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120
0 20 40 60 80 100 120 140
Time Post-Split (Hours)
0
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120
0 20 40 60 80 100 120 140
Essential 8TM Alone
+ Post-Thaw Recovery
Supplement
Essential 8TM Alone
David T. Kuninger, Lauren E. Sangenario, and Rhonda A. Newman, Cell Biology & Stem Cell Sciences, Life Technologies, Frederick, MD 21704
RESULTS
• mFreSRTM was shown to be “Best-in-Class”
• While several commercial cryopreservation solutions provide high
viability (>70%) post-thaw, these measurements are overestimates of
effective iPSC survival, as cell death from apoptosis and necrosis
following cryopreservation can take days to be realized [1-2].
• Viability 24 hours post-thaw is predictive of post-thaw recovery
•There was shown to be significant room for improvement
Figure 2. Experimental Workflow for Optimizing PSC Cryomedium
• Process workflows were used in optimization of cryomedium and
recovery medium solutions
• Assays fit for purpose were determined and used in development of
reagents using a series of screens, iterative DOEs, and titrations
• Our improved xeno-free cryomedium coupled with chemically defined post-
thaw recovery supplement provides optimum post-thaw recovery of iPSCs
• When used together with the chemically defined post-thaw supplement
being applied for 18-24 hours post passaging at each passage, iPSCs are
shown to maintain normal morphology, pluripotency, and karyotype
• Use of the post-thaw recovery supplement in conjunction with various
cryomedia provides significant improvement over the use of traditional ROCK
inhibitor, Y-27632
ABSTRACT
Pluripotent stem cells (PSCs) and primary cells are foundational tools
for basic research and applied applications including regenerative
therapy, drug discovery, and toxicological assessment. While stem
cells have a tremendous proliferative capacity, long term culture of
these cells has been shown to cause an accumulation of mutations
that result in genetic instability, increasing tumorigenicity and thus
limiting their usefulness in research and clinical applications.
Improved solutions for cryopreservation of early passage cells that
minimize loss of viability, maximize post-thaw recovery, and minimize
unwanted differentiation are essential components to PSC, as well as
primary cell workflows. While many cryopreservation reagents afford
high viability immediately post-thaw, significant apoptosis and
necrosis is often observed following the first 24 hours post-thaw,
decreasing the effective viability, reducing cell numbers and adding
additional stress and selective pressure to cultures. Further, this
extends the time post-thaw cells must be cultured prior to use in
downstream experiments. Using a series of Design of Experiments
(DOEs) and mathematical modeling methods, we describe the
development of a xeno-free cryomedium for use in cryopreservation
of PSCs and ESCs, and a chemically defined post-thaw recovery
supplement for use in recovery of PSCs, ESCs, as well as difficult-to-
preserve primary cells. When used together, we show this system
provides >80% direct post-thaw viability of PSCs with >70% cell
survival following 24 hours post-plating. As a result of increased post-
thaw survival rate, cells recover faster and are ready to passage
sooner than with current solutions, while maintaining pluripotency and
normal karyotype over 10 passages. Additionally, the post-thaw
recovery supplement was tested in combination with other
cryopreservation reagents which lead to markedly improved 24 hour
post-thaw viability of difficult-to-preserve primary cells, including
primary cortical neurons, human corneal epithelial cells, and human
epidermal keratinocytes.
INTRODUCTION
The goals of this research were (1) to provide improved, cost-effective
solutions for cryopreservation and recovery of PSCs, (2) identify
applicability of solutions to additional difficult-to-preserve cell types,
as well as, (3) assess alternative applications for the developed post-
thaw recovery supplement.
Life Technologies currently offers two ready-to-use cryopreservation
media: Synth-a-Freeze and RecoveryTM Cell Culture Freezing
Medium. During our development, these media were tested for their
applicability for cryopreservation of PSCs in comparison with the
currently recommended protocol of use of Essential 8TM + 10%
DMSO, as well as optimized solutions.
Six Sigma Design Excellence principles were used to guide
generation of improved cryopreservation and recovery systems with
an initial focus on the PSC workflow. Broader applicability of
solutions to primary cell types was subsequently assessed and is still
underway.
CONCLUSIONS• MSAs were used to define assays fit for purpose for determining cryomedia
which maximize post-thaw viability & recovery
•DOEs and mathematical modeling were used to improve Life Technologies’
solutions and associated protocols for cryopreservation and recovery of
PSCs
• Additional utility of post-thaw recovery supplement is demonstrated for
recovery of additional cryopreserved cell types, as well as for single cell
passaging of PSCs
• Prototypes will be available in Jan. 2014. Please contact Rhonda Newman
([email protected]) or Wendy Bray ([email protected])
for more information. For Research Use Only. Not for diagnostic purposes.
REFERENCES1. Baust JM, Vogel MJ, VanBuskirk RG, and Baust JG. A Molecular Basis of
Cryopreservation Failure and its Modulation to Improve Cell Survival. Cell
Transplantation (2001) 10:561-571
2. Baust JM, VanBuskirk RG, Baust JG. Cell viability improves following
inhibition of cryopreservation-induced apoptosis. In Vitro Cell. Dev. Biol.
