Stem cell banking3

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Copyright 2002 Allyn & Bacon
Customized by Marsha Blachman, LCSW

Dr /Samir Mahmoud Attia

By
Dr /Samir
Mahmoud Attia

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Aim of the work
• This essay aim to clarify the technique of
Banking of stem cells by different ways of
collection and storing of blood stem cells

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What Are Stem Cells ?
Unspecialized,
primitive Self-
renewing cells that
Can differentiate
into cells with
specific functions

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Classification of
stem cell
Embryonic,
adult
Hematopoietic,
mesenchymal
Tottipotent,
Pleuripotentl
Multipotent
unipotent

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Embryonic stem cells
• Embryonic stem cells are only found naturally
in the early stages of embryonic development
and are totipotent i.e. they can form any type
of adult cell or adult cell precursor.

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Adult stem cells
Have been found in:
Brain
Bone marrow
Blood vessels
Digestive tract
Are multipotent, e.g., hematopoietic
stem cells that form different blood
components
Skeletal muscle
Skin
Liver
Umbilical cord

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Hematopoietic stem cells
A hematopoietic stem cell is a cell isolated
from the blood or bone marrow that can
renew itself.
It can differentiate to many specialized
cells.

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Mesenchymal Stem Cells
Mesenchymal cells are distinct from
haematopoietic cells by being CD45 -.
Mesenchymal cells are a mixed cells that
are capable of supporting haematopoiesis
and differentiating into endothelial, bone,
muscle and neural cells.

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1. TOTIPOTENTIAL stem cells
Derived from embryonic stem cells
They can become any cell type
They can renew themselves indefinitely
Classification Of Stem Cells

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2. PLURIPOTENTIAL stem cells
can grow into any cell type except
totipotential stem cells. They cannot
become an embryo.
They can renew themselves indefinitely.

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3. MULTIPOTENTIAL stem cells
Generate only closely related cells, e.g.
blood cells such as white blood cells, red
blood cells, lymphocytes, platelets… etc.
They can renew themselves indefinitely.

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4. UNIPOTENTIAL (PROGENITOR) stem
cells
Produce only one cell type They also have
the ability of self renewal, which
distinguishes them from non-stem cells.

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Sources of Stem Cells

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Peripheral Blood Stem Cells (PBSC)
The bloodstream is one source of stem
cells, although not rich as a source as bone
marrow.
To have enough stem cells in a donor's
bloodstream for a transplant, the donor is
given a special drug called a "growth
factor" (filgrastim is a drug that is
commonly used for this purpose).

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When enough stem cells are present in the
bloodstream, the donor undergoes a
process called apheresis.
During an apheresis the blood stem cells
are separated from the donor's blood, and
the remaining blood goes back into the
donor's bloodstream through a sterile
needle.

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Bone Marrow Stem Cells
Bone marrow, a spongy tissue found inside
larger bones, it is a rich source of blood stem
cells.
Approximately one liter of marrow is needed for
a blood stem cell transplant, but the exact
amount needed depends on the size of the
patient.
Marrow is removed during a surgical procedure.
Sterile needles and syringes are used to remove

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Umbilical Cord Blood Stem Cells
Umbilical cord blood is another rich source of
stem cells.
Months before the baby's birth, the mother signs
an agreement to donate the umbilical cord blood
when the baby is born.
At birth, the cord blood unit is collected and
taken to a cord blood bank, where it is tissue-
typed, processed and stored frozen until needed
for a transplant.

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Cord Blood Banking
The first successful cord cell transplant to a
sibling with Fanconi’s anemia took place in
1988.
This proven utility of cord blood led to the
establishment of cord blood banks. The first
private and public banks were established in
1992.

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• CB banking provides rapid availability of
allogeneic donors for stem cell transplantation,
little donor risk or attrition, low risk of
transmitting infection, reduced or no risk of
(AGVHD) .

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Advantages and
Disadvantages of
Umbilical Cord Blood

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Advantages of UCB
1) Cord blood has been found to possess more
primitive cells.
2) Umbilical cord blood offers potential
advantages,that infectious diseases is marked
less than bone marrow or peripheral blood
despite the possibility of congenital and
perinatal transmission of infectious agents. for
example, no cord bloods would be infected with
Epstein Barr virus (EBV).

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3) CD34+ CD38- cells in umbilical cord blood also
proliferate more rapidly in response to cytokine
stimulation with IL-3, IL-6 and stem cell factor
(SCF) and generate seven times more
progenitor cells than do bone marrow .

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4) Umbilical cord blood has more haematopoietic
stem cells per volume than peripheral blood or
bone marrow. In addition, umbilical cord blood
seems more tolerant of HLA mismatches; with
less graft versus host disease
5) Cord blood is less risky to collect. The collection
of cord blood following delivery is a harmless
process that does not affect the mother or her
newborn.

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Disadvantages of UCB
1) A disadvantage of umbilical cord blood
compared with donated adult bone marrow is
that newborns may carry undiagnosed genetic
diseases.
2) BM donors can be recalled for the same patient
to provide a second donation of BM.

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3) BM donors can provide a medical history, which
is relevant to the donation, at the time of
donation. However, CB donors provide the
medical history through their mothers.
4) that bone marrow contains more mesenchymal
progenitor cells than CB.

