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Cryoprotective effects of d -allose on mammalian cells
Cryobiology 55 (2007) 87–92 www.elsevier.com/locate/ycryo
Cryoprotective effects of D-allose on mammalian cells Li Sui a
a,b
q
, Rika Nomura b, Youyi Dong a,b, Fuminori Yamaguchi a, Ken Izumori c, Masaaki Tokuda a,c,*
Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan b Kagawa Industry Support Foundation, Kagawa, Japan c The Rare Sugars Research Center, Kagawa University, Kagawa, Japan Received 23 February 2007; accepted 30 May 2007 Available online 8 June 2007
Abstract D-Allose, an aldo-hexose, is a rare sugar whose biological functions remain largely unclear. Recently, we demonstrated a novel inhibitory effect of D-allose on production of reactive oxygen species (ROS). Here, we focused on investigating cryoprotective effects of D-allose on cell viability. Mammalian cell lines including OVCAR-3 (human ovarian cancer), HeLa (human cervical cancer), HaCaT (human skin keratinocytes), HDF (human dermal fibroblasts) and NIH3T3 (murine fibroblasts) cells were frozen at 80 C in culture media with various D-allose concentrations. Cells were allowed to recover for 24h, 1 week or 1 month prior to survival assessment using the trypan blue dye exclusion test, when cell proliferation was evaluated by MTT assay. A beneficial protective role of D-allose on cell survival was found, similar to that of trehalose (disaccharide of glucose), a recognized cryoprotectant. The results suggest that D-allose as a sole additive may provide effective protection for mammalian cells during freezing. Practical studies now need to be performed with D-allose, for example to determine optimal freezing protocols and explore potential for preservation of tissues or organs at non-freezing temperatures. 2007 Elsevier Inc. All rights reserved.
Keywords: Rare sugars; D-Allose; Cell cryopreservation
Rare sugars are monosaccharides, found sparsely in nature, whose biological effects and physical functions have hitherto been little investigated because of difficulties in obtaining sufficient samples. Recently, a research group at Kagawa University has demonstrated a simple method to produce rare sugars on a large scale using two enzymes, L-rhamnose isomerase (L-RI) and D-tagatose 3-epimerase (DTE), from D-fructose [3,20]. This extends the effective strategy for mass production of rare sugars developed by the discovery of Izumoring [21], allowing production of all rare sugars from inexpensive D-glucose and D-fructose. q
This work was supported by a research grant from the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) Project of Japan. * Corresponding author. Address: Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan. Fax: +81 87 891 2096. E-mail address: [email protected] (M. Tokuda). 0011-2240/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2007.05.003
D-Allose has already been shown to exert beneficial effects as an immunosuppressant for transplantation (US Patent. No. 5620960, 1997) in line with results for rat liver transplantation and rat ischemia/reperfusion injury obtained by Hossain et al. [15–18]. We earlier found that D-allose inhibits production of ROS and has scavenging activity [26]. Since it is widely accepted that ROS generation occurs during the freezing and thawing procedures of tissues or cells [14,23], we here investigated whether D-allose, together with other rare sugars, might have cryoprotective effects on mammalian cells.
