Watch the presentation of this webinar here: https://bit.ly/3ryE5ST Optimize your mesenchymal stem cell growth. Join our webinar to learn more about our GMP-compliant xeno free media formulation that supports high performance expansions and compatibility with scalable xeno free manufacturing conditions. Optimizing ex vivo cell expansion processes in preparation for clinical use is a critical step in cell therapy manufacturing. Given the curative and lifesaving impacts these therapies can have on patients, overcoming roadblocks with scalability and supply chain, using high quality raw materials are essential for therapeutic access. The GMP-compliant Stemline® XF MSC Medium and cocktail promotes expansion of human mesenchymal stromal/stem cells (hMSCs) to high densities while maintaining cell identity and quality. This product was designed for derivation and expansion of MSCs using xeno free conditions in planar and microcarrier-based culture platforms, easing the transfer between research, clinical, and manufacturing scale cultures. In this webinar, you will: • Explore the current landscape and future trends of cell culture media for adult mesenchymal stem cells • Discover ways to derive MSC's from Bone Marrow in Xeno Free conditions from static to microcarrier-based suspension culture platforms. • Learn how Stemline® XF MSC Media provides robust performance and reduces scalability roadblocks Presented by: Kathleen Ongena, Ph.D., Head of Customer Applications and Mark Ventresco, Cell Therapy Product Manager

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Achieving High Yields
in Scalable Xeno-Free
Culture Formats with
Mesenchymal Stem
Cell Medium
Kathleen Ongena, Ph.D.
Head of Customer Applications
Mark Ventresco
Cell Therapy Product Manager
Thursday July 22, 2021

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. Agenda
1
2
3
Current Landscape and Future
Trends of hMSCs
Deriving hMSCs in Xeno-Free
Conditions
Achieving High Yields Using Xeno-Free
Microcarriers in 3L Culture Formats

4. Current
Landscape and
Future Trends

5. Human Mesenchymal Stem/Stromal Cells (hMSCs)
What are they?:
• First discovered in the early 70’s, Mesenchymal stem cells are typically found
in bone marrow, adipose, cord blood, placenta, and other tissues
• Early on the ability of MSCs to differentiate into specialized cell types attracted
clinical research to evaluate therapeutic benefits
• Later discovered were the paracrine, trophic and immunomodulatory
properties of the cells which naturally resulted in clinical research focused on
inflammatory and degenerative diseases
What are they Used For?:
• Due to the natural healing abilities, anti-inflammatory and Immunosuppressive
Responses of hMSCs, clinical targets typically include GvHD, cardiac disease,
spinal cord injury, ALS, inflammatory diseases, Crohn’s disease, and
myocardial infarction
• Recent trials have been investigating the effectiveness of MSC based therapies
on COVID-19 patients, as the leading cause of death for COVID-19 patients is
often sepsis or inflammation caused by ARDS.
5
Human Mesenchymal Stem/Stromal Cells in Cell Therapy
hMSCs are Adult Stem Cells That Offer a Wide Range of Clinical Benefit and Hold Promise
in Regenerative Medicine for a Wide Range of Indications

6. 57%
8%
8%
9%
TSSC
MSC
HSC
10%
5%
BM&CB
SVF & adipose
3%
MNC
Pluripotent
1%
Other
2016-2019
2011-2015
54%
13%
7%
9%
MSC
HSC
Bone marrow and
cord blood
3%
6%
SVF and adipose
6%
TSSC
MNC
Pluripotent & progenitor
2%
Other
2011-2019
Stem Cell
Clinical Trials
Globally: 1600+
3200+
Global Stem Cell & Immunotherapy Clinical Trials 2011-2019
24%
North America
17%
ROW
18%
Europe
41%
APAC
MSC Clinical
Trials
Globally: 800+
Clinical Trial History for Stem Cell and Immunotherapy Markets
Over Half of Stem Cell Clinical Trials Globally are Using Mesenchymal Stem Cells
10%
23%
28%
29%
3%
Phase I/II
Other
Phase III
4%
Phase I
Phase II/III
Phase II
4%
Phase IV
ImmunoTherapy
Clinical Trials
Globally:1600+
Sources: Merck KGaA, Darmstadt, Germany Commissioned Report, celltrials.org,
clinicaltrials.gov, EudraCT, and country-specific trial databases (China, Japan, Australia),
Alliance for Regenerative Medicine 2019 Annual Report
6

