Mild heat shock at 40 °C increases levels of autophagy: Role of Nrf2.

The exposure to low doses of stress induces an adaptive survival response that involves the upregulation of cellular defense systems such as heat shock proteins (Hsps), anti-apoptosis proteins, and antioxidants. Exposure of cells to elevated, non-lethal temperatures (39-41 °C) is an adaptive survival response known as thermotolerance, which protects cells against subsequent lethal stress such as heat shock (>41.5 °C). However, the initiating factors in this adaptive survival response are not understood. This study aims to determine whether autophagy can be activated by heat shock at 40 °C and if this response is mediated by the transcription factor Nrf2. Thermotolerant cells, which were developed during 3 h at 40 °C, were resistant to caspase activation at 42 °C. Autophagy was activated when cells were heated from 5 to 60 min at 40 °C. Levels of acidic vesicular organelles (AVOs) and autophagy proteins Beclin-1, LC3-II/LC3-I, Atg7, Atg5, Atg12-Atg5, and p62 were increased. When Nrf2 was overexpressed or depleted in cells, levels of AVOs and autophagy proteins were higher in unstressed cells, compared to the wild type. Stress induced by mild heat shock at 40 °C further increased levels of most autophagy proteins in cells with overexpression or depletion of Nrf2. Colocalization of p62 and Keap1 occurred. When Nrf2 levels are low, activation of autophagy would likely compensate as a defense mechanism to protect cells against stress. An improved understanding of autophagy in the context of cellular responses to physiological heat shock could be useful for cancer treatment by hyperthermia and the protective role of adaptive responses against environmental stresses.

Canadian medical student knowledge and attitudes toward female genital mutilation.

Background: Despite female genital mutilation/cutting (FGM/C) practices being an illegal form of gender-based violence in Canada, this practice impacts many Canadians. Lack of education and training among Canadian health-care providers has resulted in systematic barriers to care. Awareness and FGM/C-related education among Canadian health-care providers must be urgently assessed. Methods: Canadian medical students were recruited to complete an anonymous survey via E-mails distributed through their schools' student organization between January and March 2021. We evaluated student understanding of FGM/C, attitudes toward medicalization and legislation, and prior clinical experience using multiple choice, Likert scale, and open-response questions. Results: Respondents (n = 135) performed poorly on knowledge assessment questions (mean percent correct <50%). Only 10.4% of respondents indicated knowing how to involve appropriate authorities when necessary, and most never evaluate FGM/C in patient history (86.7%) or clinical examination (57.1%). Subgroup analysis revealed that prior education significantly improved knowledge scores and influenced students' behaviors and attitudes. About 92.2% of respondents supported the integration of FGM/C curricula in undergraduate medical education. Discussion: This study reveals that Canadian medical students have a poor understanding of FGM/C and are not prepared to identify affected patients or intervene when necessary. These results provide rationale for the implementation of FGM/C-learning modules in undergraduate medicine.

Transcriptional heterogeneity of stemness phenotypes in the ovarian epithelium.

The ovarian surface epithelium (OSE) is a monolayer of epithelial cells surrounding the ovary that ruptures during each ovulation to allow release of the oocyte. This wound is quickly repaired, but mechanisms promoting repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, but their involvement in OSE is unclear. We show that traits associated with stem cells can be increased following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. We find that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not be associated with a single, distinct population, but rather is a heterogeneous cell state that may emerge from diverse environmental cues. These findings support that the OSE may not require distinct stem cells for long-term maintenance, and may instead achieve this through transient dedifferentiation into a stem-like state.

Heat shock increases levels of reactive oxygen species, autophagy and apoptosis.

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39-41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1-3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.

Control of stress-induced apoptosis by freezing tolerance-associated wheat proteins during cryopreservation of rat hepatocytes.