Materials and methods Cell culture Cell lines including OVCAR-3, HeLa, HaCaT, HDF and NIH3T3 were cultured at 37 C under a humidified
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atmosphere of 5% CO2 in DMEM (Dulbecco’s minimum essential medium) (for HeLa, HaCaT, HDF, and NIH3T3) or RPMI 1640 medium (for OVCAR-3) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL) and streptomycin (100 lg/mL). Sugars and chemicals The rare sugars D-allose, D-altrose, D-psicose, L-psicose, and L-fructose as well as the non-rare sugars D-fructose, D-glucose, D-mannose, D-galactose and trehalose were supplied by The Rare Sugars Research Center, Kagawa University, Kagawa, Japan. All sugars were dissolved at a concentration of 1 M in media, and filter sterilized. MTT and trypan blue were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
D-sorbose, L-sorbose, D-tagatose, L-tagatose
(5 mg/mL in PBS) were added to each well and the plates were incubated for an additional 4 h at 37 C. To achieve solubilization of formazan crystals formed in viable cells, dimethylformamide (DMF) 20% sodiumdodecyl sulfate (SDS) (pH 4.7) was added to each well. The absorbance was then measured at 590 nm using a Microplate Reader (Immuno-Mini NJ-2300, Nalge Nunc International, Tokyo, Japan). Statistical analysis Statistical analyses were performed with the two-tailed unpaired t-test. A P value less than 0.05 was considered as statistically significant. Each experiment was repeated at least three times and all data are presented as mean ± SEM (standard error of measurement) values. Results
Freezing and thawing procedure After washing, centrifugation, and counting, cells (3 · 106 cells/mL/vial) in the log phase of growth were suspended in different preservation solutions: 1) 10% Me2SO (dimethyl sulfoxide) in culture media or cell banker (Commercial cell freezing preservation solution, BLC-1, Wako Pure Chemical Industries, Ltd., Tokyo, Japan) as a control; 2) 0, 0.2, 0.4, 0.6, 0.8, 1 M D-allose in culture media; 3) 0.4 or 0.6 M sugars in culture media; 4) 1–3% Me2SO with 0.4 or 0.6 M D-allose in culture media. Cryovials were frozen at 80 C in a freezer and/or liquid nitrogen by different freezing procedures. After 24 h, 1 week, or 4 weeks, individual vials were thawed quickly in a water bath at 37 C, then the survival or growth assays were performed. Trypan blue dye exclusion test Post-thaw 0.1 mL cell suspensions and 0.4% trypan blue 0.1 mL were mixed gently, and cell number was determined with a hemocytometer under a light microscope. At least 300 cells were counted in each sample, and the survival rate was defined as the ratio of viable to total cells. Plating efficiency and MTT assay Post-thaw cells were diluted (1:9) with media and after centrifugation and washing cell pellets were resuspended in 1.0 mL medium. To determine the plating efficiency, 10 or 20 · 104 cells were seeded onto 35 mm tissue culture dishes. After 3 h, dishes were washed with PBS, cells were trypsinized, and cell number was counted using a Particle Analyzer (CDA-500, Sysmex, Japan). Using MTT assays, cell viability was examined. Since MTT is reduced to colored formazan only by metabolically active cells, it exclusively detects viable cells. Cells (2–5 · 103) were seeded in 96-well plates with 0.1 mL medium/well (1 column with 8 wells per sample) and cultured in a humidified atmosphere of 5% CO2 at 37 C. After 72 h, 10 lL MTT solutions
Different freezing procedures were examined and it was found that direct freezing cells at 80 C in a freezer (other than in liquid nitrogen) without any controlled cooling, provides the best survival ratio after thawing. With this approach a period of about 40 min (average cooling rate: 2.4 C/min) is required for the temperature to fall from 25 C to 80 C. As evaluated by trypan blue dye exclusion test, almost no cells survived when frozen without any protective agent (medium only). Under the routinely used conditions, both cell banker and 10% Me2SO-containing medium were effective to give survival of more than 90% cells after freezing and thawing. For OVCAR-3 and HaCaT cells, survival rates were highest when D-allose was added at 0.4 M to the medium, and with HeLa, HDF and NIH3T3 cells, 0.6 M D-allose in the medium provided the best cell survival rate (P < 0.001, Fig. 1). Addition of D-allose beyond the optimum concentration did not favor cell survival. The survival ratio did not significantly decline with the prolongation of the freezing period (up to 1 month; data not shown). Freezing with Cell banker resulted in more than 90% survival and this was set as the positive standard for examination of the cryoprotective efficiency of monosaccharides. Trehalose, a recognized cryoprotectant, was used in our study for comparison. D-Allose always gave the highest survival among all monosaccharides tested with the 5 cell lines (all P values <0.05), with almost the same cryoprotective potential as trehalose (Fig. 2). Cell plating efficiency was examined 3 h after seeding the same number of post-thawed cells frozen in the medium only, in Cell banker, in D-allose- or trehalose-supplemented medium (D-allose 0.4 M, trehalose 0.2 M for OVCAR-3, HaCaT cells; D-allose 0.6 M, trehalose 0.3 M for HDF, HeLa, and NIH3T3 cells). D-Allose-frozen cells showed almost the same plating efficiency as those frozen with trehalose (Fig. 3a and b). To examine the functional integrity, cell growth of post-thaw cells was examined. MTT assays showed D-allose-frozen cells to have similar proliferation
L. Sui et al. / Cryobiology 55 (2007) 87–92
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Fig. 1. The effects of D-allose on survival of cryoprotective cells. Cells were frozen at different concentrations of D-allose for 24 h and then thawed, and the survival rate was determined by trypan blue dye exclusion test (n = 9).
