Microcarrier-Based Xeno-Free Expansion of Human Mesenchymal … · 2018-11-27 · David Splan,...
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Phone: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • Email: [email protected] • Web: www.pall.com/biotech © 2018 Pall Corporation. Pall, , and Allegro are trademarks of Pall Corporation. ® indicates a trademark registered in the USA and TM indicates a common law trademark. 5/18, 18.07135 ABSTRACT MATERIALS PERFORMANCE CONCLUSIONS Microcarrier-Based Xeno-Free Expansion of Human Mesenchymal Stromal Cells in a Single-Use Stirred-Tank Bioreactor David Splan, Janani Ravindhar, Kaitlynn Bayne, Alexandria Spruiel, Tariq Haq and Mark Szczypka Pall Biotech, 4370 Varsity Drive Suite B. Ann Arbor, Michigan 48108, USA u Systems for isolation and expansion of mesenchymal stem/stromal cells (hMSC) from tissues using xeno-free media are under development and some commercially available platforms are already becoming available from various vendors. u Over the past decade, much data has been generated demonstrating the feasibility of using microcarriers in suspension cultures for expansion of hMSC, however, many systems still use media that contains fetal bovine serum. Here we identified microcarriers that support xeno-free expansion of cells and demonstrate their use in a scale-down model of Pall’s single-use Allegro ™ STR bioreactor. u Performance of commercially-available microcarriers and a novel, xeno-free microcarrier type were examined in small scale spinners. Microcarriers that promoted cell attachment and growth were selected and conditions that supported growth were optimized and compared to a high-performing collagen-coated microcarrier. u The xeno-free microcarriers provided the highest cell numbers (0.46 B cells/L) followed by SoloHill collagen-coated microcarriers (0.32 B cells/L) and SoloHill Star-Plus (0.25 B cells/L). u The xeno-free microcarrier was implemented in the scale-down model of the Allegro STR bioreactor contains a bottom-mounted impeller. Cell numbers reached 0.42 B cells/L in 6 days. Standard cell characterization assays revealed that cells retained critical quality attributes indicative of cell type. u Results from this proof of concept study demonstrate the use of a complete single-use technology platform including, microcarriers and a scale-down model of Pall’s Allegro STR bioreactor. u Commercially-available bone marrow-derived human mesenchymal cells and medium. u Single-use spinners and cell factories. • Disposable spinners used for small-scale feasibility studies. • Multi-plate cell culture vessels used in seed train for bioreactor experiments. u Microcarriers: SoloHill microcarriers, including a novel xeno-free prototype. • Plastic, Plastic Plus, Star-Plus, Collagen, Hillex II, xeno-free prototype. • Microcarriers allow for direct transfer out of flatware culture and into space-efficient 3D cultures with minimal development time. • Excellent attachment and growth across multiple cell types. • No hydration needed, simply sterilize and use. • Also offered in a pre-sterilized, ready-to-use format: The Allegro Microcarrier Delivery System (AMDS). • Solid core design able to withstand mechanical harvest methods. u CerCell’s Pall-configured 3.2 L single-use bioreactor • Bottom-mounted impeller design • Ideal for use in seed train with Pall’s larger-scale Allegro STR bioreactor platforms. Cryopreserved cells Multi-plate cell culture vessel 0 20 40 60 80 100 120 CD 105 CD 73 CD 90 CD 34 CD 45 % Positive Surface Marker Expression STR A. Flatware B. Control Differentiation STR Adipogenesis Flatware u The newly-developed, xeno-free microcarriers support expansion of cells in a scale-down version of Pall’s single-use stirred tank reactor, which contains a bottom-mounted impeller. u This fed-batch, xeno-free microcarrier culture enabled cell numbers to reach 0.42 B cells/L in 6 days. u Expanded hMSC maintained critical quality attributes, including surface marker expression and multi-lineage differentiation capacity. u Results from this study demonstrate that hMSC can be successfully propagated on xeno-free microcarriers in a single-use stirred tank reactor. u These studies provide the basis for efficient and reproducible generation of high quality cells in a xeno-free system. Figure 1 Microcarrier screen performed in 125 mL disposable spinners. Expansion of hMSC on SoloHill microcarriers serial-passaged in spinners. The xeno-free microcarrier at 10 cm 2 /mL provided the highest cell numbers (4.6 x 10 5 cells/mL) followed by SoloHill collagen-coated microcarriers at 10 cm 2 /mL (3.2 x 10 5 cells/mL) and SoloHill