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2nd Tissue Engineering and Regenerative Medicine International Society World Congress 2009 31st August – 3rd September Seoul South Korea2 Tissue Engineering and Regenerative Medicine International Society World Congress 2009. 31 August – 3 September, Seoul, South Korea
C ti f Adh t C ll St t i tC ti f Adh t C ll St t i tCryopreservation of Adherent Cells: Strategies toCryopreservation of Adherent Cells: Strategies toCryopreservation of Adherent Cells: Strategies to Cryopreservation of Adherent Cells: Strategies to y p gy p gImprove PostImprove Post Thawing Viability and FunctionThawing Viability and FunctionImprove PostImprove Post--Thawing Viability and FunctionThawing Viability and FunctionImprove PostImprove Post Thawing Viability and FunctionThawing Viability and Functionpp g yg y
R Malpique1 F Ehrhart2 A Katsen Globa 2 H Zimmermann2 and P M Alves1R. Malpique1, F. Ehrhart2, A. Katsen-Globa 2, H. Zimmermann2 and P. M. Alves1
1Instituto Tecnologia Química Biológica (ITQB) / Instituto Biologia Experimental (IBET), Oeiras, Portugal; 2Frauhofer-IBMT Ensheimer (FhG-IBMT) St Ingbert GermanyInstituto Tecnologia Química Biológica (ITQB) / Instituto Biologia Experimental (IBET), Oeiras, Portugal; Frauhofer IBMT, Ensheimer (FhG IBMT), St. Ingbert, Germanyhtt //t it b l t it b l thttp://tca.itqb.unl.pt; www.itqb.unl.pt
IntroductionIntroduction Aim and StrategyAim and StrategyIntroductionIntroduction Aim and StrategyAim and StrategygygyClinical and commercial availability of cell-based products for tissue engineering and regenerative medicineClinical and commercial availability of cell based products for tissue engineering and regenerative medicinerequire effective methods for their long term storage in cryobanks which are not yet established for complex Develop optimized methodologies for therequire effective methods for their long-term storage in cryobanks, which are not yet established for complex
t h ll l ti bi th ti t t [1] - Improve cell viabilityDevelop optimized methodologies for the ti f f ti l ll
systems such as cell monolayers, tissues or biosynthetic constructs [1]. Improve cell viabilityI ll ifi f ticryopreservation of functional cell Cell entrapment in a gel is a promising cryopreservation strategy to improve post-thaw viability and function of cell - Improve cell-specific function
monolayers for cell-based therapies and in-Cell entrapment in a gel is a promising cryopreservation strategy to improve post thaw viability and function of celltypes which were shown to poorly survive the cryopreservation process [2,3] Avoid monolayer’s detachmentmonolayers for cell-based therapies and in-
it h l i l t ditypes which were shown to poorly survive the cryopreservation process [ , ].I thi k bi d t t i f th ti f dh t ll i ti t d b d ll
- Avoid monolayer s detachmentvitro pharmacological studiesIn this work, combined strategies for the cryopreservation of adherent cells were investigated based on cell - Avoid lost of cell-cell contactp g
entrapment in clinical-grade, highly purified alginate of extremely high viscosity (0.1% w/v viscosity in distilledAvoid lost of cell cell contact
p g , g y p g y g y ( ywater > 30 mPa s) uniformly cross-linked with Ba2+ [4]
STRATEGYwater > 30 mPa.s) uniformly cross linked with Ba .As model systems Neuroblastoma N2a and Caco 2 C ll M d lC ll M d l STRATEGYAs model systems, Neuroblastoma N2a and Caco-2C l Ad i ll li d d t
Cell ModelsCell Models:: STRATEGYC lt di
Colon Adenocarcinoma cell lines were used due toCacoCaco 22 N2aN2a Culture mediumtheir specific characteristics, which makes them CacoCaco--22 N2aN2a
Monolayer’s entrapment beneaththeir specific characteristics, which makes theminteresting lines for studying the cryopreservation of Monolayer’s entrapment beneath Alginate gel layerinteresting lines for studying the cryopreservation ofdiff ti t d ll [5] A th ti a layer of ultra-high viscous Cells cultured on the