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Fig. 2. Comparison of D-allose with other rare sugars or non-rare sugars with regard to cryoprotective effects on various cells (a–e). Cells were frozen in the presence of different sugars (monosaccharides 0.4 M or trehalose 0.2 M for OVCAR-3, HaCaT cells; monosaccharides 0.6 M or trehalose 0.3 M for HDF, HeLa, and NIH3T3 cells). After 24 h, the survival rate of post-thawed cells was determined by trypan blue dye exclusion test (n = 9). *P < 0.05. D-Allose vs. any monosaccharide.
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Fig. 3. The plating efficiency of cryopreserved cells. Three hours after seeding, the growth status of cells after various treatments is shown (a, 200·). The plating percentage (n = 9) is the cell number successfully plated normalized to the total number of cells seeded to the dishes (b).
characteristics as trehalose-frozen cells, with no obvious difference from cells frozen in Cell banker (P > 0.05, Fig. 4). In order to investigate whether addition of D-allose to the culture medium can reduce the necessary dose of conventional Me2SO (usually 10%), various concentrations of Me2SO was tested. OVCAR-3, HaCaT, and HDF cells were frozen with 1%, 2%, and 3% Me2SO alone, or in combination with the optimal concentration of D-allose as determined in Fig. 1. The result showed that the combination with D-allose significantly improved the cells survival ratio as compared to 1% or 2% Me2SO alone (all P values <0.05, Fig. 5). Discussion Cell damage during freezing is caused by multiple factors, of which oxygen free radical generation and membrane
damage are two major examples [1,14,23,25]. In the present study, we first demonstrated that D-allose exerts obvious cryoprotective effects during cell freezing. Although the exact mechanism remains unclear, it is worth noting that our research group has recently found that D-allose has a novel inhibitory effect on ROS production and might increase scavenging of ROS [26]. Significant inhibitory effects on ROS production were observed only with D-allose among all the sugars tested, including other rare sugars. Scavenging activity was investigated by electron spin resonance, and shown to be appreciable, albeit weaker than that of other common scavengers, such as super oxide dismutase (SOD) and carotinoids. The double effects of D-allose may result in significantly reduced ROS concentrations and thereby improve the viability of frozen cells. In addition, we speculate that D-allose might also act by stabilizing cellular membranes. In traditional cryopreservation protocols, sugars have been used exclusively as
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Fig. 4. The proliferation ability of D-allose-frozen cells. Cell growth was estimated by MTT assay (D-allose 0.4 M, trehalose 0.2 M for OVCAR-3 and HaCaT cells; D-allose 0.6 M, trehalose 0.3 M for HeLa, HDF, and NIH3T3 cells). Relative cell numbers were shown (n = 24).
Fig. 5. Combined cryopreservation effects of D-allose and Me2SO. Cells were frozen in the presence of Me2SO alone, or Me2SO combined with D-allose (D-allose 0.4 M for OVCAR-3 and HaCaT cells; D-allose 0.6 M for HDF cells) at 80 C for 24 h and then thawed. The survival rate was determined by trypan blue dye exclusion test (n = 9). *P < 0.05; **P < 0.01; ***P < 0.001.