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Bone Marrow/
Peripheral Blood
Cord Blood
-Donation requires
surgery under general
anesthesia (in B.M)
-Donors feel discomfort
and/or pain (in P.B).
-Long-term
consequences of growth
factors used in
peripheral blood stem
cell
donation posses no risk
to mother or infant.

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Large dose of stem cells.
Rapid engraftment.
Smaller dose of stem
cells. Slower
engraftment.
After a formal search
is begun, takes an
average of 4 months
to transplantion, if a
donor is available.
When a match is
found, can take only
a few days for
confirmatory and
special testing (may
reach less than 24
hours in an
emergency).

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Potential donors may
no longer be available
or may have withdrawn
consent. Donor must be
found and retested to
confirm the HLA typing
and infectious disease
results and to confirm
that the donor is still
willing and able to
donate bone marrow .
Once frozen, a cord
blood unit is available
until used.

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Bone marrow must be
used fresh (shelf-life
measured in hours).
Peripheral blood stem
cells stored for short
term (days to a few
months).
Frozen cord blood has
been transplanted
successfully up to 10
years in storage.
Latent viral infection in
the donor common (i.e.
CMV > 50% in adult
donors).
Latent viral infection in the
cord blood donor rare (i.e.
CMV <1% )
Severe graft vs host
disease (GvHD)
common.
GvHD less frequent and
easier to treat.

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Generally requires a
perfect match between
donor and recipient for
HLA-A, -B . Additional
factors HLA-C, -DQ and
-DP)are needed to
improve prognosis).
HLA-mismatched cord
blood transplantion are
possible, making it
easier to find a suitable
match.
No risk of transplanting
a genetic disease.
Cord blood has a small
risk of transplantation
rare unrecognized
genetic disease.

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Methods of umbilical cord
blood collection
• variety of potential collection methods (open,
semi- closed or closed methods) have been
proposed in order to optimize the collection
volume and reduce the risks of microbial and
maternal contamination.

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Donor selection
• The medical history interview includes a
review of the risk for HIV and hepatitis B
and C, including skin piercing and blood
transfusion, as well as the presence of
infections that can be transmitted.

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Collection of UCB
• Method of Collections :
• 1-In- utero: before delivery of
placenta
• 2-Ex- utero:After placental
delivery

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• Representation of the UCB collection system. The
delivered placenta is placed onto the funnel-shaped
supporting surface with the maternal side facing upward,
the fetal side facing downward, and the umbilical cord
passing through the central hollow cylinder of the
supporting surface. The device lid is then closed, the
pressure application system is turned on, and the UCB
collection begins.

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• Blood collection was made by puncturing
the umbilical vein after the umbilical cord
had been clamped, cut, and cleaned with
an antiseptic solution, and drainage by
gravity into a regular blood donation
triple-bag system with the anticoagulant
citrate phosphate dextrase (CPD)
reduced to 25 mL

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Collection of UCB
• Volume
80 – 120 mL. (30-40 mL..TNC > 6 x
10/9 cells)
• Transport
–By dry shipper( Refrigeration 4 C)

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Processing of UCB
• 24-36 hrs. post collection.
– Max. 48 hrs.
– Volume reduction
• depletion of erythrocytes and/or plasma
by:
• HES (hydroxy ethyl starch)
• Hespan. (1:5 ratio Hespan/UCB)

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Processing of UCB
• Analysis.
– Cell Count/Diff, Viability (>70%), ABO,
Rh,
– CD34 +, Stem Cell Assay (post
processing)
– Sterility Testing (pre and post
processing).

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Processing of UCB
• Cryopreservation
– 25 –50 mL Cryocyte Storage Container
– 10% dimethyl sulfoxide (DMSO)
– Control Rate Freeze Process
• 1 C – 2 C /min

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Most cord blood units were cryopreserved
as whole blood donations with 10% DMSO
and transfused immediately after thawing
without any attempt to remove the
cryoprotectant.

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Thawing of cord blood units is usually performed
by placing the cord blood bags in the gas phase
of liquid nitrogen for 30 min followed by a 5-min
exposure to room temperature.
The bags are then thawed in a 37C water bath
as rapidly as possible.
Immediately after thawing, each cord blood unit
is diluted with an equal volume of solution
containing 5% human serum albumin and 10%

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Dextran 40 in 0.9% NaCl, and sedimented for
10 min. The supernatant is then removed and
sedimented cells are resuspended slowly in
fresh albumin/ dextran solution.
• This procedure removes the bulk of RBC ghosts,
hemoglobin, and DMSO, thus reducing some of
the risks associated with the transplantion
procedure.

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Recommindations
• 1-Written permission should be obtained
during prenatal care and before the onset of
labour.
• 2-cord blood collection should not be done in
complicated deliveries and the cord blood
stem cell collection program should not alter
routine practice for the timing of umbilical
cord clamping.

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• 3-condition such as leukemia or severe
hemoglobinopathy may indicate the need for
directed donor cord blood banking for sibling
cord blood transplantation.
• 4-preliminary data show encouraging results
in cord blood stem cell transplantation for a
variety of genetic, hematological and
oncological diseases .

Stem cell banking3

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