After isolation and related oxidative stress, the obtained cell population is re-suspended in cryopreservation media and distributed at specific concentrations in special freezing vials. The hepatocytes are then ready for the cooling process and storage in liquid nitrogen. In this chapter, we will review the literature data for liver cell freezing solution as well the documented cooling and thawing process.
Concentration of hepatocytes and type of vials
In most studies, the hepatocyte concentration varied from 106
]. In this range, Lloyd et al[14
] did not find any significant superiority of cell concentrations investigated, when evaluating porcine hepatocytes after thawing. This was confirmed by analyzing, hepatocyte attachment, lactate dehydrogenase (LDH) leakage, bilirubin conjugation, and CYP3A4 activity. However, De Loecker et al[20,21
] revealed that a decreased cell density of rat hepatocytes correlated with an increased post-thawing viability, as estimated by viability trypan blue exclusion assay. These data suggest that higher cell densities might increase membrane-membrane contacts and subsequent cell damage. Therefore, unless high cell density will save space and is useful for the development of cell banks, cryopreservation at a low cell density (less than 107
/cell) is recommended.
Besides cell density, type and volume of vials used for liquid nitrogen storage might also influence post-thawing cell quality; however, few data are available in the literature. Based on the trypan blue exclusion test, cytochrome activity, and tetrazolium inner salt assays, bags of 50 mL seem to give better quality pig hepatocytes, post-thawing, than bags of 100 mL[22
The lack of recently published data on the concentration of hepatocytes is considered as a minor point for reaching the best post-thawing quality.
We also confirm that, in our hands, in several different volumed vials (from 2 to 100 mL) and varying cell densities, cell density does not influence post-thawing cell quality.
University of Wisconsin solution (UW) is the gold standard cryopreservation medium for isolated hepatocytes. It was originally developed as a cold storage solution for transplant organs. The principal cryoprotectants of the UW solution are Lactobionate (100 mmol/L), a large molecular weight anion impermeable to most membranes and supposed to suppress hypothermia-induced cell swelling, and Raffinose (30 mmol/L), which allows additional osmotic support[23
]. Dexamethasone, another compound in UW solution, is used to stabilise cell membranes[24
The superior beneficial effect of UW solution was demonstrated by comparing UW to three other freezing media [all were supplemented with 12% dimethylsulfoxide (DMSO)], Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and commercial solutions, Cell Banker 1 and Cell Banker 2. Parameters including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer were shown to be highly maintained in hepatocytes cryopreserved with UW solution[25
The effectiveness of UW solution as a cryoprotectant agent suggests that metabolic, as well as ultrastructural, factors might be important in the effective cryopreservation of isolated hepatocytes[26
]. However, UW solution also has limitations, principally its cost and, as demonstrated in the “Parameters for the evaluation of hepatocytes quality after cryopreservation/thawing” paragraph, its incomplete cell protection.
HypoThermosol (HTS), a recently developed freezing solution, is a dextran-based intracellular-type solution. It is used as the carrier solution of the freezing medium. Freshly isolated rat hepatocytes cryopreserved in HTS supplemented with 10% DMSO have been shown to present high viability levels, good long-term maintenance of hepatospecific functions, and good quality response to cytokine challenge at the post-thawing level compared to other supplemented culture media[27
]. Further studies confirmed the utility of HTS, allowing a decrease in the DMSO levels within the cryopreservation solution. However, no data comparing UW to HTS are available in the literature[28,29
Cryoprotectants: Cryoprotectants are essential components of freezing solutions. Two classes of cryoprotectants are described, those that permeate the cell membrane (DMSO, Glycerol) and those that do not such as polymers (Dextran), oligosaccharides (Trehalose), and sugars (Glucose, sucrose or fructose).
DMSO is an important polar permeating aprotic solvent which is less toxic than other members of this class. The use of DMSO in medicine dates from around 1963, when an University of Oregon Medical School team discovered it could penetrate the skin and other membranes without damaging them and could carry other compounds into a biological system. It is able to enter cells and reduce injury by moderating the increase in solute concentration during freezing. In most studies, DMSO is the ideal cryoprotectant, notably giving the best plating efficiency[27,30-37
]. Classically, a final concentration of 10% DMSO is described in many protocols, with some exceptions, although higher levels are potentially toxic due to high osmolarity[38
]. The rate of addition of the cryoprotectant also appears important to the outcome of cryopreservation. Freezing must be commenced as soon as possible after addition of the cryoprotectant to reduce the possibility of toxicity at ambient temperatures. Hence, DMSO should be added at 4°C, as toxicity of DMSO was demonstrated at 25°C or 37°C. Some authors proposed adding permeating cryoprotectants slowly to the cell suspension to avoid damages related to osmotic shock and cellular dehydration[33,39
]. However, this seems to be a minor point[38
The use of oligosaccharides with higher molecular weights resulted in greatest improvement in viability. Their combination with DMSO has been shown to allow efficient hepatocyte cryopreservation. Both rat and human hepatocytes exhibit significantly higher viability (as estimated by trypan blue exclusion assay) than hepatocytes previously cryopreserved without oligosaccharides. Moreover, attachment and survival rates in plastic dishes of rat hepatocytes were greater after freezing in the presence of di-, tri-, and tetrasaccharides. Such plating amelioration was not confirmed with human hepatocytes[40
]. Metabolic activity was also evaluated after cryopreservation with oligosaccharides. When trehalose was combined with DMSO for the cryopreservation of human hepatocytes, a significant increase in total protein level and secretion of albumin was observed after thawing, as well as decreased levels of aspartate aminotransferase[41
Those works were inspired from data demonstrating the influence of trehalose on cell quality on bull sperm[42,43
]. Similarly, several authors recommended adding sucrose to the cryopreservation medium, with or without trehalose, to ameliorate the quality of the cells after thawing. This allows the concentration of DMSO to be decreased while ameliorating the quality of cells. However, it was evaluated on hematopoietic stem cells and fetal liver hematopoietic stem/progenitor cells[44,45
], not on hepatocytes.