Star-Plus at 5 cm 2 /mL (2.5 x 10 5 cells/mL). Plastic, Plastic Plus and Hillex II did not support adequate growth under the conditions tested (data not shown). Figure 2 Reproducible expansion of hMSC in 3.2 L scale-down model of Pall’s Allegro STR bioreactor Expansion scheme for hMSC and on SoloHill xeno-free microcarriers using the Pall 3.2 L single-use bioreactor. Cells thawed from liquid nitrogen were seeded onto multi-plate cell culture vessels and expanded for 3 days. Cells were harvested and seeded onto xeno-free microcarriers in the Pall 3.2 L single-use bioreactor at 2.5 L volume and cultured for 6 days. Figure 3 Pall 3.2 L STR bioreactor batch-fed process hMSC on SoloHill xeno-free microcarriers were cultured in the Pall 3.2 L standard single-use bioreactor. This batch-fed process yielded 0.42 B cells/L in 6 days. Figure 4 Pall 3.2 L STR bioreactor expanded hMSC retain critical quality attributes Cell characteristics are retained when hMSCs are expanded in a multi-plate cell culture vessel or on SoloHill microcarriers in a Pall 3.2 L STR bioreactor or spinner flasks. (A) Immunophenotype analysis of hMSCs show comparable surface marker expression. The cells maintain differentiative potential. (B) Adipogenic differentiation capacity. Process Parameters Seeding density 3000 cells/cm 2 Microcarrier concentration 10 cm 2 /mL Medium exchange None Supplement Day 3 Multi-plate vessel LN 2 Pall 3.2 L STR bioreactor 0 0.5 x 10 5 1.0 x 10 5 1.5 x 10 5 2.0 x 10 5 2.5 x 10 5 3.0 x 10 5 3.5 x 10 5 4.0 x 10 5 4.5 x 10 5 5.0 x 10 5 0 1 2 3 4 5 6 Cells/mL Days Pall 3.2 L STR bioreactor Flatware XF Prototype 0 100,000 200,000 300,000 400,000 500,000 600,000 Cells/mL Day 7 Second Spinner Set, cells/mL Collagen-5 Collagen-10 Star-Plus-5 Star-Plus-10 XF-5 XF-10 Flatware LN2 Spinner Set 1 T-flasks Spinner Set 2 Process Parameters Seeding density 3000 cells/cm 2 Microcarrier concentration 5 and 10 cm 2 /mL Medium exchange None Supplement Day 3
Transcript of Microcarrier-Based Xeno-Free Expansion of Human Mesenchymal … · 2018-11-27 · David Splan,...
Phone: +800.717.7255 (USA) • +41 (0)26 350 53 00 (Europe) • Email: [email protected] • Web: www.pall.com/biotech © 2018 Pall Corporation. Pall, , and Allegro are trademarks of Pall Corporation. ® indicates a trademark registered in the USA and TM indicates a common law trademark. 5/18, 18.07135
ABSTRACT
MATERIALS
PERFORMANCE
CONCLUSIONS
Microcarrier-Based Xeno-Free Expansion of Human Mesenchymal Stromal Cells in a Single-Use Stirred-Tank Bioreactor
David Splan, Janani Ravindhar, Kaitlynn Bayne, Alexandria Spruiel, Tariq Haq and Mark Szczypka Pall Biotech, 4370 Varsity Drive Suite B. Ann Arbor, Michigan 48108, USA
u Systems for isolation and expansion of mesenchymal stem/stromal cells (hMSC) from tissues using xeno-free media are under development and some commercially available platforms
are already becoming available from various vendors.
u Over the past decade, much data has been generated demonstrating the feasibility of using microcarriers in suspension cultures for expansion of hMSC, however, many systems still use
media that contains fetal bovine serum. Here we identified microcarriers that support xeno-free expansion of cells and demonstrate their use in a scale-down model of Pall’s single-use
Allegro™ STR bioreactor.
u Performance of commercially-available microcarriers and a novel, xeno-free microcarrier type were examined in small scale spinners. Microcarriers that promoted cell attachment and
growth were selected and conditions that supported growth were optimized and compared to a high-performing collagen-coated microcarrier.
u The xeno-free microcarriers provided the highest cell numbers (0.46 B cells/L) followed by SoloHill collagen-coated microcarriers (0.32 B cells/L) and SoloHill Star-Plus (0.25 B cells/L).
u The xeno-free microcarrier was implemented in the scale-down model of the Allegro STR bioreactor contains a bottom-mounted impeller. Cell numbers reached 0.42 B cells/L in 6 days.
Standard cell characterization assays revealed that cells retained critical quality attributes indicative of cell type.
u Results from this proof of concept study demonstrate the use of a complete single-use technology platform including, microcarriers and a scale-down model
of Pall’s Allegro STR bioreactor.
u Commercially-available bone marrow-derived human mesenchymal cells and medium.
u Single-use spinners and cell factories.
• Disposable spinners used for small-scale feasibility studies.
• Multi-plate cell culture vessels used in seed train for bioreactor experiments.
u Microcarriers: SoloHill microcarriers, including a novel xeno-free prototype.