Cryopreservation is used for long-term storage of cells and tissues. Cryoprotectants such as dimethyl disulfoxide (DMSO) are used to protect cells against freeze-thaw damage. Despite the use of cryoprotectants, hepatocytes are sensitive to stresses imposed by freeze and thaw processes, which cause physical damage, loss of functionality, or cell death. As an alternative, we have developed new technology using several recombinant wheat proteins as cryoprotectants: TaENO (enolase), TaBAS1 (2-Cys peroxiredoxin), and a combination of WCS120 (dehydrin) with TaIRI-2 (inhibitor of ice recrystallization). This study aims to understand the mechanisms by which these plant proteins protect rat hepatocytes against cell death incurred during cryopreservation. Our analysis revealed that for cells cryopreserved with DMSO, cell death occurred by apoptosis and necrosis. Apoptosis was detected by activation of effector caspases-3 and -7, PARP cleavage, and nuclear chromatin condensation. These apoptotic events were inhibited when hepatocytes were cryopreserved with the different plant proteins. Cryopreservation with DMSO activated apoptosis through the mitochondrial pathway: the Bax/Bcl-2 protein ratio increased, mitochondrial membrane potential decreased, and initiator caspase-9 was activated. Furthermore, the endoplasmic reticulum pathway of apoptosis was activated: levels of the chaperone Bip/GRP78 decreased, pro-apoptotic transcription factor CHOP was induced, and initiator caspase-12 was activated. Activation of the mitochondrial and endoplasmic reticulum pathways of apoptosis was attenuated when hepatocytes were cryopreserved with the different recombinant proteins. This study improves understanding of mechanisms of cryoprotection provided by these plant proteins during freezing stress. These proteins are natural products and show promising potential by decreasing cell death during cryopreservation of hepatocytes.

Heat shock induces the cellular antioxidant defenses peroxiredoxin, glutathione and glucose 6-phosphate dehydrogenase through Nrf2.

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and/or chemotherapy. Heat (42-45 °C) can kill cancer cells. Low doses of heat at milder temperatures (39-41 °C) induce thermotolerance, an adaptive survival response that upregulates defense molecules to protect cells against subsequent exposure to toxic stress. Although hyperthermia has proven effective in clinical trials, there is still much to learn about its cellular mechanisms. This study aims to understand the role of reactive oxygen species (ROS), antioxidants and the antioxidant transcription factor Nrf2 in cellular stress responses to mild and lethal heat shock. Mild thermotolerance (40 °C) and hyperthermia (42-43 °C) caused increased expression of the antioxidants peroxiredoxin-3 (Prx3) and Prx2, and its hyperoxidized form Prx-SO3. Cellular levels of superoxide and peroxides increased at 40 °C and 42 °C. Heat shock (42 °C)-induced increases in Prx3 and Prx-SO3 were inhibited by antioxidants (PEG-catalase, MnTBAP) and a Nrf2 shRNA. Glucose metabolism by the pentose phosphate pathway produces NADPH, which maintains the antioxidant glutathione in its reduced form, GSH. Heat shock (40°C-42 °C) increased GSH levels, expression of glucose transporter GLUT1, and enzymatic activity and expression of glucose 6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose cycle. Heat-induced increases in GSH levels and G6PD expression were inhibited by antioxidants and Nrf2 knockdown. These results suggest that heat shock-generated ROS were involved in induction of cellular defense molecules Prxs, GSH and G6PD through Nrf2 activation. Our study sheds new light on the role of Nrf2 and antioxidants in cellular responses to heat shock at mild and lethal temperatures.

Inhibition of autophagy sensitises cells to hydrogen peroxide-induced apoptosis: Protective effect of mild thermotolerance acquired at 40°C.

Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threatens cellular homeostasis. Yet, lower doses of stress can stimulate defence systems allowing cell survival, whereas intense stress activates cell death pathways such as apoptosis. Mild thermal stress (40°C, 3h) induces thermotolerance, an adaptive survival response that renders cells less sensitive to subsequent toxic stress, by activating defence systems like heat shock proteins, antioxidants, anti-apoptotic and ER-stress factors. This study aims to understand how autophagy and apoptosis are regulated in response to different doses of H2O2, and whether mild thermotolerance can protect cervical carcinoma cells against apoptosis by stimulating autophagy. Autophagy was monitored through Beclin-1 and LC3 expression and acid compartment activity, whereas apoptosis was tracked by caspase activity and chromatin condensation. Exposure of HeLa and C33 A cells to H2O2 for shorter times (15-30min) transiently induced autophagy; apoptosis was activated after longer times (1-3h). Mild thermotolerance at 40°C enhanced activation of autophagy by H2O2. Disruption of autophagy using bafilomycin A1 and 3-methyladenine sensitised cells to apoptosis induced by H2O2, in non-thermotolerant cells and, to a lesser extent, in thermotolerant cells. Inhibition of autophagy enhanced apoptosis through the mitochondrial, death receptor and endoplasmic reticulum pathways. Autophagy was activated by lower doses of stress and protects cells against apoptosis induced by higher doses of H2O2. This work improves understanding of mechanisms that might be involved in toxicity of various compounds and could eventually lead to protective strategies against deleterious effects of toxic compounds.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

Plant protein 2-Cys peroxiredoxin TaBAS1 alleviates oxidative and nitrosative stresses incurred during cryopreservation of mammalian cells.

There is increasing demand for cryopreserved cells such as liver and pancreatic cells for clinical applications. Cryopreservation at ultra-low temperatures requires use of cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) to maintain cell integrity during freezing and thawing processes. Standard cryoprotectants are cytotoxic and more effective cryopreservation technologies are urgently needed for long-term storage of cells. As an alternative, soluble protein extracts (WPE) from winter wheat successfully replaced DMSO as a cryoprotectant for several mammalian cell types. To identify novel cryoactive proteins, the WPE was separated by chromatography and cryoactive fractions were analyzed by mass spectrometry. The wheat protein 2-Cys peroxiredoxin BAS1 (renamed TaBAS1) was identified as a potential cryoactive candidate. Recombinant proteins were prepared and found to possess dual functions as a peroxidase antioxidant and molecular chaperone, and display cryoprotective properties for hepatocytes and insulin-secreting INS832/13 cells. Following cryopreservation with TaBAS1, cells were plateable and showed high post-thaw viability, good adhesion properties, and well-maintained cell-specific metabolic functions. The overall quality of these cell types was equivalent or improved compared to cells that were cryopreserved with DMSO. The antioxidant and chaperone functions of TaBAS1 likely explain its efficacy in reducing oxidative/nitrosative stresses in cryopreserved cells. The plant protein TaBAS1 could be a promising molecule to include in cryostorage protocols. Biotechnol. Bioeng. 2016;113: 1511-1521. © 2015 Wiley Periodicals, Inc.

Wheat enolase demonstrates potential as a non-toxic cryopreservation agent for liver and pancreatic cells.

Cryopreservation is essential for long-term storage of cells and tissues, which can be used for clinical applications such as drug toxicity testing, human transplantation, reproductive, regenerative and transfusion medicine. It requires use of cryoprotectants (e.g. dimethyl disulfoxide (DMSO), glycerol) that protect cells and tissues from dehydration and damage caused by formation of intracellular ice during freezing. As an alternative to these cytotoxic cryoprotectants, we are developing new technology using natural substances produced by plants that survive freezing conditions. We previously showed that soluble protein extracts such as wheat protein extract (WPE) prepared from winter wheat plants can substitute for DMSO as a cryoprotectant for certain mammalian cell types. To identify novel cryoactive proteins, WPE was separated using different chromatographic procedures and cryoactive fractions were analyzed by mass spectrometry. The analysis revealed enolase as a potential wheat protein candidate. A recombinant enolase protein was prepared and was able to successfully cryopreserve rat hepatocytes and insulin-secreting INS832/13 pancreatic cells. Post-thaw cells had high viability and levels of metabolic activities. Cryopreserved cells were plateable and had good adherence and morphological properties. These results indicate that individual plant proteins such as enolase have promising potential as new, non-toxic technology for cryopreservation protocols used for clinical applications.

Winter wheat hull (husk) is a valuable source for tricin, a potential selective cytotoxic agent.