extracellular additives because of the negligible permeability of plasma membranes to sugars molecules, thus stabilizing cellular membranes during freezing, and possibly dehydrating cells so that excessive swelling and osmotic shock are avoided [7]. Previous studies in some cell types and tissues have demonstrated beneficial cryoprotective effects of sugars such as trehalose, sucrose, and raffinose [2,4,8,12,31,32]. Membrane stabilizers and bioantioxidants have been shown to possess cryoprotection qualities [5,6,24,33]. Intracellular ice crystal formation is considered to be a prime mechanism of cell damage during or after cryopreservation. Cryoprotective agents may thus reduce physical injury by preventing the formation of ice crystals and this is thought to be the mechanism of action of Me2SO [13,22]. Non-permeable sugars like D-allose may provide protection through dehydrating cells and thus reducing the amount of water present before freezing. It is also
known that the viscosity of sugar solutions rapidly increases during cooling. This is probably another advantage of D-allose, considering that increased viscosity may reduce ice crystal growth and thus potential damage. In this study, we compared the cryoprotective effects of D-allose and a recognized cryoprotectant, trehalose, a nonreducing disaccharide that stabilizes and protects cellular membranes and proteins during freezing [30]. Many studies have demonstrated protective effects of trehalose on various tissues and cells [2,4,5,8–11,19,24,28,29,31,33] during freezing. Eroglu et al. also demonstrated that intracellular trehalose greatly improved survival [10,11]. Our study showed similar protective effects of D-allose and trehalose, although the influence of intracellular D-allose was not examined. The present results suggested that D-allose might find future application as a useful cryoprotectant. Currently, Me2SO is considered to be the most effective cryoprotectant for cells. However, because of its toxic
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effects on living tissues and cells, Me2SO can cause cell damage during thawing [27]. D-Allose, a rare sugar, may have significant promise as a possible nontoxic additive or replacement for Me2SO. In order to examine whether cells uptake D-allose, 14Clabeled D-allose incorporation experiments were performed. No incorporation was observed under the freezing conditions employed (data not shown), indicating that the cryoprotective effect was caused by extra-cellular D-allose. We speculate that the presence both intra- and extracellular D-allose would further improve cell survival rates. In conclusion, our study showed that D-allose, in the absence of other protective additives, may possess beneficial cryoprotective properties. Although exact mechanisms require further investigation, the results already indicate the importance and utility of D-allose for cryopreservation of cells during freezing. Additional studies now need to be performed to check the effects of intracellular D-allose, to establish optimal freezing protocols, and explore usage for preservation of various tissues or organs at non-freezing temperatures. References [1] J.M. Baust, M.J. Vogel, R. Van Buskirk, J.G. Baust, Cell Transplant. 10 (2001) 561–571. [2] G.M. Beattie, J.H. Crowe, A.D. Lopez, V. Cirulli, C. Ricordi, A. Hayek, Diabetes 46 (1997) 519–523. [3] S.H. Bhuiyan, Y. Itami, Y. Rokui, T. Katayama, K. Izumori, J. Ferment. Bioeng. 85 (1998) 539–541. [4] T. Chen, J.P. Acker, A. Eroglu, S. Cheley, H. Bayley, A. Fowler, M. Toner, Cryobiology 43 (2001) 168–181. [5] Y. Chen, R.H. Foote, C.C. Brockett, Cryobiology 30 (1993) 423–431. [6] J.H. Crowe, L.M. Crowe, Nat. Biotechnol. 18 (2000) 145–146. [7] J.H. Crowe, L.M. Crowe, J.F. Carpenter, C. Aurell Wistrom, Biochem. J. 242 (1987) 1–10. [8] G. Erdag, A. Eroglu, J. Morgan, M. Toner, Cryobiology 44 (2002) 218–228.
[9] A. Eroglu, G. Elliott, D.L. Wright, M. Toner, T.L. Toth, Reprod. Biomed. Online 10 (2005) 503–510. [10] A. Eroglu, M.J. Russo, R. Bieganski, A. Fowler, S. Cheley, H. Bayley, M. Toner, Nat. Biotechnol. 18 (2000) 145–146. [11] A. Eroglu, M. Toner, T.L. Toth, Fertil. Steril. 77 (2002) 152–158. [12] M.R. Fernandez-Santos, F. Martinez-Pastor, V. Garcia-Macias, M.C. Esteso, A.J. Soler, P. de Paz, L. Anel, J.J. Garde, Theriogenology 67 (2007) 738–753. [13] D. Gao, J.K. Critser, ILAR. J. 41 (2000) 187–196. [14] L.A. Hernandez, N. Granger, Crit. Care Med. 16 (1988) 543–549. [15] M.A. Hossain, K. Izuishi, H. Maeta, J. Hepatobiliary Pancreat. Surg. 10 (2003) 218–225. [16] M.A. Hossain, K. Izuishi, M. Tokuda, K. Izumori, H. Maeta, J. Hepatobiliary Pancreat. Surg. 11 (2004) 181–189. [17] M.A. Hossain, H. Wakabayashi, F. Goda, S. Kobayashi, T. Maeba, H. Maeta, Transplant. Proc. 32 (2000) 2021–2023. [18] M.A. Hossain, H. Wakabayashi, K. Izuishi, K. Okano, S. Yachida, M. Tokuda, K. Izumori, H. Maeta, J. Biosci. Bioeng. 