2 Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides

There is a need for mammalian cell cryopreservation methods that either avoid or improve upon outcomes employing dimethyl sulfoxide (DMSO) as a cryoprotectant. DMSO was the second effective cryoprotectant to be discovered (Lovelock, 1959). Cell cryopreservation usually involves slow rate freezing with DMSO in culture medium and storage below -135°C for later use. Typically as long as there are enough cells surviving to start an expanding proliferating culture the yield of viable cells after thawing is not an important consideration. However, there are instances where cell yield and viability can be very important. Examples include minimization of expensive delays when starting cultures for bioreactor protein manufacturing runs and cellular therapies that involve administering cells into patients for treatment of various diseases, such as cancer. While some cells, for example fibroblasts, are easily cryopreserved other cell types like keratinocytes, hepatocytes, and cardiac myocytes do not freeze well and cell yields are often <50%. Furthermore, current opinion is that DMSO should be removed before cells are infused into patients (Caselli et al., 2009; Junior et al., 2008; Mueller et al., 2007; Otrock et al., 2008; Schlegel et al., 2009). The mechanism for DMSO cytotoxicity has not been determined, however, it is thought to modify membrane fluidity, induce cell differentiation, cause cytoplasmic microtubule changes and metal complexes (Barnett 1978; Katsuda et al., 1984, 1987; Miranda et al., 1978). DMSO also decreases expression of collagen mRNAs in a dose-dependent manner (Zeng et al., 2010).


Introduction
There is a need for mammalian cell cryopreservation methods that either avoid or improve upon outcomes employing dimethyl sulfoxide (DMSO) as a cryoprotectant. DMSO was the second effective cryoprotectant to be discovered (Lovelock, 1959). Cell cryopreservation usually involves slow rate freezing with DMSO in culture medium and storage below -135°C for later use. Typically as long as there are enough cells surviving to start an expanding proliferating culture the yield of viable cells after thawing is not an important consideration. However, there are instances where cell yield and viability can be very important. Examples include minimization of expensive delays when starting cultures for bioreactor protein manufacturing runs and cellular therapies that involve administering cells into patients for treatment of various diseases, such as cancer. While some cells, for example fibroblasts, are easily cryopreserved other cell types like keratinocytes, hepatocytes, and cardiac myocytes do not freeze well and cell yields are often <50%. Furthermore, current opinion is that DMSO should be removed before cells are infused into patients (Caselli et al., 2009;Junior et al., 2008;Mueller et al., 2007;Otrock et al., 2008;Schlegel et al., 2009). The mechanism for DMSO cytotoxicity has not been determined, however, it is thought to modify membrane fluidity, induce cell differentiation, cause cytoplasmic microtubule changes and metal complexes (Barnett 1978;Katsuda et al., 1984Katsuda et al., , 1987Miranda et al., 1978). DMSO also decreases expression of collagen mRNAs in a dose-dependent manner (Zeng et al., 2010).
One strategy for finding interesting new cryoprotectants and cryopreservation strategies is by evaluating what happens in nature . No examples of organisms synthesizing DMSO to survive freezing conditions have been found to date, however several creatures have been found that employ glycerol  the first effective cryoprotectant to be discovered (Polge, 1949). Nature has developed a wide variety of organisms and animals that tolerate low temperatures and dehydration stress by accumulation of large amounts of disaccharides, particularly trehalose, including plant seeds, bacteria, insects, yeast, brine shrimp, fungi and their spores, cysts of certain crustaceans, and some soil-dwelling animals. While the cryoprotective capabilities of sucrose and trehalose has been known for years, conventional cryopreservation protocols have generally not employed them even though early work with them demonstrated their ability to protect proteins and membrane vesicles during freezing (Rudolf & Crowe, 1985;Crowe et al., 1990). Trehalose has both major advantages and disadvantages for potential preservation of mammalian cells. On the negative side mammalian cells do not have an active trehalose transport system for uptake of trehalose from the extracellular environment, while on the plus side if you can get it in mammalian cells it is not metabolized giving the opportunity for trehalose to be accumulated to potentially effective preservation concentrations. The purpose of the studies presented here were; 1) to assess or review alternative strategies for delivery of trehalose into mammalian cells, and; 2) to determine whether the benefits were specific to trehalose by investigating alternative sugars employing the same loading strategies.

