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).