101 (2006) 369–371. [19] N. Isowa, S. Hitomi, H. Wada, Ann. Thorac. Surg. 61 (1996) 542–545. [20] H. Itoh, T. Sato, K. Izumori, J. Ferment. Bioeng. 80 (1995) 101–103. [21] K. Izumori, Naturwissenschaften 89 (2002) 120–124. [22] J.O. Karlsson, M. Toner, Biomaterials 17 (1996) 243–256. [23] J.N. Kearney, Burns 24 (1998) 507–512. [24] L.S. Limaye, V.P. Kale, J. Hematoth. Stem Cell Res. 10 (2001) 709–718. [25] G. Martin, O. Sabido, P. Durand, R. Levy, Biol. Reprod. 71 (2004) 28–37. [26] A. Murata, K. Sekiya, Y. Watanabe, F. Yamaguchi, N. Hatano, K. Izumori, M. Tokuda, J. Biosci. Bioeng. 96 (2003) 89–91. [27] D.E. Pegg, Semin. Reprod. Med. 20 (2002) 5–13. [28] C. Pellerin-Mendes, L. Million, M. Marchand-Arvier, P. Labrude, C. Vigneron, Cryobiology 35 (1997) 173–186. [29] L.L.Q. Pu, X. Cui, B.F. Fink, M.L. Cibull, D. Gao, Aesthet. Surg. J. 25 (2005) 126–131. [30] A.S. Rudolph, J.H. Crowe, Cryobiology 22 (1985) 367–377. [31] B.T. Storey, E.E. Noiles, K.A. Thompson, Cryobiology 37 (1998) 46–58. [32] J.M. Sztein, K. Noble, J.S. Farley, L.E. Mobraaten, Cryobiology 42 (2001) 28–39. [33] X.B. Zhang, K. Li, K.H. Yau, K.S. Tsang, T.F. Fok, C.K. Li, S.M. Lee, P.M. Yuen, Transfusion 43 (2003) 265–272.
Cryoprotective effects of D-allose on mammalian cells Li Sui a
a,b
q
, Rika Nomura b, Youyi Dong a,b, Fuminori Yamaguchi a, Ken Izumori c, Masaaki Tokuda a,c,*
Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan b Kagawa Industry Support Foundation, Kagawa, Japan c The Rare Sugars Research Center, Kagawa University, Kagawa, Japan Received 23 February 2007; accepted 30 May 2007 Available online 8 June 2007
Abstract D-Allose, an aldo-hexose, is a rare sugar whose biological functions remain largely unclear. Recently, we demonstrated a novel inhibitory effect of D-allose on production of reactive oxygen species (ROS). Here, we focused on investigating cryoprotective effects of D-allose on cell viability. Mammalian cell lines including OVCAR-3 (human ovarian cancer), HeLa (human cervical cancer), HaCaT (human skin keratinocytes), HDF (human dermal fibroblasts) and NIH3T3 (murine fibroblasts) cells were frozen at 80 C in culture media with various D-allose concentrations. Cells were allowed to recover for 24h, 1 week or 1 month prior to survival assessment using the trypan blue dye exclusion test, when cell proliferation was evaluated by MTT assay. A beneficial protective role of D-allose on cell survival was found, similar to that of trehalose (disaccharide of glucose), a recognized cryoprotectant. The results suggest that D-allose as a sole additive may provide effective protection for mammalian cells during freezing. Practical studies now need to be performed with D-allose, for example to determine optimal freezing protocols and explore potential for preservation of tissues or organs at non-freezing temperatures. 2007 Elsevier Inc. All rights reserved.
Keywords: Rare sugars; D-Allose; Cell cryopreservation
Rare sugars are monosaccharides, found sparsely in nature, whose biological effects and physical functions have hitherto been little investigated because of difficulties in obtaining sufficient samples. Recently, a research group at Kagawa University has demonstrated a simple method to produce rare sugars on a large scale using two enzymes, L-rhamnose isomerase (L-RI) and D-tagatose 3-epimerase (DTE), from D-fructose [3,20]. This extends the effective strategy for mass production of rare sugars developed by the discovery of Izumoring [21], allowing production of all rare sugars from inexpensive D-glucose and D-fructose. q
This work was supported by a research grant from the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) Project of Japan. * Corresponding author. Address: Department of Cell Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan. Fax: +81 87 891 2096. E-mail address: [email protected] (M. Tokuda). 0011-2240/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2007.05.003
D-Allose has already been shown to exert beneficial effects as an immunosuppressant for transplantation (US Patent. No. 5620960, 1997) in line with results for rat liver transplantation and rat ischemia/reperfusion injury obtained by Hossain et al. [15–18]. We earlier found that D-allose inhibits production of ROS and has scavenging activity [26]. Since it is widely accepted that ROS generation occurs during the freezing and thawing procedures of tissues or cells [14,23], we here investigated whether D-allose, together with other rare sugars, might have cryoprotective effects on mammalian cells.