Cell culture
Cells used in these studies are described in Table 1

Cell poration with H5
The pore-forming protein H5 was obtained from the lab of Hagan Bayley (Bayley, 1994). It is derived from the bacterial toxin -hemolysin, which forms constitutively opened pores in cell membranes. The modified bacterial toxin has been engineered to form pores in the membrane that can be opened and closed by the addition of Zn + . More specific details are www.intechopen.com presented in the discussion. Cells were plated at 10,000-20,000 cells/well the night before in 96 well microtiter plates. The next day, the cells were washed with DMEM containing 1 mM EDTA for 2 minutes and then again with DMEM to remove the EDTA. 0.2M trehalose was added and incubated for 20 minutes at 37 o C followed by the appropriate concentration of H5 for the respective cell type. Cells were porated and loaded with trehalose for 1 hour at 37 o C before addition of DMEM with 25 µM ZnSO 4 or 10% serum to close the pores. Trehalose in DMEM was then added to the wells followed by cryopreservation using a controlled rate freezer (Planar) at ~-1.0 o C/min from 4ºC to -80ºC with a programmed nucleation step at -5.0 o C. Cryopreserved cells were stored overnight at <-135 o C. The next day, the cells were placed at -20 o C for 30 minutes followed by rapid thawing at 37 o C (Campbell et al., 2003;Taylor et al., 2001). The cell cultures were washed twice and then placed at 37 o C for 1 hour to recover under normothermic cell culture conditions before assessment of cell viability.

Pretreatment (Incubation) with trehalose
Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the culture medium was replaced with EMEM or DMEM containing trehalose (0-0.6M) and cultured at 37 o C for varying periods of time. After culture, the solution was replaced with fresh medium containing trehalose (0-0.6M) and the cells were cryopreserved using a controlled rate freezer as described for H5 above.

Cell poration with ATP
Cells were plated at 10,000-20,000 cells/well and placed in culture. The next day, the cells were washed with poration buffer (phosphate-buffered saline [PBS] with 1X essential amino acids, 1X Vitastock, 5.5 mM glucose) designed to optimize binding of ATP 4-to the receptor and facilitate formation of the pore. The cells were then placed in 50 µl poration buffer, pH of 7.45, with 0.2M trehalose. A stock solution of 100 mM ATP 4-, pH of 7.45, was made fresh and added to each well to achieve a final concentration of 5 mM. After addition of the ATP 4-, the cells were left at 37 o C for 1 hour to allow sugar uptake. Following incubation, 200 µl of DMEM plus 1 mM MgCl 2 was then added to the cells at 37 o C to close the pores. After 1 hour of recovery from the loading procedure cryopreservation was initiated.

Assessment of cell viability
Cell viability was determined using the non-invasive metabolic indicator alamarBlue™ (Trek Diagnostics). A volume of 20 µl was added to cells in 200 µl of DMEM (10%FCS) and the plate was incubated at 37 o C for 3 hours. Plates were read using a fluorescent microplate reader (Molecular Dynamics) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Viability was measured before and after sugar loading, immediately after thawing and at several later time points post-thaw.