The beneficial role of a non-metabolizable glucose derivative as a cryoprotectant that mimicked the natural cryoprotective adaptations observed in freeze-tolerant frogs was also investigated. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity and cryopreserved according to a controlled rate freezer program down to -80°C before storage in liquid nitrogen. In this study, hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (< 0.2 mol/L) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes, showing that 3OMG must be considered as an interesting cryoprotectant[46
An interesting report proposed that wheat protein extracts permitted long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions, via
an osmotic modulation effect[47
]. In post-thawing culture, the morphology of hepatocytes cryopreserved with wheat protein extracts was similar to that of fresh cells. Furthermore, hepatospecific functions, such as albumin secretion and biotransformation of ammonium to urea, were well maintained during four-days post-plating. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with wheat extracts were similar to those in fresh hepatocytes. Additional data confirmed the utility of wheat extracts as efficient, non-toxic, economic natural cryoprotectants, superior to DMSO, which has limitations due to potential cellular toxicity[47-49
Finally, human application does not tolerate the use of animal origin products because of possible zoonosis contamination and/or immune response to animal proteins[50
]. Fetal calf serum (FCS) or human albumin, are classical ingredients of the cryopreservation solution, in a proportion of 10% to 90 %. No significant differences in classical viability or drug metabolizing enzyme activities were noted while varying the percentage of serum for (human, pig, and rat) hepatocyte cryopreservation in most of the published studies[26,30,33,36,51-53
]. We think that a minimal concentration of serum is required for optimal cryopreservation, even if some authors have also successfully cryopreserved porcine hepatocytes without serum. They showed that, after thawing, in appropriate conditions and without serum, the addition of conditioned medium derived from hepatic non-parenchymal cells improved attachment and function of hepatocytes (urea production and CYP activity)[54
In conclusion, UW solution remains the best and most studied freezing medium and must be supplemented with a permeating cryoprotectant; DMSO remains the gold standard. The addition of a non-permeating cryoprotectant to this solution must also be considered.
Slow freezing protocols are considered to be the best strategy for cryopreserving mature isolated hepatocytes. All the protocols described in this paragraph were developed using DMSO as the cryoprotectant. First protocols included the use of an isopropranolol cooler device, placed in a -80°C freezer, giving a constant temperature decrease of -1°C/min down to -70°C or -80°C, before storing in liquid nitrogen[55
]. Other slow freezing protocols are described in the literature, varying from -1°C/min
to -5°C/min up to -40°C or -80°C, before storing at -196°C[56
]. A decrease in temperature at -1.9°C/min from 4 to -30°C and then -30°C/min from -30°C to -150°C was also adopted by many authors[51,57,58
]. More specific protocols were developed by Diener et al and by Hengstler et al[39,59,60
]. Several cooling process protocols, where the temperatures of the vial and of the cryopreserving solution were controlled, were tested on rat hepatocytes. Firstly, shock freezing in liquid nitrogen dramatically decreased cell viability, despite the presence of 10% DMSO. Secondly, a slow freezing protocol with -2°C/min led to much better recovered viability than a cooling rate of -38°C/min. While using the slow freezing protocol, the authors determined that the cell suspension becomes supercooled around -20°C. Indeed, when crystallisation starts, the latent heat of fusion is released and the cell sample is warmed. This heat release may be deleterious; therefore, a freezing program with shock cooling was developed. Analysis of post-thawing viability did not show significant differences of hepatocyte viability (86% viability vs
79% according to the slow linear protocol). The same cooling shock can be obtained by clamping the vials, with forceps cooled in liquid nitrogen[61
]. However, studies from Lloyd et al[14
] (measuring LDH release, cell return, attachment, and biochemical assays) and from our team[62
] did not show any difference between computer-controlled freeze rate (without frozen shock), the Nalgene propan-2-ol device or simply using -20°C and -80°C freezers.
Storage of hepatocytes at -20°C or -80°C remains deleterious for cells functions as several proteases might be active at those temperatures. At -130°C, no chemical reaction can occur as there is no more thermal energy. Furthermore, at this temperature, no water, which is at the vitreous or crystalline state, is present at the liquid state. Therefore, -140/-150°C is the minimum acceptable temperature for long-term storage of cryopreserved hepatocytes[6,34,57,63
]. At -140°C (the vapour phase of liquid nitrogen) or -196°C (the liquid phase of liquid nitrogen), cells can be stored for long periods[6,34,51,57
]. A summary of freeze rate comparison studies is presented in Table . The passage of water from one state to another, IIF, is the critical point that might modulate the cell quality. The limitations of these cooling processes will be discussed later in the IIF paragraph.
Summary of freeze-rate comparison studies