml, 8 times more efficient than its nearest competitor PrepaCyte-CB (p0.05).
Finally PrepaCyte-CB processing left samples with the highest clonogenic potential after processing and more significantly after cryopreservation: 9.23 CFU/108 cells (SD2.33), 1.5 fold more effective than its nearest rival Sepax
(p0.05).
Conclusions: PrepaCyte-CB was the most flexible method; the only processing type unaffected by volume. Results indicate that processing choice is important depending on your final intended use.
Keywords: Stem cells, Bioprocessing, Umbilical cord blood, Transplantation
Introduction
The global rise in Umbilical Cord Blood (UCB) as a
transplant source, has been amazing, over 20,000 transplants already having taken place in haematopoietic reconstructions alone (1). It has become a real alternative
to bone marrow (BM) and peripheral blood (PB) as a
32
separation kit, closed separation kit, known as PrepaCyteCB which offers rapid and specific cell separation (18).
In this study we used a PrepaCyte-CB kit designed specifically for UCB units, to allow recovery of other non-erythroid subsets such as leukocytes, thrombocytes and stem
cells, including both hematopoietic stem cells (HSC) and
multi-lineage progenitor cells, without them needing to be
bound to unwanted chemicals (19) or particles. The final
separation system chosen was also the only fully automated system, provided by Biosafe, known as the Sepax
machine (20).
Sample analysis
Enumeration by differential cell counting: The cell
counts were performed using the CellDyn4000 Analyser,
a mechanical method. A sample of 1 ml was applied to
the machine at each of the three points during processing.
The WBC count and differential determine the number
of white blood cells and the percentage of each type of
WBC in each unit of blood.
Colony forming unit assays: A cell suspension was prepared with the appropriate number of cells; at 0.25
105/plate. Colony counts must then be multiplied by 4 to
give the number of colonies per 105 cells. The relevant
number of cells was added to make up a volume of IMDM
(Gibco/Invitrogen 21980-032. Paisley, UK)20% FCS
supplements to 0.5 ml, then added to a 3.5 ml aliquot of
methylcellulose culture medium, (Stem cell Technologies
Statistical analysis
Statistical analysis was carried out using the statistical
software programme, Minitab (Version 15, 2006, University of Pennsylvania, USA). The data was analysed using
Table 1. Antibody cocktails used for cell separation assessment: flow cytometry
Antibody
Type
Volume (l)
Cat. no.
Distributor
Tube no.
IgG1
IgG2b
IgG1
IgG1a
IgG1
IgG1
IgG1
IgG1
IgG2a
IgG1
IgG1
IgG1
IgG1
IgG2b
IgG1
IgG1
IgG1
IgG1
IgG1/2b
IgG2a
IgG1
IgG2a
IgG2a
IgG2a
IgG1
IgG1
5
5
5
5
20
20
5
5
20
5
20
20
5
5
5
5
5
5
20
20
5
5
5
5
5
5
557833
555570
559925
558121
333146
555360
557922
557741
555338
557708
555411
555518
348811
130-090-854
ab30418
555595
313202
ab15021
340546
558714
555392
307626
ab24265
311418
ab7758
335823
BD Biosciences
BD Pharmingen
BD Pharmingen
BD Biosciences
BD Biosciences
BD Pharmingen
BD Pharmingen
BD Pharmingen
BD Pharmingen
BD Pharmingen
BD Pharmingen
BD Pharmingen
BD Sciences
Miltenyi Biotec
AbCam
BD Biosciences
Bio legend
AbCam
BD Biosciences
BD Pharmingen
BD Pharmingen
Bio legend
AbCam
Bio legend
AbCam
BD Biosciences
b
1,
1,
1,
1,
1,
1,
1,
1,
1
1
2
2
3
3
3
3
3
3
4
4
4
4
4
4
4
4
2, 3, 4
2, 3, 4
2, 3, 4
2
2
2
2
2
Unconjugated antibodies were labelled using a Zenon labelling complex from Invitrogen; Alexa405 z25313 or Alexa430 z25301 . The
biotinylated antibodies were labelled using Quantum dots 605, also from Invitrogen, conjugated to streptavidinc.
Fig. 1. Sepax gives an increased recovery of nucleated cells than all other methods. (A) Sepax offers the highest recovery of CD45 cells
at 75.79% (SD18.58) compared to PrepaCyte CB at 72.03% (SD8.48), Plasma depletion at 71.38% (SD29.35), Hetastarch at 44.94%
(SD20.06), and Density Gradient at 19.13% (SD10.88). Sepax was only significantly more efficient than Hetastarch and density gradient
methods (p0.005). (B) After exclusion of granulocytes recovery with Sepax is still the greatest at 78.8% (SD21.36) compared to other
methods. These results were determined using differential cell counts coupled with flow cytometric analysis of CD45 cells, viability of
CD45 cells. Exclusion of granulocytes was also determined in this way to give a more accurate picture of cell recovery. Fluorochromes
used were PE Cy5 for CD45 cells, PE for CD14 / cells and 7AAD for viability.
non parametric, two-tailed Mann-Whitney U-testing to determine significance. A p0.05 was considered to be statistically significant.