Statistical methods
All experiments were repeated at least four times with four replicates in each experiment. Statistical differences were assessed by two way analysis of variance. P-values 0.05 were regarded as significant. www.intechopen.com

H5 poration
One of the first strategies for utilizing disaccharide sugars as cryoprotectants involved the use of a modified pore forming complex. In our initial studies we evaluated the H5 mutanthemolysin (Bayley, 1994) using two adherent cell lines, A10 and CPAE. The earlier studies had been done with cells in suspension (Eroglu et al., 2000). We also evaluated sucrose, another disaccharide sugar that is commonly found in nature, for its potential usefulness as a cryoprotectant. Using the protocol of Eroglu et al as a starting point, a protocol was established for adherent cells. Several parameters were evaluated and included the H5 concentration, time of poration, concentration of trehalose loaded, and time for loading trehalose. Conditions that worked best with adherent cells included 20 minutes for poration followed by 60 minutes for trehalose loading. The highest concentration of trehalose that caused the least drop in cell viability was 0.2M. The optimum H5 concentration varied according to cell type. The A10 smooth muscle cells were porated with 12.5 µg/mL of H5 while the endothelial CPAE cells were porated with 50 µg/mL. In contrast, the fibroblasts and keratinocytes in the literature were porated with 25 µg/mL (Eroglu et al., 2000). Other changes to the protocol were made that benefited viability for adherent cells specifically and included addition of trehalose prior to the addition of H5, the base solution used for poration, and the amount of EDTA (1 mM versus 10 mM) for the removal of Zn + prior to poration. After cryopreservation, however, poor viability was obtained with both cell types. A10 cells demonstrated a viability of 5.57±0.17%. The endothelial cells demonstrated similar viabilities. These values were not as good as those observed when suspended cells were cryopreserved with sugars in the literature. However, it is our experience that adherent cells are generally more difficult to cryopreserve regardless of the cryoprotectant used.

Trehalose exposure without poration
When we started adding the trehalose to cells in the H5 experiments, control cells were exposed to trehalose by addition to the culture medium prior to cryopreservation. An unanticipated observation of cell survival was made with slow rate cryopreserved CPAE cells prompting further investigation. The cells exposed to trehalose overnight were observed to develop vacuoles (Fig 1) suggesting a possible pinocytotic uptake mechanism. After these observations were made, further experiments were designed to examine cell viability after extended trehalose exposure. CPAE cells were exposed to 0.2M trehalose in Dulbecco's Modified Eagle's Medium (DMEM) buffered with 25 mM Hepes for 0-72 hours at 37 o C. After exposure the cells were left in 0.2M trehalose and cryopreserved at ~-1.0 o C/min (Fig. 2). CPAE cell viability was observed immediately after thawing. An exposure time of 24 hours provided the best overall cell survival. Extracellular exposure alone during cryopreservation failed to produce any cell survival. In contrast, A10 smooth muscle cells generally did not survive cryopreservation after trehalose exposure as well as the CPAE endothelial cells. Examination of optimal concentrations of trehalose during incubation and during cryopreservation showed that a concentration of 0.1-0.2M trehalose for incubation produced the best viability with a similar concentration being required during the freezing process. Several other parameters were also examined to further improve cell viability. Other studies have shown that not only the concentration and choice of cryoprotectant but also the vehicle solution for the cryoprotectant can have a significant impact on cell viability after cryopreservation Mathew et al., 2004;Sosef et al., 2005). Initial experiments were performed using DMEM, however, it was observed that CPAE cells, which are grown in EMEM medium, actually preferred exposure to trehalose in EMEM medium. Further experiments examined the buffers used to maintain the pH of the system. Four cell lines were evaluated. CPAE cells demonstrated decreased viability when the zwitteronic buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was used while the other 3 cell lines did not show decreased viability. Rather a combination of HEPES and sodium bicarbonate was preferred by the CPAE cells. This unusual choice of buffer prompted examination of solution pH during incubation, a pH of 7.4 was optimal for all the 4 cell lines tested. Once loaded with sugar, the cells could either be left in the extracellular sugar at another concentration or an alternative cryoprotectant for preservation.