g ate ge ayedifferentiated cells [5]. As the cryopreservation a layer of ultra high viscous (UHV) l i t
Cells cultured on the plate’s wellsmedium, serum-free CryoStorTM (Biolife Solutions®) (UHV) alginate plate s wells, y ( )
solution was compared with culture medium ( ) gsolution was compared with culture mediumsupplemented with bovine serum both containing Differentiated (5 d)Undifferentiatedsupplemented with bovine serum, both containing10% M SO
Differentiated (21 d)Undifferentiated
10% Me2SO.2
MethodsMethodsMethodsMethodsC ltC lt C 2 d N2 ll lt d 4 ll l t i ith diff ti t d f ll diff ti t d t t C 2 ll t diff ti ti i t E l t d tE l t d tCultureCulture:: Caco-2 and N2a cells were cultured on 4-well plates in either a non-differentiated or fully differentiated state. Caco-2 cells spontaneous differentiation into Evaluated parameters:Evaluated parameters:enterocyte-like cells was achieved through long-time culture. Neuronal differentiation of N2a cells was induced through retinoic acid addition to low-serum content
Diff ti tiDiff ti ti
ppy g g g
medium. After 1 or 4 days post-inoculation, a thin layer of UHV alginate cross-linked by Ba2+ ions was added over the cells on the plates. Undifferentiated DifferentiatedVersusDifferentiation Differentiation t tt tmedium. After 1 or 4 days post inoculation, a thin layer of UHV alginate cross linked by Ba ions was added over the cells on the plates.
CryopreservationCryopreservation:: After 5 days of culture (or 21 days for differentiated Caco 2 cells) cells were frozen at 1°C/min to 80°C inside the plates with either serumstatestate
CryopreservationCryopreservation:: After 5 days of culture (or 21 days for differentiated Caco-2 cells), cells were frozen at 1 C/min to -80 C inside the plates with either serum-l t d lt di C St TM CS10 (Bi Lif S l ti B th ll WA USA) b th t i i 10% M SO d t d t 80ºC d i t l t 1 k Time of alginateTime of alginatesupplemented culture medium or CryoStorTM-CS10 (BioLife Solutions, Bothell, WA, USA), both containing 10% Me2SO, and stored at -80ºC during at least 1 week. 4 days post-inoculation1 day post-inoculation VersusTime of alginate Time of alginate
addition over the cellsaddition over the cellsPostPost--thawingthawing characterizationcharacterization:: Cell viability was assessed through a membrane integrity assay and the metabolic assay alamarBlueTM. The structural integrity and addition over the cellsaddition over the cellsgg y g g y y y g ydifferentiation state of the cells was evaluated through scanning electron microscopy Maintenance of cell differentiated state after thawing was assessed through C tiC ti Serum free preservationdifferentiation state of the cells was evaluated through scanning electron microscopy. Maintenance of cell differentiated state after thawing was assessed throughbiochemical and immunohistochemical assays respectively
Cryopreservation Cryopreservation didi
Culture medium containing 10% M SO
Serum-free preservation solution CryoStorTM CS10Versusbiochemical and immunohistochemical assays, respectively. mediummedium serum + 10% Me2SO solution CryoStorTM-CS10
ResultsResultsResultsResults
Effect of alginate entrapment on cell growth and differentiationEffect of alginate entrapment on cell growth and differentiationCell seeding on the surface of 4-Day 0 Effect of alginate entrapment on cell growth and differentiationEffect of alginate entrapment on cell growth and differentiationgwell platesDay 0 well plates
CacoCaco 22 N2aN2aCacoCaco--22Day 1
CacoCaco--22 N2aN2aCacoCaco--22Alginate addition over the cells
Day 1 4 Differentiated Non-different.Non-different Differentiated AlginateAlginate entrapmententrapment 44 daysdaysAlginate addition over the cellsor 4 e e t ated
(5 days)Non different.
(5 days)Non different.
