Large-scale cell isolation is a critical step in cell therapy research. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. In this webinar, Dr. Amanda Vanden Hoek and Ms. Vesna Posarac present two novel platforms that enable efficient cell isolation from large-volume samples: the Easy 250 EasySep™ magnet and the RoboSep™-C instrument. The Easy 250 EasySep™ magnet allows users to manually isolate cells from a washed leukopak of up to 225 mL in a single procedure. Optimized negative and positive selection protocols for isolating T cells, B cells, NK cells, and monocytes ensure that isolated cells are highly purified and ready for a variety of downstream assays. The same isolation procedure for T cells can be automated in a closed system with RoboSep™-C, which performs all leukopak processing and cell isolation steps in a 75-minute workflow. By streamlining sample processing, this dedicated cell separation instrument enables scale up while maintaining sterility and compatibility of isolated cells for downstream assays. Both platforms offer efficient and user-friendly cell isolation that can be easily integrated upstream of existing workflows. Key Topics Include: - Learn about immunomagnetic cell isolation - Explore efficient platforms to scale up large-volume isolation of T cells and other immune cells - Assess the advantages of manual vs. automated cell isolation

1. Welcome!
Amanda Vanden Hoek, PhD
Business Operations
STEMCELL Technologies
Senior Product Manager
Vesna Posarac, MSc
Research & Development
STEMCELL Technologies
Senior Scientist
Quick and Easy Isolation of T
Cells and Other Immune Cells
from Large-Volume Samples

2. LUNCH & LEARN
New Systems for Bulk Cell
Isolation from Leukopaks
and Whole Blood
Presented by:
Amanda Vanden Hoek, PhD | Senior Product Manager
Vesna Posarac, MSc | Senior Scientist

3. Major Research Areas
Immunology
Pluripotent
Stem Cell Biology
Hematopoietic
Stem Cell Biology
Intestinal
Biology
Neural
Biology
Endothelial
Cell Biology
Pulmonary
Research
Mesenchymal Stromal
Cell Biology
Mammary
Research
Pancreatic
Research
CHO Cells &
Hybridomas
Prostate
Research
STEMCELL Technologies
Global Biotech Leader Supporting Life Science Research

4. Source Isolate
● Fresh human cells
● Frozen human cells
STEMCELL Products
For Every Step of Your Immune Cell-Based Research
Activate, Edit, Expand &
Differentiate
Analyse &
Cryopreserve
● EasySep™
● RoboSep™
● RosetteSep™
● SepMate™
● Lymphoprep™
● ImmunoCult™
● STEMdiff™
● StemSpan™
● Cytokines
● ArciTect™
● Antibodies
● ELISA kits
● Dyes & stains
● CryoStor™
● Contract Assay Services

5. STEMCELL Products
EasySep™ For Immunomagnetic Immune Cell Isolation
EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free
magnetic system for fast and easy isolation of highly purified cell populations that are immediately
ready for downstream assays.

6. STEMCELL Products
EasySep™ For Immunomagnetic Immune Cell Isolation
Starting Sample
Species
● Human
● Mouse
● Rat
● Non-human primate
● Other
● Whole blood
● Buffy coat
● Cord blood
● Bone marrow
● PBMC
● Leukapheresis
● Spleen
● Non-hematopoietic
tissues
● Other
Selection Method
● Negative
● Positive
● Depletion

7. Key Applications
● Biobanking
● Drug discovery
● Cell therapy research
Primary human immune cells are an important tool for
conducting physiologically relevant immunology research.
● Reflect inherent variability in donors/samples
● Facilitate translation of basic research into preclinical and
clinical applications
● Leukopaks provide large number of single-donor MNCs
from peripheral blood
Large-Volume Cell Isolation in
Immunology Research
CONTENTS

8. Key Applications
● Biobanking
● Drug discovery
● Cell therapy research
Key Cell Isolation Considerations
● Efficient, smooth scale up to full Leukopak capacity
○ Ease of use
○ Protocol speed
○ Performance (cell purity, recovery, viability, function)
○ Integration with downstream applications
● Manual vs. automated, closed systems
○ Risk of operator error or contamination
○ Need for robustness and reproducibility
○ Need for efficiency and flexibility
Large-Volume Cell Isolation in
Immunology Research
CONTENTS