• Plastic, Plastic Plus, Star-Plus, Collagen, Hillex II, xeno-free prototype.
• Microcarriers allow for direct transfer out of flatware culture and into space-efficient 3D cultures with minimal development time.
• Excellent attachment and growth across multiple cell types.
• No hydration needed, simply sterilize and use.
• Also offered in a pre-sterilized, ready-to-use format: The Allegro Microcarrier Delivery System (AMDS).
• Solid core design able to withstand mechanical harvest methods.
u CerCell’s Pall-configured 3.2 L single-use bioreactor
• Bottom-mounted impeller design
• Ideal for use in seed train with Pall’s larger-scale Allegro STR bioreactor platforms.
Cryopreserved cells
Multi-plate cell culture vessel
0
20
40
60
80
100
120
CD 105 CD 73 CD 90 CD 34 CD 45
% P
ositi
ve
Surface Marker Expression
STR
A. B.
Flatware
Con
trol
Diff
eren
tiatio
n
STR
Adipogenesis
Flatware
0
20
40
60
80
100
120
CD 105 CD 73 CD 90 CD 34 CD 45
% P
ositi
ve
Surface Marker Expression
STR
A. B.
Flatware
Con
trol
Diff
eren
tiatio
n
STR
Adipogenesis
Flatware
u The newly-developed, xeno-free microcarriers support expansion of cells in a scale-down version of Pall’s single-use stirred tank reactor, which contains a bottom-mounted impeller.
u This fed-batch, xeno-free microcarrier culture enabled cell numbers to reach 0.42 B cells/L in 6 days.
u Expanded hMSC maintained critical quality attributes, including surface marker expression and multi-lineage differentiation capacity.
u Results from this study demonstrate that hMSC can be successfully propagated on xeno-free microcarriers in a single-use stirred tank reactor.
u These studies provide the basis for efficient and reproducible generation of high quality cells in a xeno-free system.
Figure 1
Microcarrier screen performed in 125 mL disposable spinners.
Expansion of hMSC on SoloHill microcarriers serial-passaged in spinners. The xeno-free
microcarrier at 10 cm2/mL provided the highest cell numbers (4.6 x 105 cells/mL) followed
by SoloHill collagen-coated microcarriers at 10 cm2/mL (3.2 x 105 cells/mL) and SoloHill
Star-Plus at 5 cm2/mL (2.5 x 105 cells/mL). Plastic, Plastic Plus and Hillex II did not support
adequate growth under the conditions tested (data not shown).
Figure 2
Reproducible expansion of hMSC in 3.2 L scale-down model of Pall’s Allegro STR bioreactor
Expansion scheme for hMSC and on SoloHill xeno-free microcarriers using the Pall 3.2 L
single-use bioreactor. Cells thawed from liquid nitrogen were seeded onto multi-plate
cell culture vessels and expanded for 3 days. Cells were harvested and seeded onto
xeno-free microcarriers in the Pall 3.2 L single-use bioreactor at 2.5 L volume and
cultured for 6 days.
Figure 3
Pall 3.2 L STR bioreactor batch-fed process
hMSC on SoloHill xeno-free microcarriers were cultured in the Pall 3.2 L standard
single-use bioreactor. This batch-fed process yielded 0.42 B cells/L in 6 days.
Figure 4
Pall 3.2 L STR bioreactor expanded hMSC retain critical quality attributes
Cell characteristics are retained when hMSCs are expanded in a multi-plate cell culture
vessel or on SoloHill microcarriers in a Pall 3.2 L STR bioreactor or spinner flasks.
(A) Immunophenotype analysis of hMSCs show comparable surface marker expression.
The cells maintain differentiative potential. (B) Adipogenic differentiation capacity.
Process Parameters
Seeding density 3000 cells/cm2
Microcarrier concentration
10 cm2/mL
Medium exchange None
Supplement Day 3
Multi-platevesselLN2
Pall 3.2 L STRbioreactor
0
0.5 x 105
1.0 x 105
1.5 x 105
2.0 x 105
2.5 x 105
3.0 x 105
3.5 x 105
4.0 x 105
4.5 x 105
5.0 x 105
0 1 2 3 4 5 6
Cel
ls/m
L
Days
Pall 3.2 L STR bioreactor
Flatware
XF Prototype
0
100,000
200,000
300,000
400,000
500,000
600,000
Cel
ls/m
L
Day 7
Second Spinner Set, cells/mL
Collagen-5
Collagen-10Star-Plus-5
Star-Plus-10
XF-5XF-10
Flatware
LN2 SpinnerSet 1T-�asks Spinner
Set 2
Process Parameters
Seeding density 3000 cells/cm2
Microcarrier concentration
5 and 10 cm2/mL
Medium exchange None
Supplement Day 3
