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2 nd Tissue Engineering and Regenerative Medicine International Society World Congress 2009 31 st August – 3 rd September Seoul South Korea 2 Tissue Engineering and Regenerative Medicine International Society World Congress 2009. 31 August – 3 September, Seoul, South Korea C ti f Adh tC ll St t i t C ti f Adh tC ll St t i t Cryopreservation of Adherent Cells: Strategies to Cryopreservation of Adherent Cells: Strategies to Cryopreservation of Adherent Cells: Strategies to Cryopreservation of Adherent Cells: Strategies to Improve Post Improve Post Thawing Viability and Function Thawing Viability and Function Improve Post Improve Post-Thawing Viability and Function Thawing Viability and Function Improve Post Improve Post Thawing Viability and Function Thawing Viability and Function R Malpique 1 F Ehrhart 2 A Katsen Globa 2 H Zimmermann 2 and P M Alves 1 R. Malpique 1 , F. Ehrhart 2 , A. Katsen-Globa 2 , H. Zimmermann 2 and P. M. Alves 1 1 Instituto Tecnologia Química Biológica (ITQB) / Instituto Biologia Experimental (IBET), Oeiras, Portugal; 2 Frauhofer-IBMT Ensheimer (FhG-IBMT) St Ingbert Germany Instituto Tecnologia Química Biológica (ITQB) / Instituto Biologia Experimental (IBET), Oeiras, Portugal; Frauhofer IBMT, Ensheimer (FhG IBMT), St. Ingbert, Germany htt //t it b l t it b l t http://tca.itqb.unl.pt ; www.itqb.unl.pt Introduction Introduction Aim and Strategy Aim and Strategy Introduction Introduction Aim and Strategy Aim and Strategy Clinical and commercial availability of cell-based products for tissue engineering and regenerative medicine Clinical and commercial availability of cell based products for tissue engineering and regenerative medicine require effective methods for their long term storage in cryobanks which are not yet established for complex Develop optimized methodologies for the require 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 viability Develop optimized methodologies for the ti ff ti l ll systems such as cell monolayers, tissues or biosynthetic constructs [1] . Improve cell viability I ll ifi f ti cryopreservation 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 cell types which were shown to poorly survive the cryopreservation process [2,3] Avoid monolayer’s detachment monolayers for cell-based therapies and in- it h l i l t di types 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 td b d ll - Avoid monolayer s detachment vitro pharmacological studies In this work, combined strategies for the cryopreservation of adherent cells were investigated based on cell - Avoid lost of cell-cell contact entrapment in clinical-grade, highly purified alginate of extremely high viscosity (0.1% w/v viscosity in distilled Avoid lost of cell cell contact water > 30 mPa s) uniformly cross-linked with Ba 2+ [4] STRATEGY water > 30 mPa.s) uniformly cross linked with Ba . As model systems Neuroblastoma N2a and Caco 2 C llM d l C llM d l STRATEGY As model systems, Neuroblastoma N2a and Caco-2 Cl Ad i ll li d d t Cell Models Cell Models : STRATEGY C lt di Colon Adenocarcinoma cell lines were used due to Caco Caco 2 N2a N2a Culture medium their specific characteristics, which makes them Caco Caco-2 N2a N2a Monolayer’s entrapment beneath their specific characteristics, which makes them interesting lines for studying the cryopreservation of Monolayer’s entrapment beneath Alginate gel layer interesting lines for studying the cryopreservation of diff ti t d ll [5] A th ti a layer of ultra-high viscous Cells cultured on the differentiated cells [5] . As the cryopreservation a layer of ultra high viscous (UHV) li t Cells cultured on the plate’s wells medium, serum-free CryoStor TM (Biolife Solutions ® ) (UHV) alginate plate s wells solution was compared with culture medium solution was compared with culture medium supplemented with bovine serum both containing Differentiated (5 d) Undifferentiated supplemented with bovine serum, both containing 10% M SO Differentiated (21 d) Undifferentiated 10% Me 2 SO. 2 Methods Methods Methods Methods C lt C lt C 2 d N2 ll lt d 4 ll lt i ith diff ti t d f ll diff ti t d tt C 2 ll t diff ti ti it E l td t E l td t Culture Culture : 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 ti Diff ti ti medium. After 1 or 4 days post-inoculation, a thin layer of UHV alginate cross-linked by Ba 2+ ions was added over the cells on the plates. Undifferentiated Differentiated Versus Differentiation Differentiation tt tt medium. 