Materials and methods Cell culture Cell lines including OVCAR-3, HeLa, HaCaT, HDF and NIH3T3 were cultured at 37 C under a humidified
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atmosphere of 5% CO2 in DMEM (Dulbecco’s minimum essential medium) (for HeLa, HaCaT, HDF, and NIH3T3) or RPMI 1640 medium (for OVCAR-3) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL) and streptomycin (100 lg/mL). Sugars and chemicals The rare sugars D-allose, D-altrose, D-psicose, L-psicose, and L-fructose as well as the non-rare sugars D-fructose, D-glucose, D-mannose, D-galactose and trehalose were supplied by The Rare Sugars Research Center, Kagawa University, Kagawa, Japan. All sugars were dissolved at a concentration of 1 M in media, and filter sterilized. MTT and trypan blue were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
D-sorbose, L-sorbose, D-tagatose, L-tagatose
(5 mg/mL in PBS) were added to each well and the plates were incubated for an additional 4 h at 37 C. To achieve solubilization of formazan crystals formed in viable cells, dimethylformamide (DMF) 20% sodiumdodecyl sulfate (SDS) (pH 4.7) was added to each well. The absorbance was then measured at 590 nm using a Microplate Reader (Immuno-Mini NJ-2300, Nalge Nunc International, Tokyo, Japan). Statistical analysis Statistical analyses were performed with the two-tailed unpaired t-test. A P value less than 0.05 was considered as statistically significant. Each experiment was repeated at least three times and all data are presented as mean ± SEM (standard error of measurement) values. Results
Freezing and thawing procedure After washing, centrifugation, and counting, cells (3 · 106 cells/mL/vial) in the log phase of growth were suspended in different preservation solutions: 1) 10% Me2SO (dimethyl sulfoxide) in culture media or cell banker (Commercial cell freezing preservation solution, BLC-1, Wako Pure Chemical Industries, Ltd., Tokyo, Japan) as a control; 2) 0, 0.2, 0.4, 0.6, 0.8, 1 M D-allose in culture media; 3) 0.4 or 0.6 M sugars in culture media; 4) 1–3% Me2SO with 0.4 or 0.6 M D-allose in culture media. Cryovials were frozen at 80 C in a freezer and/or liquid nitrogen by different freezing procedures. After 24 h, 1 week, or 4 weeks, individual vials were thawed quickly in a water bath at 37 C, then the survival or growth assays were performed. Trypan blue dye exclusion test Post-thaw 0.1 mL cell suspensions and 0.4% trypan blue 0.1 mL were mixed gently, and cell number was determined with a hemocytometer under a light microscope. At least 300 cells were counted in each sample, and the survival rate was defined as the ratio of viable to total cells. Plating efficiency and MTT assay Post-thaw cells were diluted (1:9) with media and after centrifugation and washing cell pellets were resuspended in 1.0 mL medium. To determine the plating efficiency, 10 or 20 · 104 cells were seeded onto 35 mm tissue culture dishes. After 3 h, dishes were washed with PBS, cells were trypsinized, and cell number was counted using a Particle Analyzer (CDA-500, Sysmex, Japan). Using MTT assays, cell viability was examined. Since MTT is reduced to colored formazan only by metabolically active cells, it exclusively detects viable cells. Cells (2–5 · 103) were seeded in 96-well plates with 0.1 mL medium/well (1 column with 8 wells per sample) and cultured in a humidified atmosphere of 5% CO2 at 37 C. After 72 h, 10 lL MTT solutions
Different freezing procedures were examined and it was found that direct freezing cells at 80 C in a freezer (other than in liquid nitrogen) without any controlled cooling, provides the best survival ratio after thawing. With this approach a period of about 40 min (average cooling rate: 2.4 C/min) is required for the temperature to fall from 25 C to 80 C. As evaluated by trypan blue dye exclusion test, almost no cells survived when frozen without any protective agent (medium only). Under the routinely used conditions, both cell banker and 10% Me2SO-containing medium were effective to give survival of more than 90% cells after freezing and thawing. For OVCAR-3 and HaCaT cells, survival rates were highest when D-allose was added at 0.4 M to the medium, and with HeLa, HDF and NIH3T3 cells, 0.6 M D-allose in the medium provided the best cell survival rate (P < 0.001, Fig. 1). Addition of D-allose beyond the optimum concentration did not favor cell survival. The survival ratio did not significantly decline with the prolongation of the freezing period (up to 1 