Optimization of Slow Cooling Cryopreservation for Human Pluripotent Stem Cells Miyazaki T., Nakatsuji N., Suemori H. Dezember 2013

Masterseminar Viola Schweitzer, 17.12.14 Dr. Stephanie Bur, Fraunhofer IMBT

Overview • Introduction • Stem cells • Cryopreservation

• Analysis • • • • •

Viability Recovery Adhesion Karyotype Differentiation

• Summary • Literature

biokryo.de 2

totipotent

Introduction: Stem cells embryonic (ESC) and induced pluripotent stem cells (iPSC)

• in culture, stem cells need complex surfaces to grow on

stemcellresearchfoundation.org

• pluripotent stem cells pluripotent

differentiation

(feeder layers, matrix proteins)

• the cells adhere and form colonies (100 – 1000 µm ø) cell-cell contacts are vital, dead cells detach from the colonies (Chen et al., 2010) (Amit et al., 2000)

• stem cells need to remain undifferentiated in culture but still be able to differentiate upon stimulation 3

Introduction: Cryopreservation • two basically different strategies

osm. damage survival

intracell. ice

optimum

slow

cooling rate

slow cooling (- 1 °C/ min) vs. vitrification (- 1K – 10K °C/ min) (Heng et al., 2005) (Reubinoff et al., 2001)

fast

Mazur‘s two-factor hypothesis

• cryoprotectants are used to incease survival rates (Ha et al., 2005) DMSO, glycerol, ethylene glycol, various sugars (some are potentially toxic)

• for each cell type, techniques have to be developed empirically depending on size, water content, robustness ect. e. g. cryopreserved spermatozoa (5 µm) retain much higher viability than cryopreserved ova (120 µm)

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• stem cell culture is elaborate, expensive and research is strictly regulated development of cryopreservation methods is highly experimental • vitrification is a tested approach for adherent cells (Suemori et al., 2006) (Li et al., 2010) throughput is too low for therapeutic applications of stem cells (T'Joen et al., 2012) • size is a crucial factor for cryopreservation success stem cells grow best in colonies of several hundred µm the cryopreservation of stem cells by slow cooling as single cells seems to provide a simple and efficient approach 5

Analysis: Viability • assessment of membrane integrity before and after cryopreservation by trypan blue exclusion (left) and flow cytometry (right) before cryo

after cryo

hESC

hiPSC

hESC colonies

hESC single cells 6

Analysis: Recovery • assessment of the influence of the seeding density (left) and the presence of the ROCK inhibitor Y-27632 at seeding (right) (Watanabe et al., 2007)

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Analysis: Adhesion • assessment of „stem cell-like“ behaviour after thawing and seeding rinsing of dead cells from the colonies, detachment and dissociation of the colonies, counting of living cells in the hemocytometer (counting chamber) subculture of freeze-thawed cells

hESC single cells

hESC single cells (xeno-free)

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Analysis: Karyotype • assessment of chromosome intactness colcemid treatment, fixation of the cells, giemsa staining of chromatin hESC single cells

hESC single cells (xeno-free)

• chromosome damages would affect the stem cells‘ ability to differentiate (Suemori et al., 2006) 9

Analysis: Differentiation • Flow cytometric analysis of undifferentiated markers (upper) and reverse transcription(EB: embryoid bodies) PCR of differentiation markers (lower) (Kumagai et al., 2013) (Miyazaki et al., 2012) hESC single cells

hESC single cells (xeno-free)

undifferentiated markers

differentiated markers 10

Summary • The cryopreservation of human ESC by slow cooling leads to up to 50 – 60 % recovery if the clumps are dissociated to single cells and seeded in the presence of the ROCK inhibitor Y-27632 or at optimal seeding density. • The cells do not differentiate in subculture (three passages), but retain their ability to differentiate upon stimulation. • What still needs to be investigated is: Does the cryopreservation of other ESC and hiPSC lines lead to similar results? Can we use these results to improve the slow cooling of stem cells as clumps? 11

Literature • Chen, G., Hou, Z., Gulbranson, D.R., and Thomson, J.A. (2010). Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell stem cell 7, 240-248. • Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz-Eldor, J., and Thomson, J.A. (2000). Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Developmental biology 227, 271-278. • Heng, B.C., Kuleshova, L.L., Bested, S.M., Liu, H., and Cao, T. (2005). The cryopreservation of human embryonic stem cells. Biotechnology and applied biochemistry 41, 97-104.

• Reubinoff, B.E., Pera, M.F., Vajta, G., and Trounson, A.O. (2001). Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Human reproduction 16, 2187-2194. • Ha, S.Y., Jee, B.C., Suh, C.S., Kim, H.S., Oh, S.K., Kim, S.H., and Moon, S.Y. (2005). Cryopreservation of human embryonic stem cells without the use of a programmable freezer. Human reproduction 20, 1779-1785. • Suemori, H., Yasuchika, K., Hasegawa, K., Fujioka, T., Tsuneyoshi, N., and Nakatsuji, N. (2006). Efficient establishment of human embryonic stem cell lines and longterm maintenance with stable karyotype by enzymatic bulk passage. Biochemical and biophysical research communications 345, 926-932. • Li, T., Mai, Q., Gao, J., and Zhou, C. (2010). Cryopreservation of human embryonic stem cells with a new bulk vitrification method. Biology of reproduction 82, 848-853. • T'Joen, V., De Grande, L., Declercq, H., and Cornelissen, M. (2012). An efficient, economical slow-freezing method for large-scale human embryonic stem cell banking. Stem cells and development 21, 721-728. • Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature biotechnology 25, 681-686. • Kumagai, H., Suemori, H., Uesugi, M., Nakatsuji, N., and Kawase, E. (2013). Identification of small molecules that promote human embryonic stem cell self-renewal. Biochemical and biophysical research communications 434, 710-716. • Miyazaki, T., Futaki, S., Suemori, H., Taniguchi, Y., Yamada, M., Kawasaki, M., Hayashi, M., Kumagai, H., Nakatsuji, N., Sekiguchi, K., et al. (2012). Laminin E8 fragments 12 support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nature communications 3, 1236.