(5 days)e e t ated
(5 days)gg pp yy
postpost inoculationinoculation::Metabolic Activity (alamarBlue assay) ( y )(5 days)(5 days) ( y ) postpost--inoculationinoculation::y ( y)
25000.) ut e 25000
.U ou nate
Caco-2 cells proliferation rateAssessment of cell viability and 20000(A ith gin Caco-2 cells proliferation rate
Day 5 Assessment of cell viability and diff ti ti t t
15000e (
Wi
alg
and metabolic activity are notDay 5 differentiation state
15000nce and metabolic activity are not
+ 10000en affected;C ll f i 4 ll l t+ 1 10000
sc affected;Cell freezing on 4-well plates
ate y
5000res
C ffand storage at -80ºC na da
y
5000
uor Cell morphology is not affected;
g
lgi d
0Flu p gy ;
A
5 10 15 20 25
F
Cell differentiation state/Th iDay 12 4 5 10 15 20 25Ti (d )
Cell differentiation state/Thawing+
Day 12
ate s Time (day) capacity is not affected;+ na ay capacity is not affected;
Assessment of cell viability and Al i t 4 dAlginate 1 dayWithout alginate lgi dAssessment of cell viability and
differentiation state/capacityAlginate 4 daysAlginate 1 dayWithout alginate A
differentiation state/capacity
PostPost thaw viability and differentiation state ofthaw viability and differentiation state ofP tP t th fth f diff ti t ddiff ti t d PostPost--thaw viability and differentiation state of thaw viability and differentiation state of PostPost--thaw recovery of nonthaw recovery of non--differentiateddifferentiated yydifferentiated monolayersdifferentiated monolayers
PostPost thaw recovery of nonthaw recovery of non differentiated differentiated ll differentiated monolayersdifferentiated monolayersmonolayersmonolayers
CC 22yyo o aye so o aye s
CacoCaco--22Metabolic Activity:Membrane integrity: Metabolic Activity:Membrane integrity:M t b li A ti it S i l t iMetabolic Activity: Scanning electron microscopy:
Before 3 da s post1 day posty g py
Before f i 160
Undamaged cell surface with Shrinkage of the whole3 days post-h i
1 day post-th iCacoCaco--22 Control Day 1 after Day 3 after
freezing140160
gthick microvilli carpet
Shrinkage of the whole “tissue-like” structurethawingthawingCacoCaco--22 thawing thawing
140
%) thick microvilli carpet tissue-like structure
Immed. after Day 2 after
m
120 (%thawingy
thawing
ut
te
um
100
ery
tou
nat
200diu 80
ove
Culture MediumWih
tlg
in
%)ed 60co 0Culture MediumW al
150(%Me 40R
e
100ry
e M
20
R
4 100veure
0Damaged cells at the Multiple cell layersat
e s
50cov
ltu Without alginate Alginate 4dDamaged cells at the
l ’ fMultiple cell layers
ina
day
50
Rec
Cu Without alginate Alginate 4d monolayer’s surface
Alg
i d
0
RC Additional factors related to the three A
Control Day 1 after Day 10 after0 dimensional arrangementControl Day 1 after
thawingDay 10 afterthawing
Without alginate Alginate 4dg
Immed after
g g
Day 3 after
0 out
ate Immed. after
thawingDay 3 afterthawing Sl h d t200S1 CryoStorTM-CS10ht
oin
a g g Slower recovery when compared to non-diff ti t d ll200
)CS y
Wih
alg differentiated cells
150%)
M-C W a
150
y (
rTM N2aN2a
100ery
tor N2aN2a
4
100
ove
St 160 Alginate entrapment:Immediately 1 day post- 1 day post-ate
ys
50eco
yoS
140
%)
Alginate entrapment:after thawing thawing thawing
gina day
50
Re
Cry
100120(% Maintenance ofA
lg d
0C
80100ry
Maintenance of
ut e β- -IIIA
0Without alginate Alginate 4d 60
80
ve neuronal networkstou
nat
ti-β
ulin
Without alginate Alginate 4d4060
cov neuronal networks
N2aN2a Wih
tgi
n
Ant
ubu
2040
Rec integrity;N2aN2a W al A
Tu
00R g y;
Culture MediumWithout alginate Alginate 4d Expression ofCulture Medium
120 2m ut
te 4 Without alginate Alginate 4d
t i l l120
AP2m to
una
t 4
typical neuronal100
%)
MAiu
Wih
tgi
n
Control Day 1 after Day 3 after ate
ays
markers80(%
nti-Med W al
Day 1 afterthawing
ythawing in
ada markers.