7. 0
20
40
60
80
100
120
140
160
2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020
MSC Clinical Trials Started by Year
123% Growth Between 2019
and 2020 Driven by COVID
Related Clinical Trials
hMSC Clinical Trials and Recent Market Trends
Overall hMSC Market Shows Growth in 2020 and 2021, Primarily Driven by
COVID-19 Related Clinical Trials
Take Home Points
41 out of 134 Clinical Trials started in 2020 are
COVID Related, 93 non-COVID related
indicates 55% Growth over 2019 for non-
COVID related Trials
2021 YTD 40 clinical trials have been
started, this indicates a slightly lower run
rate than 2020 (non COVID related), but a
faster run rate than 2019
2020 and 2021 Clinical Trial data suggests
significant growth in the MSC market
26%
40%
25%
4%
Phase 1
Early Phase 1
Phase 1|Phase 2
Phase 2
3%
Phase 2|Phase 3
2%
Phase 3
0%
Phase 4
Sources: Merck KGaA, Darmstadt, Germany Commissioned Report, clinicaltrials.gov, Alliance for Regenerative
Medicine 2020 Annual Report
Current Active
MSC Clinical
Trials By Phase
7

8. Clinical Trials Started or Scheduled in 2020+
hMSC Clinical Trials and Recent Market Trends
COVID-19 Indications Occupy the Majority Market Share for New hMSC Clinical
Trials
Clinical Trials Started in 2020 &
2021 COVID Related Studies
46%
22%
54%
Other
ARDS
Pneumonia
Sources: clinicaltrials.gov, Alliance for Regenerative Medicine 2020 Annual Report
The Majority of COVID Related Clinical Trials in 2020/21 were
focused on short- and long-term Lung complications as well as
the immunomodularity properties of hMSCs and their potential
ability to strike balance in the immune cells of patients with
hyperactive immune systems (e.g. cytokine storm)
Clinical Trials Started or Scheduled Between
2010-2019
9%
5%
70%
Crohn’s
COVID-19
0%
Arthritis
Ligament Repair
2%
6% Myocardial Infarction
Alzheimers
Diabetes
4% GVHD
1%
1%
Psoriasis
1%
2%
ALS
Other
28%
6%
47%
2%
5%
COVID-19
Arthritis
5%
Myocardial Infarction
3%
Crohn’s
1%
Diabetes
GVHD
Alzheimers
2%
Psoriasis
2%
Ligament Repair
Other
8

9. Seed Train Production
Planar Process
•Manually Driven
•Open process
•Labor intensive
Seed Train Production
Bioreactor Process
• Greater control
• Partially closed
• Production Scale
Human Mesenchymal Stem/Stromal Cells in Cell Therapy
How are they Manufactured: Typical Seed Train Strategies for Production of hMSCs
Manufacturing of Allogeneic hMSC
based therapies can require an
excess of 100e9 cells often
requiring a scale up which can
exceed 50 Ls.
Closed Bioreactor
Process
•Reduced Safety &
Sterility Risk
Seed Train
9

10. Deriving hMSCs
in Xeno-Free
Conditions

11. Deriving hMSCs in Xeno-Free Conditions
Transition to Xeno-Free Conditions Provides Reduced Safety and Sterility
Risk for hMSC Therapy Development
 Use of Fetal Bovine Serum in media:
➢Undefined: lot to lot variability
➢Future availability of Fetal Bovine Serum
➢Concern of pathogen contamination
• Downstream processing
 Transition to serum-free, xeno-free conditions
 Need for cGMP grade and high-quality raw
material
Current need
Current challenge
11

12. *Our Global Quality Management System is certified as ISO 9001:2015, and all sites voluntarily comply
with the Joint IPEC-PQG Guide on Good Manufacturing Practices (GMP) for Excipients and applicable
sections of Annex 1 of the EU Guidelines to Good Manufacturing Practice for Medicinal Products.
12
Deriving hMSCs in Xeno-Free Conditions
Stemline® XF MSC Medium
Stemline® XF MSC Medium
• Developed to promote the expansion of human
mesenchymal stem/stromal cells (hMSCs) in both planar and
suspension culture formats
• 500 mL bottle and 5 mL vial of supplement (also available in
5L bag formats)
• Serum-free, xeno-free formulation
• Carefully selected raw materials to support clinical
manufacturing of hMSCs
• GMP-manufactured*

13. 13
Xeno-free process:
• Derivation of hMSCs in
xeno-free conditions
• Use of xeno-free medium
for planar and suspension
culture
• Use of xeno-free
microcarriers
Derivation from bone marrow,
umbilical cord, adipose tissue, …
Seed Train
Deriving hMSCs in Xeno-Free Conditions
Transition to Xeno-Free Reagents and Processes
Production