9. ● Designed to isolate cells from large-volume human whole blood and
leukapheresis samples
○ negative selection: 25 - 225 mL
○ positive selection: 40 - 125 mL
● ~20 - 40 minute protocols
○ same steps as our other EasySep™ magnets (pipette off)
○ protocol times are typically similar to the comparable Easy50 protocol
○ large-volume sample can be processed in a single experiment
(no splitting the sample across multiple magnets)
● Uses a standard T-75 cm2 cell culture flask
● Optimized protocols and larger format reagents
○ available now for a broad range of cell types
○ based on existing EasySep™ reagent formulations
Easy 250 EasySep™ Magnet
New EasySep™ Magnet for Processing Large-Volume Samples

10. Easy 250 EasySep™ Magnet
Typical Cell Isolation Protocol

11. Cell Type Selection Method Catalog #
T cell Negative 100-0695
CD4⁺ T cell Negative 100-0696
CD8⁺ T cell Negative 100-0710
Monocyte Negative 100-0697
CD3⁺ Positive 100-0692
CD4⁺ Positive 100-0693
CD8⁺ Positive 100-0699
CD14⁺ Positive 100-0694
PBMC Negative 19654
RBC depletion Depletion 18170
Easy 250 EasySep™ Magnet
EasySep™ Reagents Optimized for Large-Format Use

12. Cell Type Selection Method Catalog #
T cell Negative 100-0695
CD4⁺ T cell Negative 100-0696
CD8⁺ T cell Negative 100-0710
Monocyte Negative 100-0697
CD3⁺ Positive 100-0692
CD4⁺ Positive 100-0693
CD8⁺ Positive 100-0699
CD14⁺ Positive 100-0694
PBMC Negative 19654
RBC depletion Depletion 18170
New large-format reagent kits
(for processing 10 B cells)
Easy 250 EasySep™ Magnet
EasySep™ Reagents Optimized for Large-Format Use

13. Cell Type Selection Method Catalog #
T cell Negative 100-0695
CD4⁺ T cell Negative 100-0696
CD8⁺ T cell Negative 100-0710
Monocyte Negative 100-0697
CD3⁺ Positive 100-0692
CD4⁺ Positive 100-0693
CD8⁺ Positive 100-0699
CD14⁺ Positive 100-0694
PBMC Negative 19654
RBC depletion Depletion 18170
Optimized protocols using
existing reagents
Easy 250 EasySep™ Magnet
EasySep™ Reagents Optimized for Large-Format Use

14. Don’t see your cell type or
kit of interest? Let us know!
Additional protocols are in
development.
Cell Type Selection Method Catalog #
T cell Negative 100-0695
CD4⁺ T cell Negative 100-0696
CD8⁺ T cell Negative 100-0710
Monocyte Negative 100-0697
CD3⁺ Positive 100-0692
CD4⁺ Positive 100-0693
CD8⁺ Positive 100-0699
CD14⁺ Positive 100-0694
PBMC Negative 19654
RBC depletion Depletion 18170
Easy 250 EasySep™ Magnet
EasySep™ Reagents Optimized for Large-Format Use

15. Negative Selection Positive Selection
Easy 250 EasySep™ Magnet: Performance Data
High Purity of Isolated Cells from Leukopaks

16. ● EFFICIENT. Isolate human immune cells from large-volume samples (up to 225 mL) in a single experiment.
● FAST. Go from full Leukopak to isolated cells in as little as 20 minutes.
● HIGH PURITY. Achieve up to 99% purity of viable, functional cells.
● FLEXIBLE. Optimized and customizable protocols compatible with many EasySep™ cell isolation kits.
Want to try it in your lab?
Request a demonstration today!
Easy 250 EasySep™ Magnet
New EasySep™ Magnet for Processing Large-Volume Samples

17. STEMCELL Products
RoboSep™ For Automated Immunomagnetic Immune Cell Isolation
RoboSep™ instruments fully automate all cell labeling and separation steps of the
EasySep™ procedure, minimizing sample handling and freeing up technician time.

18. RoboSep™-S RoboSep™-16 RoboSep™-C
NEW
STEMCELL Products
RoboSep™ For Automated Immunomagnetic Immune Cell Isolation

19. RoboSep™-C enables automated, closed, large-scale isolation
of human immune cells from leukapheresis samples in ~75 minutes.
RoboSep™-C
Isolation Kit
RoboSep™-C
Tubing Set
RoboSep™-C
Buffer
RoboSep™-C System Overview

20. Fresh Human Peripheral Blood Leukopaks
● RoboSep™-C can process a sample between
2.5 - 20 billion cells.
RoboSep™-C Cell Isolation Kits
● Kits contain 10 mL Cocktail + 10 mL Particles
● For processing 1 x 10^10 cells
● Negative selection kits currently available:
○ Human T Cell Isolation Kit
○ Human CD4+ T Cell Isolation Kit
○ Human CD8+ T Cell Isolation Kit
Reagents & Consumables