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These studies were then extended to include other sugars, sucrose, raffinose, and stachyose (Fig. 3). The potential cryoprotective benefits of these sugars were evaluated and it was found that stachyose was as good as trehalose using an identical protocol, sucrose was not quite as good and raffinose had very little benefit. All cell lines showed evidence of some cell survival days after cryopreservation and thawing. The second smooth muscle cell line, A7R5, demonstrated low levels of viability with stachyose. Both endothelial cell lines, CPAE and BCE, showed good viability after exposure and freezing with sucrose. Overall, the CPAE cell line had the best viability in these experiments. Use of an optimized protocol with trehalose produced excellent post cryopreservation results with 10-14mM intracellular trehalose . Conditions included 24 hours of cell culture with 0.2M trehalose followed by cryopreservation with 0.2-0.4M trehalose in sodium bicarbonate buffered EMEM at pH 7.4 resulting in ~75% post-preservation cell viability .These experiments confirmed that this technique is more effective for endothelial cells than smooth muscle cells and demonstrated that stachyose is effective for cryopreservation.

ATP poration
In addition to the H5 mutant -hemolysin poration strategy, we sought other poration techniques that could be used to permeate mammalian cells with disaccharides. Cells expressing the P2 X7 purinergic cell surface receptor, also known as the P2 z receptor, may be permeabilized by the formation of a channel/pore that allows passage of molecules into and out of the cell when the active form of ATP (ATP 4-) binds to the receptor. Our initial studies focused on determination of whether or not the P2 X7 was expressed on smooth muscle and endothelial cells. Experiments using the ELICA assay demonstrated the presence of the P2 X7 receptor on both endothelial and smooth muscle cell lines to varying degrees (Fig 4). The smooth muscle cell lines demonstrated the greatest density of the receptor. ATP-permeabilized cells retained better viability than untreated cells both immediately after thawing and five days later (Fig 5). Immediate metabolic activity in A7R5 and CPAE cells demonstrated dependence upon increasing ATP concentrations, while for A10 and BCE cells immediate metabolic activity was increased at all ATP concentrations with only slight improvement at the higher concentrations tested. However, survival at five days demonstrated that intermediate concentrations of ATP (0.5-2.5mM) were best. Further cryopreservation studies were performed to optimize cell survival resulting in at least 25% cell survival for both endothelial cell lines but only low levels of survival for the smooth muscle cells. www.intechopen.com