14. 14
Passaging
Outputs
• Cell yield
• Cell viability
• Cell morphology
• Surface marker
expression
Test conditions:
Stemline® XF MSC Medium
• Without coating
• Different xeno-free substrates
Control condition:
DMEM/FBS media
• 0.1% Gelatin
Bone marrow
aspirates
Colony Forming
Unit Assay
Culture
Deriving hMSCs in Xeno-Free Conditions
Experimental Design
Xeno-Free Substrate/Surface Vendor
ECMatrixTM -511 E8 Laminin Substrate
Merck KGaA, Darmstadt,
Germany
Human recombinant Fibronectin
Merck KGaA, Darmstadt,
Germany
CellBind® Surface Corning
No coating NA
EmbryoMax® 0.1% Gelatin (control) Merck KGaA, Darmstadt,
Germany

15. 15
Multiple Substrate/Surfaces Supported Colony Formation in Stemline®
XF MSC Medium
Colony Forming Unit Assay (CFU)
10000 20000 40000
0
5
10
15
20
25
DMEM/FBS - Gelatin Stemline® - no coat Stemline® - Laminin Stemline® - Fibronectin Stemline® - CellBind
AV
number
of
colonies
Substrate/surface
AV number of colonies per substrate/surface
Experimental Conditions
Cell type bone marrow derived hMSCs
Format T25 with different substrates/surfaces
Duration 10 days, media exchange at day 3 and 6
Seed density 10 000, 20 000, 40 000 cells/cm2
Media Stemline® XF MSC
Characterization Microscopy: crystal violet stain, colony counting

16. 16
Good Cell Growth Performance over Multiple Passages with hMSCs Derived
in Xeno-Free conditions
Planar Passaging Study in Stemline® XF MSC Medium
Experimental Conditions
Cell type bone marrow derived hMSCs
Format T75
Duration 4 days per passage
Seed density 3000 cells/cm2
Media Stemline® XF MSC, DMEM/FBS
Characterization Cell count and % viability, surface marker flow cytometry, microscopy
Data not shown:
Viability >95% for every
condition and timepoint
0.00
5.00
10.00
15.00
20.00
25.00
30.00
P1 P2 P3 P4
AV
Doubling
time
(hrs)
Passage
DMEM/FBS - derived on Gelatin (control)
Stemline® - derived with no coating
Stemline® - derived on Fibronectin
Stemline® - derived on Laminin
Stemline® - derived on CellBind

17. 17
Planar Passaging Study in Stemline® XF MSC Medium
hMSC Surface Phenotype is Maintained When Using Xeno-Free
Reagents and Processes
0
10
20
30
40
50
60
70
80
90
100
CD105 CD90 CD73 CD11b CD34 CD45 HLA-DR
%
Gated
Cells
Surface Markers
Flow Cytometry Surface Markers at Passage 4
DMEM/FBS - derived on Gelatin
Stemline® - derived with no coating
Stemline® - derived on Laminin
Stemline® - derived on Fibronectin
Stemline® - derived on CellBind

18. 18
Planar Passaging Study in Stemline® XF MSC Medium
hMSC Morphology is Maintained When Using Xeno-Free Reagents and Processes
Stemline® - Cellbind
DMEM/FBS - gelatin
Stemline® - Fibronectin
Stemline® - no coat
Stemline® - Laminin

19. 19
Multiple Options for Xeno-Free Substrates/Surfaces Supporting the
Derivation of hMSCs
Deriving hMSCs in Xeno-Free Conditions
Substrate
Pre-coating
Requirement
Ease-0f-
Use
Cell Growth
Performance
Effect on Surface
Marker Expression
511 E8-Laminin NA √ √ √
Fibronectin √ √ √ √
CellBind® NA √ √ √
No coating NA √ √ √
Gelatin √ √ √ √

20. Achieving High
Yields Using Xeno-
Free Microcarriers
in 3L Culture
Formats

21. Scaling Up Human Mesenchymal Stem/Stromal Cell Manufacturing
➢ Maintaining closed system processing from hMSC source through
bioreactor production
➢ Typical patient doses often require large scale expansion formats
to meet cell count targets for manufacturing
➢ Commercially available media does not always maintain
performance from planar culture through bioreactor suspension
culture formats using xeno-free conditions
➢ Manufacturing process that supports scale up of hMSCs using
xeno-free conditions (reagents, substrates, & microcarriers)
➢ A scalable cell culture media that supports high yield in planar
and suspension bioreactor formats
Current need
Current challenge
21
Current Challenges and Industry Needs

22. 22
Experimental Conditions
Feasibility Study: Expansion in Mobius® 3L Bioreactor using
Stemline® XF MSC Medium
Experimental Conditions
Cell type bone marrow derived hMSCs
Volume/process 2.4 L, fed-batch
Duration 8 days
Seed density 3000 cells/cm2
Microcarriers Collagen-coated polystyrene, 15g/L (360cm2/g)
Media Stemline® XF MSC, ɑMEM/hPL, XF-X (commercial available xeno-free formulation)
Temperature 37°C
pH 7.4 (±0.05)
DO 50%
Agitation rate 35 rpm (day 0 – feed 1), 61 rpm onwards
Characterization Cell count and % viability, surface marker flow cytometry, microscopy, IDO, differentiation
Thaw

