mesenchymal stem cells (msc) · mesenchymal stem cells • the international society for cellular...
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Mesenchymal Stem Cells (MSC)
Ian McNiece, PhD
Professor of Medicine
Director, Cell Therapy Laboratories
The University of Texas MD Anderson
Cancer Center
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MESENCHYMAL STEM CELLS
• Multipotent stem cells originally defined in the bone marrow
• Equivalent to stromal cells identified back in the 1960’s by Dexter and colleagues
• Grown from BM mononuclear cells by their adherence to plastic in tissue culture flasks
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MESENCHYMAL STEM CELLS
• The International Society for Cellular Therapy position paper:
• Defined the minimal criteria for defining multipotent mesenchymal stromal cells.
• Plastic-adherent cells expressing CD105, CD73 and CD90, but not CD45, CD34, CD14, CD11b, CD79alpha, CD19 or HLA-DR.
• MSC must differentiate to osteoblasts, adipocytes and chondrocytes in vitro.
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MSC Manufacture
Autologous versus Allogeneic
• Autologous cells considered safer
because there are no issues with immune
rejection of graft versus host
• Autologous MSCs require a minimum of 5
weeks for isolation, expansion and
release. This limits their application
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CMC Considerations
Source Control
source of cells
donor screening
Production of MSCs:
Heterogeneity of patient products
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0
100
200
300
400
500
600
20 40 60 80
MS
C Y
ield
(1E
6)
AGE
Series1
Linear (Series1)
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CMC Considerations
Process controls
validation of production process
cGMPs
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MSC Manufacture
Bone Marrow Aspirate
Ficol Gradient Separation
Mononuclear fraction
Culture in Flasks
10 x T162cm2
P1
2 weeks Culture in Flasks
60 x T162cm2
P0
1 week
* Target for manufacture 250 million MSC
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MSC Manufacture
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MSC Manufacture Cat. No. 165250 167695 140004 164327 170009 139446
Number of
trays
1 2 4 10 10 40
Culture
area, cm²
632 1264 2528 6320 6320 25280
Suggested
working
volume, ml
200 400 800 2000 2000 8000
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MSC Manufacture
Volume of BM 25ml
Starting cell count (x106) 588
Post ficol cell count (x106)
90
P0 – total cells (x106)
142
P1 - total cells (x106)
514
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CMC Considerations
Product testing
- should ensure product safety
- should ensure consistency of process
and final product
- should predict in vivo activity
- is guided by detailed understanding of
the manufacturing process and product
= CHARACTERIZATION
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Final MSC Preparation Testing
• Release testing
– Sterility
– Endotoxin
– Mycoplasma
– Viability
– Cell Concentration
– Purity (FACS)
Purity (FACS)
CD45- CD105+ CD166+
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CMC Considerations
Identity
Is the product what you say it is?
For MSCs can visually confirm identity by
microscopy
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ADHERENT MSC IN CULTURE
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CFU-F Colony
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CMC Considerations
Quality
Potency
For MSCs – CFU-F
Flow analysis
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0
100
200
300
400
500
600
0 20 40 60 80 100 120 140
MS
C Y
ield
(1E
6)
CFU-F/1E6 BM MNC
CFU-F Assays
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CMC Considerations
Purity
Ideal product has high levels of desired
cells with a low level of unwanted cells
Typically MSC products > 95% CD105+
> 95% CD45 –ve
< 1% CD3+ cells
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ADHERENT MSC IN CULTURE
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CMC Considerations
Strength
How much?
How will you dose?
Dose finding studies needed to identify
effective dose.
Studies to date have given up to 200M
MSCs without safety issues
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Delivery of Cell Products
• Intravenous injection (IV) – BMT
products
• Sub cutaneous (subQ) – drugs
• Direct injection to tissue
– Heart – catheter delivery
» post by-pass surgery
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Surgical Injection of MSCs
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DELIVERY OF CELL PRODUCTS TO HEART
TISSUE
• Ideally we want the volume to be delivered to
be minimal
• To deliver a large number of cells in a small
volume means the cell must be prepared at a
very high cell concentration. Eg 40M MSC/ml
• This can result in a viscous cell product which
can result in clumping and other complications
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DELIVERY OF CELL PRODUCTS TO HEART
TISSUE
• Preparing cell products results in cell loss
- transfer to a sterile cup to fill syringes
- filling syringes, removing air
- priming catheters (200 ul deadspace = 4%
of the product)
• With a minimal volume of cells, will you inject
the same number of sites with a smaller
volume OR inject the same number of cells
into fewer sites??
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CMC Considerations
Lot release
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Final MSC Preparation Testing
• Release testing
– Sterility
– Endotoxin
– Mycoplasma
– Viability
– Cell Concentration
– Purity (FACS)
Purity (FACS)
CD45- CD105+ CD166+
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MANUFACTURING ISSUES
• Different cell yields with different patients
• Some patients fail to grow
• Excess product – should this be stored for
future use of the patient, or discarded?
• BM products for placebo patients – should
these be stored for the patients future use?
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0
100
200
300
400
500
600
20 40 60 80
MS
C Y
ield
(1E
6)
AGE
Series1
Linear (Series1)
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0
20
40
60
80
100
120
140
0 20 40 60 80 100
Normal donors
Patients
AGE
CFU-F
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Initial Observations
• Many patients requiring CABG surgery are unable to wait for
production of MSC. One option could be to use allogeneic MSC
for this patient group.
• Delivery of concentrated cell products (40 million cells per ml)
can result in clumping of products.
• Delivering cell doses offers challenges.
– Losses with thawing and washing
– Losses with transfer to syringes and elimination of air bubbles
– Loss of cells at the site of injection
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Sources of MSC
• Bone Marrow
• Adipose Tissue
• Cord Blood Products
• Placenta
• Warten’s Jelly
• Amniotic Fluid
• Other tissues