21. RoboSep™-C Tubing Set
● Single-use biocompatible tubing set provides a
closed, sterilized fluid path that enables efficient
cell washing and cell isolation.
RoboSep™-C PBS/EDTA Buffer
● 3L bag of PBS + 1 mM EDTA
○ PIS recommends addition of FBS
(2% Final)
Reagents & Consumables

22. Tubing Cassettes
Easy to install cassettes
organize all lines of tubing
Cell Wash Cartridge
Spinning filter gently washes
and concentrates your sample
Magnet Housing
Specially designed magnet
array removes labelled cells
Scale Tower
Weight scales ensure accurate
liquid volume movement
RoboSep™-C

23. RoboSep™-C enables automated isolation of human T cells with high purity,
recovery and viability from Leukopak samples.
T cells
CD4+
T cells
CD8+
T cells
Start
Isolated
%
Viability
T cells
CD4+
T cells
CD8+
T cells
%
Recovery
T cells
CD4+
T cells
CD8+
T cells
%
Purity
RoboSep™-C
Evaluation of Purity, Recovery and Viability of Isolated T Cells

24. RoboSep™-C
Evaluation of Purity of Isolated T Cells
RoboSep™-C enables automated isolation of
human T cells with high purity from Leukopak samples.
RoboSep™-C Human T Cell Isolation Kit
RoboSep™-C Human CD4+ T Cell Isolation Kit
RoboSep™-C Human CD8+ T Cell Isolation Kit
95.4%
97.5%
93.3%

25. RoboSep™-C
Evaluation of CD45+ Contaminants in Isolated T Cell Fraction
Isolated cell populations are highly pure with minimal CD45+ contaminants.
RoboSep™-C Human
T Cell Isolation Kit
RoboSep™-C Human
CD4+ T Cell Isolation Kit
RoboSep™-C Human
CD8+ T Cell Isolation Kit

26. RoboSep™-C
RoboSep™-C is highly effective at removing both red blood cells (RBCs)
and platelets from Leukopak samples.
3 log reduction
T cells CD4+
T cells
CD8+
T cells
Start Isolated
Evaluation of RBC and Platelet Depletion Efficacy

27. Start Isolated
T Cell % (CD3+) 55.2 95.0
CD4:CD8 Ratio 2.1:1 2.2:1
Number of T cells (109) 1.5 1.1
% Viability 99.0 99.0
Isolated T cells show robust activation and expansion when stimulated
with ImmunoCult™ Human T Cell Activators in ImmunoCult™-XF T Cell Expansion Medium.
RoboSep™-C
Evaluation of Downstream T Cell Activation and Expansion

28. RoboSep™-C
Coming Soon: Monocyte and NK Cell Isolation (Negative Selection)
RoboSep™-C also enables automated isolation of human monocytes and NK cells
with high purity, recovery and viability from Leukopak samples.

29. Benefits
• Automated. Minimal hands-on time for consistent walk-
away cell isolations.
• Closed System. Easy to install single-use tubing sets
increases product and operator safety.
• Fast & Easy. Go from a full leukopak to isolated cells in
as little as 75 minutes.
• Gentle. Obtain viable, functional cells without the need
to wash or concentrate your sample.
Applications & Timelines
• Research Use Only
• STEMCELL is committed to supporting a GMP workflow
and will communicate timelines when appropriate.
RoboSep™-C

30. RoboSep™-C Easy 250 EasySep™ Magnet
Column-free immunomagnetic cell isolation system
Fully automated, closed system Manual, open system
~75 minute protocols
(including upstream and downstream sample washing)
~20-40 minute protocols
(upstream and downstream sample washing performed separately)
Optimized for T cell isolation
(with protocols and reagents for additional cell types in development)
Optimized for a broad range of cell types
Optimized for use with fresh (and frozen) Leukopaks Optimized for use with Leukopaks & whole blood
RoboSep™-C and Easy 250 EasySep™ Magnet
Better Solutions for Cell Isolation from Large-Volume Samples

31. Starting Sample
STEMCELL Products for Cell Isolation
Highly Purified Cells for Your Research Applications
Species
● Human
● Mouse
● Rat
● Non-human primate
● Other
Selection Method
● Negative
● Positive
● Depletion
● Sequential
● Whole blood
● Buffy coat
● Cord blood
● Bone marrow
● PBMC
● Leukapheresis
● Spleen
● Non-hematopoietic tissues
(e.g. lung, tumor)
● Other

32. Questions?
Scientists Helping Scientists™ | WWW.STEMCELL.COM

33. Thank you for participating!
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