Trehalose preincubation increases mesenchymal (CD271+) stem cells post-cryopreservation viability

  • Indra Kusuma Faculty of Medicine, Universitas YARSI, Jakarta
  • Restu S. Hadi Faculty of Medicine, Universitas YARSI, Jakarta
  • Bambang Kiranadi Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor
  • Arief Boediono Department of Anatomy, Physiology, and Pharmacology, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor
Keywords: cryopreservation, mesenchymal (CD271 ) stem cells, trehalose preincubation


Background: Dimethyl sulfoxide (Me2SO) is a common cryoprotective agent widely used in cell preservation system. Me2SO is currently known to cause epigenetic changes which are  critical in stem cells development and cellular differentiation. Therefore, it is imperative to develop cryopreservation techniques that protect cellular functions and avert Me2SO adverse effect. Trehalose was able to protect organism in extreme condition such as dehydration and cold. This study aimed to verify the protective effect of trehalose preincubation procedure in cryopreservation.

Methods: The study was conducted using experimental design. Thawed mesenchymal (CD271+) stem cells from YARSI biorepository were used for the experiment. Trehalose preincubation was performed for 1 hour, internalized trehalose was confirmed by FTIR-ATR measurement. Three groups consisted of (1) cryopreserved without trehalose preincubation, (2) cryopreserved with trehalose preincubation, and (3) did not undergo cryopreservation were evaluated after 24 hours in LN2 for viability in culture. The absorbance from each group was measured at 450 nm. The analysis performed using paired student t test.

Results: Viability of thawed mesenchymal (CD271+) stem cells that undergo trehalose preincubation prior cryopreservation was significantly higher (p<0.05) compared to group without trehalose preincubation. Higher viability observed between group with trehalose preincubation compared with controlled group suggests protection to trypsinization. Mesenchymal (CD271+) stem cells incubated for 1 hour in 100 mM trehalose supplemented medium results in 15% trehalose loading efficiency.

Conclusion: These findings confirm the protective effect of trehalose preincubation in cryopreservation. Future research should be directed to elucidate the trehalose internalization mechanism and eventually the protective mechanism of trehalose in mammalian cell cryopreservation.


chromosomes stability affected by cryopreservation conditions? Cytotechnology. 2008;58(1):11-6.

Zhou XL, Zhu H, Zhang SZ, Zhu FM, Chen GM, Yan LX. Freeze-drying of human platelets: influence of intracellular trehalose and extracellular protectants. Cryo Letters. 2006;27(1):43-50.

Streeter JG. Effect of trehalose on survival of Bradyrhizobium japonicum during desiccation. J Appl Microbiol. 2003;95(3):484-91.

Jain NK, Roy I. Effect of trehalose on protein structure. Protein Sci. 2009;18(1):24-36.

Emanuele E, Bertona M, Sanchis-Gomar F, Pareja-Galeano H, Lucia A. Protective effect of trehalose-loaded liposomes against UVB-induced photodamage in human keratinocytes. Biomed Rep. 2014;2(5):755-9.

Elliott GD, Liu XH, Cusick JL, Menze M, Vincent J, Witt T, et al. Trehalose uptake through P2X7 purinergic channels provides dehydration protection. Cryobiology. 2006;52(1):114-27.

Lynch AL, Chen R, Slater NK. pH-responsive polymers for trehalose loading and desiccation protection of human red blood cells. Biomaterials. 2011;32(19):4443-9.

Tsuchiyama K, Wakao S, Kuroda Y, Ogura F, Nojima M, Sawaya N, et al. Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts. J Invest Dermatol. 2013;133(10):2425-35.

Simerman AA, Perone MJ, Gimeno ML, Dumesic DA, Chazenbalk GD. A mystery unraveled: nontumorigenic pluripotent stem cells in human adult tissues. Expert Opin Biol Ther. 2014;14(7):917-29.

Lynch AL, Slater NK. Influence of intracellular trehalose concentration and pre-freeze cell volume on the cryosurvival of rapidly frozen human erythrocytes. Cryobiology. 2011;63(1):26-31.

Rodrigues J, Paraguassú-Braga F, Carvalho L, Abdelhay E, Bouzas LF, Porto LC. Evaluation of trehalose and sucrose as cryoprotectants for hematopoietic stem cells of umbilical cord blood. Cryobiology. 2008;56(2):144-51.

Oliver AE, Jamil K, Crowe JH, Tablin F. Loading human mesenchymal stem cells with trehalose by fluid-phase endocytosis. Cell Preserv Tech. 2004;2(1):35-49.

McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2011;12(8):517-33.

Sakurai M, Furuki T, Akao K, Tanaka D, Nakahara Y, Kikawada T, et al. Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki. Proc Nat Acad Sci. 2008;105(13):5093-8.

Lynch AL, Slater NK. Mediated trehalose un-loading for reduced erythrocyte osmotic fragility and phosphatidylserine translocation. Cryo Letters. 2011;32(5):415-24.

Zhang S, Qian H, Wang Z, Fan J, Zhou Q, Chen G, et al. Preliminary study on the freeze-drying of human bone marrow-derived mesenchymal stem cells. J Zheijang Univ. 2010;11 (11):889-94.

How to Cite
Kusuma I, Hadi RS, Kiranadi B, Boediono A. Trehalose preincubation increases mesenchymal (CD271+) stem cells post-cryopreservation viability. Med J Indones [Internet]. 2016Oct.14 [cited 2024Mar.4];25(3):128-35. Available from:
Basic Medical Research