Anim. (2000) 36(4):262-270
ACKNOWLEDGEMENTSWe would like to thank Rene Quintanilla, Tori Barron, Marian Piekarczyk,
Connie Lebakken, David Piper, Alexandria Sams, Kirsten Amaral, Stephen
Ferguson for helpful discussions.
TRADEMARKS/LICENSING© 2013 Life Technologies Corporation. All rights reserved. The trademarks mentioned
herein are the property of Life Technologies Corporation and/or its affiliate(s) or their
respective owners. mFreSR is a trademark of StemCell Technologies, Inc. Essential 8 is a
registered trademark of Cellular Dynamics International, Inc. pZerve is a trademark of
Protide Pharmaceuticals, Inc. Stem-CellBanker is a registered trademark of Nippon
Zenyaku Kogyo Co., Ltd. hESFreeze is a trademark of GlobalStem. Accutase is a
registered trademark of Innovative Cell Technologies.
Improved Cryopreservation & Recovery Solutions for
Pluripotent Stem Cells & Difficult-to-Preserve Primary Cells
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
B167
A. Input-Output Control Diagram for Cryomedium Development
B. Process Workflow for Cryomedium & Recovery Medium Development
0
25
50
75
100
%S
YT
OX
AA
Dva
nce
dT
M
Neg
ati
ve
17
23
29
35
41
47
53
59
66
72
78
84
90
%Viability
Each Pair
Student’s t
0.05
BL3 Sytox AADvanced 640-H
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Assessment of Direct Post-Thaw Viability
200,000
Pre
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600,000
800,000
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11
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33
38
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54
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65
70
%Viability
Each Pair
Student’s t
0.05
76
82
Assessment of 24 Hour Post-Thaw Viability
Determined Assays Fit for
Purpose By Screening
Various Cell Health
Reagents for Accurate
Discrimination of
Differences in Cell Viability
Conducted Iterative DOEs
to Determine Reagnets with
the Largest Impact on Cell
Viability
Conducted Small Molecule
Library Screens to Identify
Prosurvival Small Molecules
Compared Optimum
Formulations w/ Commercial
& Homebrew Solutions
Assessed
Commercial
Solutions Using
Assays Fit for
Purpose
Control
Factors
Cryopreservation
of Feeder-Free
PSCs
Critical
Functional
Responses
Noise
Factors
1. Basal Medium
2. Cryoprotective Agents A. Type
B. Concentration
3. Medium Additives
A. Membrane Stabilizers
B. Caspase Inhibitors
C. Antioxidants
D. Non-permeating solutes
4. Freezing Protocol
A. Temperature of
Cryopreservation Medium
B. Time of exposure prior to
freezing
C. Freezing Rate
1. Donor Variability
2. Size of Stem Cell clusters
3. Chemical Impurities
in Reagents
1. Post-Thaw Viability
2. Post-Thaw Recovery
3. Maintenance of Normal
Pluripotency
4. Maintenance of Normal
Karyotype
Final Formulation(s)
Assessment of
Post-Thaw
Viability (Direct & 24 Hour)
Assessment of
Post-Thaw Recovery
Maintenance of Normal
Karyotype & Pluripotency
C. Defining Assays Fit for Purpose
D. Example DOE Used in Optimization of Post-Thaw Recovery
Figure 3. Analysis of Existing Cryomedium Solutions for induced PSC (iPSC)
Cryopreservation
A. Direct Post-Thaw Viability
Re
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B. 24 hours Post-Thaw Viability
1,000,000
Pre
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2,000,000
3,000,000
4,000,000
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10% DMSO
Synth-a-
Freeze® CryoStor10
pZerve
Stem-
CellBanker®
CryoStor5RecoveryTM
hESFreezeTM mFreSRTM
LIV
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Re
pre
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nta
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Im
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C. Post-Thaw Recovery
(Confluence Monitored 6 Days Post-Thaw)
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All Pairs
Tukey-Kramer
0.05
20
40
60
80
100
D. Maintenance of PluripotencyEssential 8TM +
10% DMSO Synth-a-Freeze®
Alk
ali
ne
Ph
os
ph
ata
se
Liv
e S
tain
Oc
t-4/
-tu
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EA
4/N
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Blu
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mFreSRTM