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. Cryopreservation Cryopreservation: 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 state state Cryopreservation Cryopreservation : 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 td lt di C St TM CS10 (Bi Lif S l ti B th ll WA USA) b th tii 10% M SO d t d t 80ºC d i t l t 1 k Time of alginate Time of alginate supplemented culture medium or CryoStor TM -CS10 (BioLife Solutions, Bothell, WA, USA), both containing 10% Me 2 SO, and stored at -80ºC during at least 1 week. 4 days post-inoculation 1 day post-inoculation Versus Time of alginate Time of alginate addition over the cells addition over the cells Post Post-thawing thawing characterization characterization : Cell viability was assessed through a membrane integrity assay and the metabolic assay alamarBlue TM . The structural integrity and addition over the cells addition over the cells differentiation state of the cells was evaluated through scanning electron microscopy Maintenance of cell differentiated state after thawing was assessed through C ti C ti Serum free preservation differentiation state of the cells was evaluated through scanning electron microscopy . Maintenance of cell differentiated state after thawing was assessed through biochemical and immunohistochemical assays respectively Cryopreservation Cryopreservation di di Culture medium containing 10% M SO Serum-free preservation solution CryoStor TM CS10 Versus biochemical and immunohistochemical assays, respectively . medium medium serum + 10% Me 2 SO solution CryoStor TM -CS10 Results Results Results Results Effect of alginate entrapment on cell growth and differentiation Effect of alginate entrapment on cell growth and differentiation Cell seeding on the surface of 4- Day 0 Effect of alginate entrapment on cell growth and differentiation Effect of alginate entrapment on cell growth and differentiation well plates Day 0 well plates Caco Caco 2 N2a N2a Caco Caco-2 Day 1 Caco Caco-2 N2a N2a Caco Caco-2 Alginate addition over the cells Day 1 4 Differentiated Non-different. Non-different Differentiated Alginate Alginate entrapment entrapment 4 days days Alginate addition over the cells or 4 (5 days) Non different. (5 days) Non different. (5 days) (5 days) post post inoculation inoculation: Metabolic Activity (alamarBlue assay) (5 days) (5 days) post post-inoculation inoculation: 25000 .) ut e 25000 .U ou nate 9 Caco-2 cells proliferation rate Assessment of cell viability and 20000 (A ith gin 9 Caco-2 cells proliferation rate Day 5 Assessment of cell viability and diff ti ti tt 15000 e ( Wi alg and metabolic activity are not Day 5 differentiation state 15000 nce and metabolic activity are not + 10000 en affected; C llf i 4 ll lt + 1 10000 sc affected; Cell freezing on 4-well plates ate y 5000 res 9 C ff and storage at -80ºC na day 5000 uor 9 Cell morphology is not affected; lgi d 0 Flu A 5 10 15 20 25 F 9 Cell differentiation state/ Th i Day 12 4 5 10 15 20 25 Ti (d ) 9 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 d Alginate 1 day Without alginate lgi d Assessment of cell viability and differentiation state/capacity Alginate 4 days Alginate 1 day Without alginate A differentiation state/capacity Post Post thaw viability and differentiation state of thaw viability and differentiation state of P t P t th f th f diff ti t d diff ti t d Post Post-thaw viability and differentiation state of thaw viability and differentiation state of Post Post-thaw recovery of non thaw recovery of non-differentiated differentiated differentiated monolayers differentiated monolayers Post Post thaw recovery of non thaw recovery of non differentiated differentiated l differentiated monolayers differentiated monolayers monolayers monolayers C 2 Caco Caco-2 Metabolic Activity: Membrane integrity: Metabolic Activity : Membrane integrity : Mtbli A ti it S i l t i Metabolic Activity: Scanning electron microscopy: Before 3 da s post 1 day post Before f i 160 Undamaged cell surface with Shrinkage of the whole 3 days post- h i 1 day post- th i Caco Caco-2 Control Day 1 after Day 3 after freezing 140 160 thick microvilli carpet