Discussion
As cryopreservation has been applied to cells and tissues for clinical use, concerns about toxicity relating to the various cryoprotectants being used, particularly DMSO, have developed. Because of this, there has been renewed interest in finding less toxic cryoprotectants. The cryoprotective capabilities of some sugars, disaccharide sugars in particular, has been known for years and early work with them demonstrated their ability to protect proteins and membrane vesicles during freezing (Crowe et al., 1990;Rudolph & Crowe, 1985). Coupled with these early studies are observations made in nature regarding organisms that can survive extremes in temperature and desiccation due to their ability to accumulate large amounts of disaccharide sugars, specifically trehalose and sucrose, until more favorable conditions are available. The protective effects of trehalose and sucrose have been determined and may be classified under two general mechanisms: (1) "the water replacement hypothesis" or stabilization of biological membranes and proteins by direct interaction of sugars with polar residues through hydrogen bonding, and (2) stable glass formation (vitrification) by sugars in the dry state (Crowe et al., , 1988(Crowe et al., , 1998(Crowe et al., , 2001Slade & Levine, 1991).
Two primary stresses that destabilize membranes have been defined, fusion and lipid phase transition. Studies have shown that when the water that hydrates the phospholipid molecules of the membrane is removed, packing of the head groups increases. The result is an increase in van der Waals interactions and a dramatic increase in the phase transition temperature (T m ) (Crowe et al., , 1988(Crowe et al., , 1990(Crowe et al., , 1991. At the phase transition the phospholipid bilayer shifts from a gel phase to a liquid crystalline phase, the state normally observed in fully hydrated cells. For example, the T m of a cell membrane might be -10 o C when fully hydrated but when water is removed the T m increases to over 100 o C. Thus, the membrane is in the gel phase at room temperature. As the membrane shifts between the gel phase and the liquid crystalline phase it becomes transiently leaky allowing its intracellular contents to leak out. Therefore it would be advantageous to avoid the lipid phase transition as this can compromise the health of a rehydrated cell. Addition of disaccharide sugars, in particular trehalose, depresses T m allowing the membrane to remain in the liquid crystalline state even when dried, so that upon rehydration no phase transition takes place and no transient leaking. During cryopreservation water is not necessarily lost, but it undergoes a phase change forming ice as the temperature drops and depending upon the rate of cooling, the cells become more or less dehydrated rendering the cells vulnerable to damage by mechanisms similar to those proposed for desiccated cells. The stabilizing effect of these sugars has been shown in a number of model systems including liposomes, membranes, viral particles, and proteins. The mechanism by which disaccharide sugars are able to decrease the T m for a given bilayer has been elucidated. Interactions take place between the sugars and the -OH groups of the phosphate in the phospholipid membrane preventing interaction or fusion of the head groups as the structural water is removed (Crowe et al., , 1988(Crowe et al., , 1989a(Crowe et al., , 1989b. Although not as well understood, a similar mechanism of action stabilizes proteins during drying (Carpenter et al., 1986(Carpenter et al., , 1987a(Carpenter et al., , 1987b(Carpenter et al., , 1989. Despite their protective qualities, the use of these sugars in mammalian cells has been somewhat limited mainly because mammalian cell membranes are impermeable to disaccharides or larger sugars and there is strong evidence that sugars need to be present on both sides of the cell membrane in order to be effective (Crowe et al., 2001;Eroglu et al., 2000;Beattie et al., 1997). This is why, in addition to loading sugars, we added sugars to the cryopreservation solution just before initiating cooling.
In addition to trehalose and sucrose, we were interested in other sugars that could be used as cryoprotectants avoiding monosacharides that would likely be degraded in the cell. Larger more complex sugars such as disaccharides or larger would be less likely to be degraded and utilized inside cells and might therefore be more stable as cryoprotectants.