60ry
An
Me Immed. after Day 2 after A
lg
60
vee M
4
thawing thawing A
40
cov
ure
e 4
s
20ecltu nat
ays
0R
Cul gi
n da CONCLUSIONSCONCLUSIONS0Witho t alginate Alginate 4d
C Al CONCLUSIONSCONCLUSIONSWithout alginate Alginate 4d CONCLUSIONSCONCLUSIONS
CryoStorTM CS10 • Monolayer entrapment beneath an alginate layer improves cell recovery by avoiding detachment from theCryoStorTM-CS10
0 ut
ate • Monolayer entrapment beneath an alginate layer improves cell recovery by avoiding detachment from the
f d b k f ll ll i t ti12010 hto
ina surface and breakage of cell-cell interactions.
100%)
CS Wih
algi
Th f C St TM l ti i th ti f b th ll li ll i th80
100
(%-C W a • The use of CryoStorTM solution improves the cryopreservation process for both cells lines, allowing the
6080ry
TM-
maintenance of high post-thaw recovery of viability and differentiation state.60veorT
4
maintenance of high post thaw recovery of viability and differentiation state.40co
v
Sto
e 4
s
20RecoS nate
ays An efficient novel strategy for successful cryopreservation of readyAn efficient novel strategy for successful cryopreservation of ready--toto--use celluse cell
020R
ryo
gin da An efficient novel strategy for successful cryopreservation of readyAn efficient novel strategy for successful cryopreservation of ready--toto--use cell use cell
0With t l i t Al i t 4dC
r Al monolayers was validated based on cell entrapment in clinical grade, UHVmonolayers was validated based on cell entrapment in clinical grade, UHVWithout alginate Alginate 4dC monolayers was validated based on cell entrapment in clinical grade, UHV monolayers was validated based on cell entrapment in clinical grade, UHV
l i t d th f C Stl i t d th f C St TMTM l til tialginate and the use of CryoStoralginate and the use of CryoStorTMTM solutionsolutionAlginate entrapment improves recovery of culture medium cryopreserved
g yg yAlginate entrapment improves recovery of culture medium cryopreserved
S t th i l t ti f ti ti ti f i d llcells by minimizing membrane damage and cell detachment after thawing. Supports the implementation of routine cryopreservation practices for engineered cellsy g g g y gand tissues and their immediate availability for cell-based therapies
N th l U t 50% d th ithi 24 h ft th i !and tissues and their immediate availability for cell-based therapies.
Nevertheless…Up to 50% death within 24 hours after thawing!AcknowledgmentsAcknowledgmentsReferencesReferences
p gAcknowledgmentsAcknowledgmentsReferencesReferences
The authors acknowledge the financial support received from the European[1] Baust, J. G. and Baust, J. M., Advances in Biopreservation CRC Press:63-87(2006).CryoStorTM-CS10 solution allows full recovery of metabolic activity and commission (“Cell Programming by Nanoscaled Devices" NMP4-CT-2004-500039[2] Mahler, S. M. et al., Cell Transplant. 12 (6): 579-92 (2003).
y y yinitiation of proliferation within 24 hours post-thawing
and the Fundação para a Ciência e Tecnologia (FCT), Portugal[3] Inaba, K. et al., Transplantation 61 (2): 175-9 (1996).initiation of proliferation within 24 hours post-thawing.ç p g ( ) g
(PTDC/BIO/69407/2006). R Malpique acknowledges FCT for finantial support[4] Zimmermann, H. et al., J. Mat. Sci.: Mat. Med. 16(6): 491 – 501 (2005). ( ) p q g pp(Grant SFRH/BD/22647/2005).[5] Malpique, R. et al., Biotechnol. Bioeng. 98(1): 155-66 (2007). (Grant SFRH/BD/22647/2005).