The document presents a study that evaluated the use of the CellSeal® Closed-System Cryogenic Vial for short-term low volume storage of regulatory T cells for clinical trials involving organ transplantation. The study found that the CellSeal® vial maintained cell viability, phenotype, and potency after 12 weeks of cryopreservation. In conclusion, the CellSeal® vial is highly suitable for low volume storage in clinical trials of cellular therapies like regulatory T cells.

1. www.cookgbt.com
Evaluation of Closed System Medical
Device for Low Volume Storage for Clinical
Studies Involving Regulatory T Cells
Presenter: Evonne R. Fearnot, MS
June 2, 2014, Savannah, GA

2. Overview
Objectives for this topic are to:
– Present elementary information related to organ
transplant problems, and immunotherapy as a
potential solution.
– Present the need for appropriate closed system
medical devices as new therapies emerge.
– Expand understanding on available cryogenic storage
container systems.
– Evaluate a closed system medical device which has
proven to be highly suitable for low volume storage in
clinical trial settings.

3. Transplant Problems
• Organ transplantation has been utilized since the
1950’s.
• While success rates of transplant surgery have
improved remarkably since it’s discovery, transplant
problems do exist and multiple factors influence
which and when post-operative problems arise.
• Of particular interest, there are long-term risks
associated with the use of immunosuppressive drugs.
• Immunosuppressive drugs may harm the transplant
or other body’s organs, and do not guarantee long-
term successful engraftment of the transplant.

4. Transplant Problems
• Transplant problems is a real clinical issue that Cook General
BioTechnology has witnessed first-hand. Two years ago, one of my
colleagues received a kidney transplant.
• Since, my colleague continues to be affected by the many
associated problems and the lack of successful, alternative
treatment options available.

5. Transplant Problems
• My colleague continues to suffer and had to take an
extended leave of absence from work.
• As such, the discovery for new treatment options, such as
cellular therapy, to mitigate transplant problems is underway.

6. Emerging Cellular Therapies
The study of cellular therapy allows us to take a new
perspective on human disease, a new perspective for which
scientists have not yet fully explored, and for which discovery
to further benefit patients is possible;

7. Emerging Cellular Therapies
• Regulatory T cells have been considered as key regulators in
various immunological processes and therefore there has been
a great interest in using them for immunotherapy.
• Thus, regulatory T cells may provide a successful, alternative
treatment option to mitigate transplant problems, specifically
for reducing or replacing the need for immunosuppressive
drugs, as well as to successfully prolong organ allograft survival.
• With the discovery of potential new therapies, such as the use
of regulatory T cells to mitigate transplant problems, the need
for appropriate closed system medical devices for storage,
transport, and delivery of low volume cellular therapeutics is
evident.

8. State of the Art for Container Systems
• In the past, biological materials have generally
been stored in either vials or cryogenic bags.
• Each container system type has its own benefits
and drawbacks.
vs.

9. State of the Art for Container Systems
Cryogenic bags are beneficial because cryogenic bags:
• are “closed-systems”, and
• allow for integral segments.
– “Closed-systems” decrease the risk of contamination
and integral segments allow for subsequent or
confirmatory testing.
On the other hand, vials are beneficial because vials:
• are associated with lower risk of breakage and thus a
lower breakage rate, and
• are useful for storing smaller volumes.

10. State of the Art for Container Systems
Conversely, cryogenic bags present drawbacks because cryogenic bags:
• may be prone to cracking or breaking, and are not as useful for smaller
volumes.
– Cracking or breaking container systems compromise the integrity
and sterility of the stored cellular therapies.
– Cracking or breaking often leads to longer and thus, delayed
treatment times, as well as the need for the use of prophylactic
antibiotics where it is otherwise unnecessary.
– Cracking or breaking may also lead to complete loss of the stored
cellular therapy.
Also, vials present drawbacks because vials:
• are generally “open-systems”, and
• do not allow for integral segments.

11. • Yet, the CellSeal® Closed-System Cryogenic Vial is
designed to incorporate the benefits of bag storage,
while still retaining the benefits of vial storage.

12. • The CellSeal Closed-System Cryogenic Vial is a
newly CE Marked, Health Canada registered, and
TGA approved medical device.
• The CellSeal Closed-System Cryogenic Vial is
intended for the freezing and storage of tissue,
cells, blood and blood components for the purpose
of introduction into the body by means of infusion
or other means of administration.