month; data not shown). Freezing with Cell banker resulted in more than 90% survival and this was set as the positive standard for examination of the cryoprotective efficiency of monosaccharides. Trehalose, a recognized cryoprotectant, was used in our study for comparison. D-Allose always gave the highest survival among all monosaccharides tested with the 5 cell lines (all P values <0.05), with almost the same cryoprotective potential as trehalose (Fig. 2). Cell plating efficiency was examined 3 h after seeding the same number of post-thawed cells frozen in the medium only, in Cell banker, in D-allose- or trehalose-supplemented medium (D-allose 0.4 M, trehalose 0.2 M for OVCAR-3, HaCaT cells; D-allose 0.6 M, trehalose 0.3 M for HDF, HeLa, and NIH3T3 cells). D-Allose-frozen cells showed almost the same plating efficiency as those frozen with trehalose (Fig. 3a and b). To examine the functional integrity, cell growth of post-thaw cells was examined. MTT assays showed D-allose-frozen cells to have similar proliferation
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Fig. 1. The effects of D-allose on survival of cryoprotective cells. Cells were frozen at different concentrations of D-allose for 24 h and then thawed, and the survival rate was determined by trypan blue dye exclusion test (n = 9).
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HaCaT
Survival rate (%)
100 80 60 40
Cell banker D-allose D-psicose D-sorbose D-tagatose L-fructose D-glucose D-galactose
Medium D-altrose L-psicose L-sorbose L-tagatose D-fructose D-mannose Trehalose
20 0
Fig. 2. Comparison of D-allose with other rare sugars or non-rare sugars with regard to cryoprotective effects on various cells (a–e). Cells were frozen in the presence of different sugars (monosaccharides 0.4 M or trehalose 0.2 M for OVCAR-3, HaCaT cells; monosaccharides 0.6 M or trehalose 0.3 M for HDF, HeLa, and NIH3T3 cells). After 24 h, the survival rate of post-thawed cells was determined by trypan blue dye exclusion test (n = 9). *P < 0.05. D-Allose vs. any monosaccharide.
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Fig. 3. The plating efficiency of cryopreserved cells. Three hours after seeding, the growth status of cells after various treatments is shown (a, 200·). The plating percentage (n = 9) is the cell number successfully plated normalized to the total number of cells seeded to the dishes (b).
characteristics as trehalose-frozen cells, with no obvious difference from cells frozen in Cell banker (P > 0.05, Fig. 4). In order to investigate whether addition of D-allose to the culture medium can reduce the necessary dose of conventional Me2SO (usually 10%), various concentrations of Me2SO was tested. OVCAR-3, HaCaT, and HDF cells were frozen with 1%, 2%, and 3% Me2SO alone, or in combination with the optimal concentration of D-allose as determined in Fig. 1. The result showed that the combination with D-allose significantly improved the cells survival ratio as compared to 1% or 2% Me2SO alone (all P values <0.05, Fig. 5). Discussion Cell damage during freezing is caused by multiple factors, of which oxygen free radical generation and membrane
damage are two major examples [1,14,23,25]. In the present study, we first demonstrated that D-allose exerts obvious cryoprotective effects during cell freezing. Although the exact mechanism remains unclear, it is worth noting that our research group has recently found that D-allose has a novel inhibitory effect on ROS production and might increase scavenging of ROS [26]. Significant inhibitory effects on ROS production were observed only with D-allose among all the sugars tested, including other rare sugars. Scavenging activity was investigated by electron spin resonance, and shown to be appreciable, albeit weaker than that of other common scavengers, such as super oxide dismutase (SOD) and carotinoids. The double effects of D-allose may result in significantly reduced ROS concentrations and thereby improve the viability of frozen cells. In addition, we speculate that D-allose might also act by stabilizing cellular membranes. In traditional cryopreservation protocols, sugars have been used exclusively as
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Fig. 4. The proliferation ability of D-allose-frozen cells. Cell growth was estimated by MTT assay (D-allose 0.4 M, trehalose 0.2 M for OVCAR-3 and HaCaT cells; D-allose 0.6 M, trehalose 0.3 M for HeLa, HDF, and NIH3T3 cells). Relative cell numbers were shown (n = 24).