E8 + 10% DMSO;
E8 +10 µM Y-27632
FBS + 10% DMSO;
E8 +10 µM Y-27632
mFreSRTM; E8 +
10 µM Y-27632
PSC Cryomedium;
E8 + Post-Thaw Recovery
Supplement
Figure 4. iPSC Cryopreservation and Recovery Using Optimized Solutions
A. Optimized Cryomedium & Post-Thaw Recovery Solutions Provide Maximum Recovery of
Cryopreserved iPSCs
D. Post-Thaw Recovery Supplement Can
Improve Recovery of iPSCs Cryopreserved
in Multiple Cryomedia Solutions
Pre
sto
Blu
e
Flu
ore
sc
en
ce
(24
Ho
urs
Po
st-
Th
aw
)
IncuCyte ZOOM Phase Contrast
Images w/ Confluence Mask
Pre
sto
Blu
e
Flu
ore
sc
en
ce
(24
Ho
urs
Po
st-
Th
aw
)
B. Optimized Solutions Provide
Efficient Post-Thaw Recovery of iPSCs
0
20
40
60
80
100
0 50 100 150 200
Essential 8 Alone
Essential 8 + Post-Thaw Recovery Supplement
Inc
uC
yte
TM
Mo
nit
ore
d C
on
flu
en
ce
Time Post-Thaw (Hours)
C. Optimized Solutions Provide Maintenance of
Normal Morphology, Pluripotency, & Karyotype of
iPSCs Over 10 Passages Post-Thaw
Figure 5. Post-Thaw Recovery Supplement Demonstrates Applicability for
Recovery of Cryopreserved Primary Cells
Calcein/EthD-1
Calc
ein
Ob
ject
Co
un
t/
Eth
D-1
Ob
ject
Co
un
t
Cell Body/Neurite
A. Experimental Workflow
B. HEKn 24 Hour Post-Thaw Viability
C. HCEC 24 Hour Post-Thaw Viability
Pre
sto
Blu
e
Flu
ore
sc
en
ce
(24 H
ou
rs P
ost-
Th
aw
)
Pre
sto
Blu
e
Flu
ore
sc
en
ce
(24 H
ou
rs P
ost-
Th
aw
)
Quickly Thawed
Cryopreserved
Cells at 37 °C
Performed Viable
Cell Counts
Plated at
Recommended
Seeding Densities in
Growth Medium +/-
post-thaw recovery
supplement
Incubated Cells at
37 °C, 5% CO2
for 24 Hours
Assessed Viability 24
Hours Post-Thaw
Using PrestoBlue
Cell Viability Reagent
OR LIVE/DEAD
Viability/Cytotoxicity
Kit
Fed Neurons According to
Recommended Protocol
w/ Growth Medium
Lacking post-thaw
recovery supplement
6 Days Post-
Thaw Assessed
Neurite LengthOR
D. Rat Cortical Neuron 24 Hour Post-Thaw Viability
E. Rat Cortical Neuron Neurite Length
(6 Days Post-Thaw)
Ca
lce
in/E
thD
-1
Ob
ject
Co
un
tN
eu
rite
Len
gth
(mm
/[C
ell B
od
y C
luste
r]
Figure 6. Post-Thaw Recovery Supplement Demonstrates Applicability for
Survival of PSCs Following Single Cell Passaging
• Our chemically defined post-thaw recovery
supplement shows utility in recovery from
cryopreservation of a variety of primary cell
types, including human epidermal
keratinocytes, neonatal (HEKn), human
corneal epithelial cells (HCECs), and rat
cortical neurons
A. StemPro Accutase Passaged iPSC Recovery
Time Post-Split (Hours)
B. TrypLETM Select Passaged iPSC Recovery
Inc
uC
yte
Mo
nit
ore
d
%C
on
flu
en
ce
)
• Use of the Post-Thaw Recovery Supplement for 18-24 hours post-single
cell passaging via StemPro Accutase or TrypLE TMSelect passaging
provides vast improvement of cell survival compared to recovery in
Essential 8TM Alone
• Multi passage study is currently underway using single cell passaging
Essential 8TM Alone
+ Post-Thaw Recovery
Supplement
Essential 8TM Alone
Figure 1. Life Technologies Provides a Broad Array
of Solutions for Pluripotent Stem Cell Research
Culture Reprogram Characterize Expand Cryopreserve Differentiate Measure
CytoTuneTM
Lentiviral iPSC
Reprogramming
Particles
KnockOutTM
ESC/iPSC Media
Kit
KnockOutTM
CTS XenoFree
ESC/iPSC Media
Kit
Gibco® Primary
Cell MediaAlkaline
Phosphatase
Live Stain
ELF® 97
Endogenous
Phosphatase
Detection Kit
Conjugated
Antibodies (e.g.,
anti-TRA-1-60 &
anti-SSEA4)
TaqMan® Stem
Cell Pluripotency
Array
KnockOutTM
ESC/iPSC Media
Kit
KnockOutTM
CTS XenoFree
ESC/iPSC Media
Kit
StemPro® hESC
SFM
StemPro® Differentiation
Kits
Human
Recombinant
Growth Factors
(e.g., BMP-4,
bFGF, DKK-1,
Noggin)
TaqMan® Ready
Made Probes
Primary &
Secondary
Antibodies for
Immunocytoche
mistry
Goal
Cryopreservation
of PSCs
Pre
sto
Blu
eA
ctu
al
PrestoBlue Predicted
Inc
uC
yte
Mo
nit
ore
d
%C
on
flu
en
ce
)
E8 + Post-Thaw Recovery
Supplement 24 Hour Recovery
3 Hours 24 Hours
48 Hours 72 Hours
96 Hours 120 Hours
E8 + Post-Thaw Recovery
Supplement 24 Hour Recovery
3 Hours 24 Hours
48 Hours 72 Hours
96 Hours 120 Hours
IncuCyte ZOOM Phase Contrast
Images w/ Confluence Mask
Phase Oct4
NanogTra1-60
Karyotype
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