13. • The CellSeal Closed-System Cryogenic Vial is made of materials that are
stable and durable during rapid temperature changes associated with
controlled rate freezing and at low temperatures.
• The design of the CellSeal Closed-System Cryogenic Vial has:
– a loading port for ease of loading,
– a microbial barrier vent to prevent pressure from building up in the
vial during loading and to allow sterile air back into the vial during
retrieval,
– tubing that can be sealed to create a functionally closed system and
to include an integral segment, and
– a retrieval port allowing maximum recovery of the stored volume.
• Also, the retrieval port on the CellSeal Closed-System Cryogenic Vial
offers seamless transfer at the point of care to standard delivery
needles or infusion sets for delivery to patients.

14. • Insert CellSeal video here.

15. Study Introduction
The subject of the current study is the CellSeal®
Closed-System Cryogenic Vial for storage of T cells.

16. Study Objective
The objective of the current study was to evaluate
the use of the CellSeal® Closed-System Cryogenic
Vial for the application of short-term low volume
storage of separated and expanded regulatory T
cells in preparation for clinical studies involving
organ transplantation.

17. Study Methods
For cryopreservation, separated and expanded
regulatory T cells were counted and resuspended
in Cryostor CS10 (BioLife Solutions Inc, WA, USA).
Expansion
Purity
Suppression
0.5x106
Beads (4:1) / (1;1)
IL-2 (500U)
Rapa (100nM)
Stimulation 1 2, 3
+ +++ +
Feed 1 2 3 4 5

18. Study Methods
The cell concentration per single dose was calculated so
the volume of the final dose (3x106/kg of patient’s body
weight) was frozen and stored in 2 mL CellSeal Closed-
System Cryogenic Vials using a controlled rate freezer and
stored in vapor phase of liquid nitrogen.

19. Study Methods
After 12 weeks of cryopreservation, the cells were
thawed, resuspended in 5% human serum
albumin, and evaluated for viability, phenotype,
potency and recovery.

20. Study Results
• The phenotype and the potency of the cells
remained stable.
• The viability of the cells complied with the release
criteria of the study (>70%).
• The cells also had a high degree of recovery.
Purity Potency Viability Recovery
CD4/CD25/FoxP3 CD8 Treg:Teff(1:1)
≥60% ≤10% ≥60% ≥70% NA
Pre-freeze 88.5 1.44 91.3 95 NA
Post-thaw 89 6.35 95 85 110

21. Discussion
This study corroborates the results previously seen and reported
when CellSeal Closed-System Cryogenic Vial was used to store
other cellular therapy products, including but not limited to:
– Mesenchymal stem cells from multiple tissue sources (skin,
bone marrow, adipose, endometrium, dental pulp, umbilical
cord, amnion)
– Peripheral blood stem cells
– Stromal vascular fraction
– Dendritic cells
– Umbilical cord blood
– Sperm

22. Study Conclusion
• The study results provides supportive data for
use of a single dose of autologous regulatory T
cells cryopreserved using CellSeal® Closed-
System Cryogenic Vials to be injected in vivo to
induce prolongation of organ allograft survival.
• Also, the study results indicate that the CellSeal®
Closed-System Cryogenic Vial met expectations in
preparation for two clinical trials using regulatory
T cells.

23. Summary
• In summary, understanding unmet clinical needs is critical to
developing new solutions within the healthcare field.
• The study of cellular therapy, such as for an alternative
treatment option to mitigate transplant problems such as
immunotherapy, allows us to take a new perspective on human
disease for which discovery to further benefit patients is
possible.
• With these new discovered therapies, the need for appropriate
closed-system medical devices for storage, transport, and
delivery of low volume cellular therapeutics is evident.
• Finally, the CellSeal® Closed-System Cryogenic Vial is highly
suitable for low volume storage in clinical trial settings in cellular
therapy.

24. Acknowledgements
I want to acknowledge my co-authors listed on this slide who
conducted the study:
• Henrieta Fazekasova, Immunoregulation Laboratory, Kings College
London, London, England, UK
• Sarah Thirkell, Nuffield Department of Surgical Sciences,
University of Oxford, Oxford, England, UK
• Katie Lowe, Immunoregulation Laboratory, Kings College London,
London, England, UK
• Andrew Bushell, Nuffield Department of Surgical Sciences,
University of Oxford, Oxford, England, UK
• Giovanna Lombardi, Immunoregulation Laboratory, Kings College
London, London, England, UK

25. Thank you for your time and attention.
Questions?
Evonne R. Fearnot, MS
evonne.fearnot@cookgbt.com
www.cookgbt.com