Fig. 5. Combined cryopreservation effects of D-allose and Me2SO. Cells were frozen in the presence of Me2SO alone, or Me2SO combined with D-allose (D-allose 0.4 M for OVCAR-3 and HaCaT cells; D-allose 0.6 M for HDF cells) at 80 C for 24 h and then thawed. The survival rate was determined by trypan blue dye exclusion test (n = 9). *P < 0.05; **P < 0.01; ***P < 0.001.
extracellular additives because of the negligible permeability of plasma membranes to sugars molecules, thus stabilizing cellular membranes during freezing, and possibly dehydrating cells so that excessive swelling and osmotic shock are avoided [7]. Previous studies in some cell types and tissues have demonstrated beneficial cryoprotective effects of sugars such as trehalose, sucrose, and raffinose [2,4,8,12,31,32]. Membrane stabilizers and bioantioxidants have been shown to possess cryoprotection qualities [5,6,24,33]. Intracellular ice crystal formation is considered to be a prime mechanism of cell damage during or after cryopreservation. Cryoprotective agents may thus reduce physical injury by preventing the formation of ice crystals and this is thought to be the mechanism of action of Me2SO [13,22]. Non-permeable sugars like D-allose may provide protection through dehydrating cells and thus reducing the amount of water present before freezing. It is also
known that the viscosity of sugar solutions rapidly increases during cooling. This is probably another advantage of D-allose, considering that increased viscosity may reduce ice crystal growth and thus potential damage. In this study, we compared the cryoprotective effects of D-allose and a recognized cryoprotectant, trehalose, a nonreducing disaccharide that stabilizes and protects cellular membranes and proteins during freezing [30]. Many studies have demonstrated protective effects of trehalose on various tissues and cells [2,4,5,8–11,19,24,28,29,31,33] during freezing. Eroglu et al. also demonstrated that intracellular trehalose greatly improved survival [10,11]. Our study showed similar protective effects of D-allose and trehalose, although the influence of intracellular D-allose was not examined. The present results suggested that D-allose might find future application as a useful cryoprotectant. Currently, Me2SO is considered to be the most effective cryoprotectant for cells. However, because of its toxic
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effects on living tissues and cells, Me2SO can cause cell damage during thawing [27]. D-Allose, a rare sugar, may have significant promise as a possible nontoxic additive or replacement for Me2SO. In order to examine whether cells uptake D-allose, 14Clabeled D-allose incorporation experiments were performed. No incorporation was observed under the freezing conditions employed (data not shown), indicating that the cryoprotective effect was caused by extra-cellular D-allose. We speculate that the presence both intra- and extracellular D-allose would further improve cell survival rates. In conclusion, our study showed that D-allose, in the absence of other protective additives, may possess beneficial cryoprotective properties. Although exact mechanisms require further investigation, the results already indicate the importance and utility of D-allose for cryopreservation of cells during freezing. Additional studies now need to be performed to check the effects of intracellular D-allose, to establish optimal freezing protocols, and explore usage for preservation of various tissues or organs at non-freezing temperatures. References [1] J.M. Baust, M.J. Vogel, R. Van Buskirk, J.G. Baust, Cell Transplant. 10 (2001) 561–571. [2] G.M. Beattie, J.H. Crowe, A.D. Lopez, V. Cirulli, C. Ricordi, A. Hayek, Diabetes 46 (1997) 519–523. [3] S.H. Bhuiyan, Y. Itami, Y. Rokui, T. Katayama, K. Izumori, J. Ferment. Bioeng. 85 (1998) 539–541. [4] T. Chen, J.P. Acker, A. Eroglu, S. Cheley, H. Bayley, A. Fowler, M. Toner, Cryobiology 43 (2001) 168–181. [5] Y. Chen, R.H. Foote, C.C. Brockett, Cryobiology 30 (1993) 423–431. [6] J.H. Crowe, L.M. Crowe, Nat. Biotechnol. 18 (2000) 145–146. [7] J.H. Crowe, L.M. Crowe, J.F. Carpenter, C. Aurell Wistrom, Biochem. J. 242 (1987) 1–10. [8] G. Erdag, A. Eroglu, J. Morgan, M. Toner, Cryobiology 44 (2002) 218–228.
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