Stem cell banking3

transcript

Dr /Samir Mahmoud Attia

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

• This essay aim to clarify the technique of

Banking of stem cells by different ways of collection and storing of blood stem cells

What Are Stem Cells ?

Unspecialized, primitive Self-renewing cells that Can differentiate into cells with specific functions

Classification of stem cell

Embryonic , adult

Hematopoietic, mesenchymal

Tottipotent,PleuripotentlMultipotentunipotent

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.

• 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.

Adult stem cells

Have been found in:BrainBone marrowBlood vesselsDigestive tract

Are multipotent, e.g., hematopoietic stem cells that form different blood

components

Have been found in:BrainBone marrowBlood vesselsDigestive tract

Are multipotent, e.g., hematopoietic stem cells that form different blood

components

Skeletal muscleSkinLiverUmbilical cord

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.

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.

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.

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.

1. TOTIPOTENTIAL stem cells

Derived from embryonic stem cells

They can become any cell type

They can renew themselves indefinitely

1. TOTIPOTENTIAL stem cells

Derived from embryonic stem cells

They can become any cell type

They can renew themselves indefinitely

Classification Of Stem Cells

2. PLURIPOTENTIAL stem cells

can grow into any cell type except

totipotential stem cells. They cannot

become an embryo.

They can renew themselves indefinitely.

2. PLURIPOTENTIAL stem cells

can grow into any cell type except

totipotential stem cells. They cannot

become an embryo.

3. MULTIPOTENTIAL stem cells

Generate only closely related cells, e.g.

blood cells such as white blood cells, red

blood cells, lymphocytes, platelets… etc.

3. MULTIPOTENTIAL stem cells

Generate only closely related cells, e.g.

blood cells such as white blood cells, red

blood cells, lymphocytes, platelets… etc.

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.

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.

Sources of Stem Cells

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).

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).

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.

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.

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 the

marrow from the pelvic bone.

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 the

marrow from the pelvic bone.

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.

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.

Cord Blood Banking

The first successful cord cell transplant to a

sibling with Fanconi’s anemia took place in

This proven utility of cord blood led to the

establishment of cord blood banks. The first

private and public banks were established in

The first successful cord cell transplant to a

sibling with Fanconi’s anemia took place in

This proven utility of cord blood led to the

establishment of cord blood banks. The first

private and public banks were established in

• 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) .

• 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) .

Advantages and Disadvantages of

Umbilical Cord Blood

Advantages and Disadvantages of

Umbilical Cord Blood

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).

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).

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 .

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 .

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

• 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.

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.

• 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.

Collection of UCB

• Method of Collections :

• 1-In- utero: before delivery of placenta

• 2-Ex- utero:After placental delivery

• Method of Collections :

• 1-In- utero: before delivery of placenta

• 2-Ex- utero:After placental delivery

• 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.

• 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

Collection of UCB

• Volume

80 – 120 mL. (30-40 mL..TNC > 6 x 10/9 cells)

• Transport

–By dry shipper( Refrigeration 4 C)

• Volume

80 – 120 mL. (30-40 mL..TNC > 6 x 10/9 cells)

• Transport

–By dry shipper( Refrigeration 4 C)

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)

• 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)

Processing of UCB

• Analysis.– Cell Count/Diff, Viability (>70%), ABO,

– CD34 +, Stem Cell Assay (post processing)

– Sterility Testing (pre and post processing).

• Analysis.– Cell Count/Diff, Viability (>70%), ABO,

– CD34 +, Stem Cell Assay (post processing)

– Sterility Testing (pre and post processing).

Processing of UCB

• Cryopreservation

– 25 –50 mL Cryocyte Storage Container

– 10% dimethyl sulfoxide (DMSO)

– Control Rate Freeze Process

• 1 C – 2 C /min

• Cryopreservation

– 25 –50 mL Cryocyte Storage Container

– 10% dimethyl sulfoxide (DMSO)

– Control Rate Freeze Process

• 1 C – 2 C /min

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.

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.

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%

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%

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.

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.

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.

• 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.

• 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 .

• 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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