Results
For this study, 80 UCB units were processed. Of those
samples, 46% were from female infants (n=37) and 54%
were from male infants (n=43). The birth weights of the
infants involved ranged from 2.41 kg to 4.42 kg with an
average of 3.52 kg (SD0.38). The volume of blood collected ranged from 46.6 ml to 194.3 ml with an average
volume of 98.72 ml (SD31.56).
Fig. 2. PrepaCyte-CB provides the most efficient recovery of haematopoietic stem cells than other methods tested. (A) Recovery of late
stage haematopoietic (CD34/CD133) stem cells was greater with PrepaCyte-CB at 74.41% (SD8.89) than Sepax; 73.91% (SD16.86),
plasma depletion; 73.88% (SD22.26) Hetastarch; 56.48% (SD21.99) and density gradient separation methods 22.83% (SD18.56).
However, results are only significant when compared to HES (p=0.05). (B) Recovery of mid stage haematopoietic (CD34/CD133) stem
cells stem cells was greater with PrepaCyte-CB at 65.83% (SD14.22) than Hetastarch; 64.21% (SD17.89), Sepax; 60.98% (SD14.90),
plasma depletion; 54.30% (SD26.79) and density gradient separation methods 27.43% (SD13.37). However, results are only significant
when compared to density gradient (p=0.01). (C) Recovery of early stage haematopoietic (CD34/CD133) stem cells was greater
with PrepaCyte-CB at 62.59% (SD16.23) than plasma depletion; 60.10% (SD28.5), Sepax; 48.50% (SD15.06) Hetastarch; 48.40%
(SD26.2) and density gradient separation methods 13.06% (SD9.36). However, results are only significant when compared to density
gradient (p=0.01). CD34 cell numbers and viability were calculated using flow cytometric analysis of CD34 antibody and 7AAD uptake.
Flourochromes used were FITC for CD45 cells, PE for CD34 cells and 7AAD for viability.
Fig. 3. PrepaCyte-CB gives the greatest recovery of T Cells (CD45/CD3) and B Cells (CD45/CD19). (A) Recovery of T Cells (CD45
/CD3) was greater with PrepaCyte-CB at 71.84% (SD18.06) than plasma depletion; 65.54% (SD35.0), Sepax; 65.26% (SD35.19)
Hetastarch; 56.85% (SD23.79) and density gradient separation methods 36.62% (SD26.32). (B) Recovery of B Cells (CD45/CD19)
was greater with PrepaCyte-CB at 75.68% (SD24.0) than Sepax; 71.75% (SD16.84), plasma depletion; 71.74% (SD8.66) Hetastarch;
46.47% (SD25.69) and density gradient separation methods 19.13% (SD10.88). However, results are only significant when compared
to HES (p=0.05).
Fig. 4. Density gradient separation is the most effective method for volume reduction by removal of either; (A) Red blood cells and (B)
Haemoglobin. (A) Compared to whole blood, which has an average of 2.9106 cell/ml (SD0.75), post processing, density gradient is
the most effective method for the removal of red blood cells, it leaves 0.03106 cell/ml (SD0.02), PrepaCyte-CB leaves 0.24106 cell/ml
6
6
(SD0.08), Hetastarch leaves 1.9310 cell/ml (SD0.22), plasma depletion leaves 2.0110 cell/ml (SD1.53) and Sepax leaves
6
3.3310 cell/ml (SD0.95), all results are significant (p=0.05). Post thaw density gradient is again the most effective method for the
removal of red blood cells, it leaves 0.02106 cell/ml (SD0.01), PrepaCyte-CB leaves 0.11106 cell/ml (SD0.10), Hetastarch leaves
6
6
6
0.2210 cell/ml (SD0.16), plasma depletion leaves 0.5410 cell/ml (SD0.22) and Sepax leaves 2.0110 cell/ml (SD0.94), all
results are significant (p=0.05). (B) Compared to whole blood, which has an average of 10.58 g/dl (SD4.11), post processing, density
gradient is the most effective method for the removal of haemoglobin, it leaves 0.17 g/dl (SD0.12), PrepaCyte-CB leaves 0.68 g/dl
(SD0.31), Hetastarch leaves 6.94 g/dl (SD4.74), plasma depletion leaves 7.23 g/dl (SD5.56) and Sepax leaves 13.07 g/dl (SD7.03),
all results are significant (p=0.05). Post thaw PrepaCyte-CB is the most effective method for the removal of haemoglobin, it leaves 0.10
g/dl (SD0.04), density gradient leaves 0.13 g/dl (SD0.05), Hetastarch leaves 0.46 g/dl (SD0.29), plasma depletion leaves 1.94 g/dl
(SD1.05) and Sepax leaves 7.57 g/dl (SD3.70), all results are significant (p=0.05).