The comparative structures of the sugars we considered for preservation of mammalian cells are illustrated in Figure 6. Three other sugars were evaluated besides trehalose and included sucrose, raffinose and stachyose. Sucrose and trehalose are both non-reducing sugars, so they do not react with amino acids or proteins and should be relatively stable under low pH conditions and at temperature extremes. Raffinose is a trisaccharide and stachyose is a tetrasaccharide.  , 2001). We have since used these conditions to cryopreserve several adherent cell types (Campbell et al., , 2010. Our rationale for using this adherent model was twofold. First, due to our interest in regenerative medicine we thought that adherent cells more closely mimicked cells in tissue engineered devices. Second, we thought there might be a market for cells cryopreserved on plates for research and cytotoxicity testing, CryoPlate™. More recently another group has been using adherent cells for investigation of preservation by vitrification and drying and have reported on cryopreservation of adherent pluripotent stem cells (Katkov et al., 2006;Katkov et al., 2011;). Katkov et al. presented results for preservation of human embryonic stem cells in 4-well plates and pointed out several advantages of cryopreservation in adherent mode. These included elimination of possible bias due to selective pressure within a pluripotent stem cell line after cryopreservation and distribution of multiwell plates for immediate use for embryotoxicity and drug screening in pluripotent stem cell-based toxicology in vitro kits (Katkov et al., 2011).
There are several methods in the literature that could be employed for intracellular delivery of these sugars including those already discussed ; Table 2). Many drugs, therapeutic proteins and small molecules have unfavorable pharmacokinetic properties and do not readily cross cell membranes or other natural physiological barriers within the body. This has resulted in the search for and discovery of alternative methods to transport materials, like sugars, across mammalian cell membranes.
Some of these strategies have been presented in depth in the results sections. The first involved the use of the Staphylococcus aureus toxin, -hemolysin. This toxin is produced as a monomer by the bacteria. It then oligomerizes to form pores on mammalian cell membranes. Hagan Bayley and his group modified the wild type -hemolysin protein by replacing 4 native residues with histidines, termed H5. In addition to pore formation on cell membranes, the H5 mutant also enabled it to be opened and closed at will. When inserted into the membrane, it is open and molecules up to 3000 daltons are able to pass through. Then the pores are closed in the presence of Zn + . To reopen the pore, addition of a chelating agent such as EDTA will remove the Zn + and the pore is ready to be used again (Bayley, 1994;Walker et al., 1995). Early studies showed that H5 could create pores in mammalian cell membranes and that they could be used for efficient intracellular loading of trehalose (Eroglu et al, 2000;Acker et al., 2003). Our experiments with H5 worked well initially using adherent cells. The results demonstrated good poration and loading of trehalose into cells. However, after adherent cells were cryopreserved, their viability was not very good (<6%). At this point in our studies, several issues arose that prevented further studies using H5. First, the H5 pore was derived from the bacterial toxin -hemolysin so there were concerns raised whether regulatory approval could be obtained if it was ever to be used clinically with human cells and tissues. There were some indications during these studies that the pores were shed from the membrane over time. However, H5 was still detectable in picogram quantities after 7 days in culture. Finally, as new batches of H5 were delivered the activity varied greatly and more H5 was required to achieve the same level of poration compared with earlier batches. Ultimately the batch variation was attributed to a protein stability issue. When these issues were not resolved other strategies for introducing trehalose into cells were explored.