23. 23
Good Cell Growth with Stemline® XF MSC Medium in Stirred System
Expansion in Mobius® 3L Bioreactor
0.0E+00
2.0E+08
4.0E+08
6.0E+08
8.0E+08
1.0E+09
0 2 4 6 8 10
Total
Cells
Days
Total Cells
Stemline® XF MSC ɑMEM/hPL XF-X
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10
Glucose
(g/L)
Days
Glucose
0
0.5
1
1.5
2
0 2 4 6 8 10
Lactate
(g/L)
Days
Lactate

24. 24
Tri-Lineage Differentiation is Maintained
Expansion in Mobius® 3L Bioreactor
Osteoblasts Chondroblasts Adipocytes

25. 25
Expansion in Mobius® 3L Bioreactor
Immunomodulatory Functions Confirmed by IDO Activity
*hMSCs activated with TNF-ɑ and INF-ϒ
L-tryptophan
in media
L-kynurenine
IDO
hMSC*
0
10
20
30
40
50
60
Non
licensed
Licensed Non
licensed
Licensed
Pre-3L Post-3L
L-tryptophan
(μM)
Day 0 Day 3
*
0
2
4
6
8
10
12
14
16
18
Non licensed Licensed Non licensed Licensed
Pre-3L Post-3L
L-kynurenine
(μM)
Day 0 Day 3
*

26. 26
Good Cell Growth with Stemline® XF MSC Medium Media and Xeno-Free
Microcarriers
Selection of Xeno-Free Microcarriers (Spinner Study)
0.0E+00
4.0E+06
8.0E+06
1.2E+07
1.6E+07
2.0E+07
0 1 2 3 4 5 6 7
Total
Cells
Days
Stemline® - Synthemax
Stemline® - Plastic Plus
Stemline® - Plastic Plus +
Laminin
Microcarrier Vendor
Low Concentration Synthemax® II Corning
Plastic Plus Sartorius
Collagen Coated Sartorius

27. 27
Experimental Conditions
Feasibility Study: Expansion in Mobius® 3L Bioreactor
using Xeno-Free conditions
Experimental Conditions
Cell type bone marrow derived hMSCs
Volume/process 2.4 L, fed-batch
Duration 9 days
Seed density 3000 cells/cm2
Microcarriers Low Concentration Synthemax® II
Media Stemline® XF MSC
Temperature 37°C
pH 7.4 (±0.05)
DO 50%
Agitation rate 35 rpm (day 0 – feed 1), 61 rpm onwards
Characterization Cell count and % viability, surface marker flow cytometry, microscopy
Thaw

28. 28
Expansion in Mobius® 3L Bioreactor
Good Cell Growth with Stemline® XF MSC Medium and Xeno-Free
Microcarriers in Stirred System
0.00E+00
4.00E+08
8.00E+08
1.20E+09
1.60E+09
2.00E+09
0 2 4 6 8 10
Total
Cells
Days
Total Cells
Stemline® - Synthemax rep 1
Stemline® - Synthemax rep 2
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10
Glucose
(g/L)
Days
Glucose
0
1
2
3
4
0 2 4 6 8 10
Lactate
(g/L)
Days
Lactate

29. 29
hMSCs Surface Phenotype is Maintained after Expansion on
Xeno-Free Microcarriers
Expansion in Mobius® 3L Bioreactor
0
20
40
60
80
100
CD105 CD90 CD73 CD11b CD19 CD34 CD45 HLA-DR
%
Gated
Cells
Surface Markers
Flow Cytometry Surface Markers Stemline® - Synthemax
Day 6: DAPI Staining
Day 1: Calcein Staining

30. • Multiple xeno-free substrate/surfaces support the
derivation of hMSCs in Stemline® XF MSC Medium
• The performance of the Stemline® XF MSC medium was
confirmed across a xeno-free process using xeno-free
reagents
• High cell yield was achieved in 3L suspension culture with
Stemline® XF MSC Medium maintaining multilineage
differentiation and immunomodulatory properties
• Expansion on xeno-free microcarriers resulted in high cell
growth while preserving hMSCs surface phenotypes
29
Achieving High Yields in Scalable Xeno-Free Culture
Formats with Mesenchymal Stem Cell Medium
Summary & Conclusions

31. The vibrant M, SAFC, Stemline, Mobius are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners.
Detailed information on trademarks is available via publicly accessible resources. © 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.