Shrinkage of the whole “tissue-like” structure thawing thawing Caco Caco-2 thawing thawing 140 %) thick microvilli carpet tissue-like structure Immed. after Day 2 after m 120 (% thawing thawing ut te um 100 ery tou nat 200 diu 80 ove Culture Medium Wiht lgin %) ed 60 co 0 Culture Medium W al 150 (% Me 40 Re 100 ry e M 20 R 4 100 ve ure 0 Damaged cells at the Multiple cell layers ate s 50 cov ltu Without alginate Alginate 4d Damaged cells at the l ’ f Multiple cell layers ina day 50 Rec Cu Without alginate Alginate 4d monolayer’s surface Algi d 0 R C Additional factors related to the three A Control Day 1 after Day 10 after 0 dimensional arrangement Control Day 1 after thawing Day 10 after thawing Without alginate Alginate 4d Immed after Day 3 after 0 out ate Immed. after thawing Day 3 after thawing Sl h dt 200 S1 CryoStor TM -CS10 hto ina Slower recovery when compared to non- diff ti t d ll 200 ) CS Wih alg differentiated cells 150 %) M -C W a 150 y ( r TM N2a N2a 100 ery tor N2a N2a 4 100 ove St 160 Alginate entrapment: Immediately 1 day post- 1 day post- ate ys 50 eco yoS 140 %) Alginate entrapment : after thawing thawing thawing gina day 50 Re Cry 100 120 (% 9 Maintenance of Alg d 0 C 80 100 ry 9 Maintenance of ut e β- -III A 0 Without alginate Alginate 4d 60 80 ve neuronal networks tou nat ti-β ulin Without alginate Alginate 4d 40 60 cov neuronal networks N2a N2a Wiht gin Ant ubu 20 40 Rec integrity; N2a N2a W al A Tu 0 R 9 Culture Medium Without alginate Alginate 4d 9 Expression of Culture Medium 120 2 m ut te 4 Without alginate Alginate 4d t i l l 120 AP2 m tou nat 4 typical neuronal 100 %) MA iu Wiht gin Control Day 1 after Day 3 after ate ays markers 80 (% nti-M ed W al Day 1 after thawing thawing ina da markers. 60 ry An Me Immed. after Day 2 after Alg 60 ve e M 4 thawing thawing A 40 cov ure e 4 s 20 ec ltu nat ays 0 R Cul gin da CONCLUSIONS CONCLUSIONS 0 Witho t alginate Alginate 4d C Al CONCLUSIONS CONCLUSIONS Without alginate Alginate 4d CONCLUSIONS CONCLUSIONS CryoStor TM CS10 • Monolayer entrapment beneath an alginate layer improves cell recovery by avoiding detachment from the CryoStor TM -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 it ti 120 10 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 th 80 100 (% -C W a • The use of CryoStor TM solution improves the cryopreservation process for both cells lines, allowing the 60 80 ry TM - maintenance of high post-thaw recovery of viability and differentiation state. 60 ve or T 4 maintenance of high post thaw recovery of viability and differentiation state. 40 cov Sto e 4 s 20 Rec oS nate ays An efficient novel strategy for successful cryopreservation of ready An efficient novel strategy for successful cryopreservation of ready-to to-use cell use cell 0 20 R ryo gin da An efficient novel strategy for successful cryopreservation of ready An efficient novel strategy for successful cryopreservation of ready-to to-use cell use cell 0 With t li t Al i t 4d Cr Al monolayers was validated based on cell entrapment in clinical grade, UHV monolayers was validated based on cell entrapment in clinical grade, UHV Without alginate Alginate 4d C monolayers was validated based on cell entrapment in clinical grade, UHV monolayers was validated based on cell entrapment in clinical grade, UHV li t d th fC St li t d th fC St TM TM l ti l ti alginate and the use of CryoStor alginate and the use of CryoStor TM TM solution solution 9 Alginate entrapment improves recovery of culture medium cryopreserved 9 Alginate entrapment improves recovery of culture medium cryopreserved 9 S t th i l t ti f ti ti ti f i d ll cells by minimizing membrane damage and cell detachment after thawing. 9 Supports the implementation of routine cryopreservation practices for engineered cells and 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! Acknowledgments Acknowledgments References References Acknowledgments Acknowledgments References References 9 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). 9 CryoStor TM -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). initiation 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. (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). (Grant SFRH/BD/22647/2005). [5] Malpique, R. et al., Biotechnol. Bioeng. 98(1): 155-66 (2007). (Grant SFRH/BD/22647/2005).