An unexpected outcome of our H5 experiments was the development of a new, simple strategy for introduction of trehalose into cells which involved incubating cells in sugar for www.intechopen.com extended periods of time at physiological temperature . One possible mechanism to explain this observation was that the trehalose is substituting for water molecules in the cell membranes keeping the membrane stable and preventing it from going through a phase transition (Crowe et al., 1988. A second mechanism is most likely an active uptake mechanism involving endocytosis similar to that proposed for loading of trehalose by Oliver et al (Oliver et al., 2004). Her results suggested that human MSCs are capable of loading trehalose from the extracellular space by a clathrin-dependent fluidphase endocytotic mechanism that is microtubule-dependent but actin-independent (Oliver et al., 2004). Further research is required to elucidate the mechanism by which culture in the presence of trehalose facilitates cell cryopreservation and determine the degree of cell viability retention under different storage conditions.
The last method presented was poration using the P2 z receptor and ATP. This was a somewhat unique strategy in that it took advantage of the cell's own machinery. It was shown that cells expressing the P2 X7 purinergic cell surface receptor, also known as the P2 z receptor, could be permeabilized when the receptor binds to ATP 4-. The interaction with ATP resulted in the formation of a non-selective pore that allows molecules up to ~900 Daltons to pass through (Nihei et al., 2000). The P2 X7 receptor selectively binds to only ATP 4whose presence in solution is dependent on temperature, pH, and the concentration of divalent cations such as Mg 2+ . Closure of the pore after activation by ATP is achieved by simply removing ATP from the system or adding exogenous Mg 2+ that has a high affinity for the active form of ATP, ATP 4-. The P2 z receptor is found on a number of cell types including cells of hematopoietic origin (Nihei et al, 2000). There were several factors that likely affected the cell viability and survival of cells after ATP poration. First, is the density of the receptor on the cells which directly affects the amount of trehalose that can be loaded into the cells and how long it takes. Another factor is that poration with ATP tends to promote the detachment of adherent cells from their substrate. Part of the protocol requires a recovery period of 1 hour at 37 o C to allow cells that may have been perturbed by the poration process the chance to settle back onto their substrate. Finally, cell loss is at least in part due to apoptosis. There is evidence in the literature that poration with ATP induces apoptosis in some cell types (Murgia et al., 1992).
In marked contrast the human stem cell line, TF-1, demonstrated excellent post cryopreservation survival (Buchanan et al., 2004;. We have exposed TF-1 cells to ATP with trehalose for 1 hour followed by a 10-fold dilution of the ATP and inactivation of the active form of ATP (ATP 4-) by the addition of 1 mM MgCl 2 followed by a 1-hour recovery period at 37 o C . When the cells were compared to cells cryopreserved with 10% DMSO, the DMSO group demonstrated greater initial viability close to 100% that steadily declined over days in culture post thaw. However by day 4 of culture postcryopreservation cells cryopreserved in disaccharides were similar to the viability of cells cryopreserved in DMSO. Similarly colony forming assays with TF-1 cells demonstrated similar outcomes compared with DMSO. Furthermore, the use of disaccharides, trehalose and sucrose, appeared to result in similar results at both slow (1°C/min) and rapid (100°C/min) cooling rates. Buchanan et al (Buchanan et al., 2010) have extended these studies obtaining excellent TF-1 cell line and cord blood-derived multipotential hematopoietic progenitor cell survival after freeze drying and storage at room temperature for 4 weeks! It is studies such as Buchanan's that keep us optimistic that disaccharide introduction/preservation strategies can be developed for preservation of other mammalian www.intechopen.com cell types. Further development work is required with the cell culture and P2 x7 methods with the promise of preservation by freezing and freeze-drying.