Discussion
Recently there has been an explosion of clinical applications for UCB. This escalation leads to an increased need
for optimising separation, whether to reduce volume to
make storage more efficient or to increase stem cell yield
for transplant. UCB potential is well documented, with
enough clinical value to warrant further investigation.
Since the advent of regular UCB transplantation in the
1990s, and the potential of the units increased, it was necessary to increase banking to meet the demand. It may
also be linked to limitations of BM registries. The process
of finding a donor can be long; potential donors may
change address without notifying their registry; ethnicity
can also be an issue with ethnic minorities often struggling to find a suitable donor. UCB can help towards this
problem as it is easier to store samples from donors of
many ethnicities (21). UCB is an accessible option, with
samples already stored. However this increase is not in
line with sample demand, and the contrast between these
two resources is significant. There are currently 11 million
patrons registered worldwide for BM donation, only an estimated 300,000 UCB units are publically banked. Some
of these banks are struggling financially so it is of critical
importance to find the most effective and economical
processing, and storage methods (22). Many factors affect
the success of processing, not least the physicality of normal birthing (13), so it remains an important priority to
find the best possible methods of separation, so that even
smaller units may prove clinically viable.
Our results show that Sepax depletion gives a higher recovery of nucleated cells, crucial for successful engraftment. However, recovery using Sepax is reduced as the
size of unit processed increases. Hetastarch, density gradient and plasma depletion separation were also affected
in this way, but PrepaCyte-CB processing was not affected
by the initial volume of the collected unit, and recovery
of both TNC and CD34 progenitor cells was as efficient
with smaller volumes as it was with larger units. Density
gradient separation shows a reverse correlation: as UCB
volume increase, so does recovery. Although interesting,
it does not fairly compare to the other methods as the
maximum volume processed with density gradient was 90
Fig. 6. Initial cord blood volume negatively correlates with recovery of CD45 and CD34 haematopoietic progenitor cells when processing with Hetastarch, Plasma Depletion and Sepax. For CD45 cell recovery: (A) Hetastarch processing negatively correlates with initial
volume (p0.01). (B) Initial volume has no significant impact on recovery of TNC when processing with PrepaCyte-CB (p0.05). (C) Density
gradient processing has a positive correlation with an increase in collected volume (p0.01). (D) Plasma Depletion separation negatively
correlates with initial UCBV volume (p0.01). (E) Sepax processing also negatively correlates with initial UCB volume but not significantly
(p0.05). For CD34 recovery results are the same. (F) Hetastarch (p0.01). (G) PrepaCyte-CB (p0.05). (H) Density gradient (p0.05).
(I) Plasma Depletion (p0.01). (J) Sepax processing negatively correlates with initial UCB volume (p0.05).
Fig. 6. Continued.
of T cells transplanted can increase bone marrow reconstitution and therefore haematopoiesis and also eliminate
residual leukemic disease in the transplanted mice. However, it is important to get the fullest picture of the unit
quality that we can.
When looking at the volume reduction of the physical
size of the unit, it would seem that plasma depletion
would be of particular benefit, as a smaller volume reduces the space needed for storage and also means less
DMSO is added to the sample in preparation for cryopreservation (20). This means that it could even save the
need to wash samples before infusion (for haematopoietic
transplant only) as it has been previously shown that TNC
recovery after cryopreservation is greater without a wash
step (30). However, if you measure volume reduction as
the ability to remove RBC and Haemoglobin; then it is
actually a simple and economic density gradient separation which is the most efficient. Removal of RBC is also
PrepaCyte-CB
Sepax
Plasma Depletion
Density gradient
Hetastarch
1st
2ndb
3rdc
4
2
2
2
0
4
3
1
1
1
1
2
4
0
3
Points
21
14
12
8
5
cess to a clean room may not be available. The PrepaCyte-CB system does not require expensive laboratory
equipment and this is a significant issue in the developing
countries where transplants are rarely carried out due to
lack of necessary equipment. It is worth noting that UCB
transplants are currently unavailable to large sections of
the second and third worlds.
In conclusion we can say that, depending on your primary use for UCB, different processing methods may be
more applicable than others, but that while futureproofing is not easy in medical sciences, the choice of processing method must be carefully addressed so that patients
have cord blood units available in a suitable form to treat
their disease.
Acknowledgments
With thanks to the staff at Newcastles Royal Victoria
Infirmary Womans Directorate and Maternity Unit. We
are grateful to One North East regional development agency of the British Government for their financial support
to our laboratories.
Potential Conflict of Interest
The authors have no conflicting financial interest.
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21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.