H5
Derived from -hemolysin, which normally forms a constitutively opened pore in the membrane. Engineered to close in the presence of Zn+ or serum.
Derived from a bacterial toxin. Batch to batch variation and instability.
Acker Bayley, 1994Eroglu et al. 2000 ATP The naturally occurring p2 x7 receptor forms a nonspecific pore upon binding of ATP 4-able to allow molecules <900 daltons to pass through.
P2 x7 receptor found on some but not all cell types. Buchanan et al. 2005 Culture methods There are still other methods in the literature that could lead to intracellular delivery of disaccharides in addition to those already discussed  Table 2). One method takes advantage of the lipid phase transition described above when the cell membrane is exposed to changes in temperature. As the membrane changes from the liquid crystalline phase to the gel phase it becomes leaky providing an opportunity to introduce molecules into the cell that would not normally cross like trehalose. Beattie used this method to cryopreserve pancreatic islets by introducing DMSO and trehalose into the islets during the thermotropic phase transition between 5 and 9 o C. The islets were then cryopreserved in combination with DMSO and the viability of the islets after thawing was greater than when DMSO alone was used, 94% versus 58% (Beattie et al., 1997). In a related study, Mondal et al, cryopreserved kidney cells (MDBK) using 264 mM trehalose. The cells were suspended in trehalose with 20% fetal bovine serum in culture medium then incubated at 40 o C for 1 hour before slow rate cooling for storage at -80 o C. Viability was measured using Trypan Blue exclusion at 74% upon thawing (Mondal, 2009).
In another variation for loading molecules into cells, a number of proteins have been discovered that possess the ability to cross the cell membrane. These protein transduction domains (PTDs) generally correspond to portions of native proteins. Examples of PTDs include the Tat protein from the human immunodeficiency virus type I, the envelope glycoprotein E rns from the pestivirus and the DNA binding domains of leucine zipper proteins such as c-fos, c-jun and yeast transcription factor GCN4 (Futaki et al., 2001(Futaki et al., , 2004Langedijk, 2002;Langedijk et al., 2004;Lindgren et al., 2000;Richard et al., 2003;Vives et al., 1997). These PTDs are short cationic peptides that cross the cell membrane in a concentration-dependent manner that is independent of specific receptors or other transporters. The exact mechanism of translocation has not been defined. Enrichment of basic amino acids, particularly arginine and in some instances lysine, have been shown to be important for the translocation activity (Futaki et al., 2001(Futaki et al., , 2004Vives et al., 1997). Some studies have suggested that endocytosis is involved (Lundberg & Johansson, 2002;Richard et al., 2003), however, the current theory includes interaction with glycosaminoglycans and uptake by a non-endocytotic mechanism that may involve the charged heads of the phospholipid groups within the cell membrane. (Langedijk, 2002;Langedijk et al., 2004;Mai et al., 2002).
While most of these peptides need to be cross linked to the molecule of interest, there are peptides that can move proteins and other peptides across the membrane without the requirement for cross-linking. Examples include Pep-1, a 21-residue peptide which contains three domains; a tryptophan rich region (5 residues) for targeting the membrane and forming hydrophobic interactions; a lysine rich domain to improve intracellular delivery whose design was taken from other nuclear localization sequences from other proteins like the simian virus 40 large T antigen, and, a spacer region with proline that provides flexibility and maintenance of the other two regions. When mixed with other peptides or proteins, Pep-1 rapidly associates and forms a complex with the protein of interest by noncovalent hydrophobic interactions to form a stable complex. Once in the cytoplasm the peptide dissociates from the protein that has been carried across the membrane causing little if any interference regarding the protein's final destination or function. The process occurs by an endocytosis independent mechanism (Morris et al., 1999(Morris et al., , 2001. We anticipate that such peptides may eventually lead to methods for introduction of disaccharides into mammalian cells . Another alternative method is electroporation, also called electropermeabilization, which involves the application of an electric pulse that briefly permeabilizes the cell membrane. Since its introduction in the 1980's it has been primarily used to transfect mammalian cells and bacteria with genetic material. Initially electroporation tended to kill most cells. However, further work and development of the electroporation process, such as alternate electrical pulses like the square wave pulse, have refined the process so that better permeabilization and cell viability can be achieved (Gehl, 2003;Hapala, 1997;Heiser, 2000). The formation of pores, their size and the recovery of the membrane are important factors that influence the success of an electroporation protocol (Gehl, 2003;Hapala, 1997;Heiser, 2000). Most importantly, electroporation is applicable to all cell types.
It was hypothesized that trehalose provided protection during electropermeabilization in a manner similar to chelating agents such as EDTA or lipids like cholesterol (Katkov, 2002; www.intechopen.com Mussauer et al., 2001). Effective electroporation protocols are a balance between how much material can be loaded into the cells and cell survival after membrane permeabilization. So, while it cannot be predicted how well certain cell types will respond to electroporation, there is ample evidence that electroporation can be used with a reasonably certainty of success. A short culture period may be all that is required to permit restabilization of membranes post-electroporation. Additionally, like trehalose which interacts with membranes under stressful conditions such as drying, other compounds, such as cholesterol and unsaturated fatty acids, can also interact with membranes and may facilitate resealing of the membranes increasing overall cell survival (Katkov, 2002). Efficient resealing of cell membranes after permeabilization is thought to be essential for promoting cell recovery (Gehl et al., 1999) and compounds such as Poloxamer 188 facilitate membrane resealing (Lee et al., 1992).

Conclusion
In conclusion, there are multiple potential ways to introduce trehalose into mammalian cells and in some cases excellent cell preservation can be achieved. However, it is clear that methods for each cell type will need to be diligently developed and many years of work remain before we can replace DMSO as the lead cryoprotectant. In the mean time, we must not forget that there are other relatively low molecular weight sugars available. Preliminary evidence suggests that with further work sucrose and stachyose may, in some cases, be equally effective for cell preservation.

Acknowledgements
We would like to thank Elizabeth Greene for her assistance in the preparation of this manuscript. This work was supported by a cooperative agreement (No. 70NANB1H3008) between the U.S. Department of Commerce, National Institute of Standards and Technology-Advanced Technology Program, and Organ Recovery Systems, Inc www.intechopen.com