The flavone, tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) has great potential as an anticancer agent, due to its specific chemopreventive activity. In spite of these characteristics, its use in preclinical studies is still limited, mainly because of its limited availability and high production cost. Tricin is found mainly in cereal grains, such as wheat, rice, barley, oat and maize. However, its concentration in these plants is not sufficient for commercial use. To find a reliable, rich source of tricin, we investigated its distribution in different parts of wheat (Triticum aestivum) and designed an efficient method for its isolation and purification. The highest amount (770 ± 157 mg/kg dry weight) was found in the husks of winter wheat. This concentration is one of the highest in any plant species and is considered as a cheap source of natural tricin. The purified wheat husks tricin was found to be a selective potent inhibitor of two cancer cell lines of liver and pancreas, while having no side effects on normal cells. This selective action suggests that tricin could be considered as a potential candidate for pre-clinical trials as a chemopreventive agent. In addition, fibre-rich crude wheat husk could be used as a natural chemopreventive agent in food supplement.

Cryopreservation of insulin-secreting INS832/13 cells using a wheat protein formulation.

Diabetes is a global epidemic that affects about 285million people worldwide. For severely-ill patients with type I diabetes, whole pancreas or islet transplantation is the only therapeutic option. Islet transplantation is hindered by the scarce supply of fresh functional islets and limitations in cryopreservation procedures. Thus, improved cryopreservation procedures are needed to increase the availability of functional islets for clinical applications. Towards this goal, this work developed a cryopreservation protocol for pancreatic cells using proteins that accumulate naturally in freezing-tolerant plants. A preincubation of cells with 1% lecithin-1% glycerol-1% N-methylpyrrolidone followed by cryopreservation with partially purified proteins from wheat improved the viability and insulin-secreting properties of INS832/13 cells, compared to cryopreservation with 10% dimethyl sulfoxide (Me2SO). The major factor that enhanced the cryoprotective effect of the wheat protein formulation was preincubation with the lipid lecithin. Expression profiles of genes involved in metabolic and signaling functions of pancreatic cells (Ins, Glut1/2/3, Pdx1, Reg1α) were similar between fresh cells and those cryopreserved with the plant protein formulation. This novel plant-based technology, which is non-toxic and contains no animal material, is a promising alternative to Me2SO for cryopreservation of insulin-secreting pancreatic cells.

Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good expression of these adhesion molecules. These promising results could lead to a new and improved cryopreservation technology for applications such as clinical transplantation of hepatocytes.

Wheat proteins improve cryopreservation of rat hepatocytes.

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze-thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance-associated proteins such as WCS120, TaTIL, WCS19, and TaIRI-2. These improved procedures enhance long-term storage (2-12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77-93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non-toxic, natural products that do not require animal serum, and are economical and easy to prepare.

Metabolic activity of cytochrome p450 isoforms in hepatocytes cryopreserved with wheat protein extract.

The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 (P450) isoforms, drug-drug interactions, and establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethyl sulfoxide (DMSO). However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. Therefore, it is necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major P450 isoforms both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2, and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively show that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical P450 inducers when compared with freshly isolated hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for rat hepatocytes. They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity.

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria.

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12. Caspase-12 is localized to the cytoplasmic side of the endoplasmic reticulum and is involved in apoptosis mediated by the endoplasmic reticulum. Tri-n-butyltin also caused translocation of the pro-apoptotic proteins Bax and Bad from the cytosol to mitochondria, as well as changes in mitochondrial membrane permeability, events which can activate the mitochondrial death pathway. Tri-n-butyltin induced downstream apoptotic events in rat hepatocytes at the nuclear level, detected by chromatin condensation and by confocal microscopy using acridine orange. We investigated whether the tri-n-butyltin-induced pro-apoptotic events in hepatocytes could be linked to perturbation of intracellular calcium homeostasis, using confocal microscopy. Tri-n-butyltin caused changes in intracellular calcium distribution, which were similar to those induced by thapsigargin. Calcium was released from a subcellular compartment, which is likely to be the endoplasmic reticulum, into the cytosol. Cytosolic acidification, which is known to trigger apoptosis, also occurred and involved the Cl(-)/HCO(3)(-) exchanger. Pro-apoptotic events in hepatocytes were inhibited by the calcium chelator, Bapta-AM, and by a calpain inhibitor, which suggests that changes in intracellular calcium homeostasis are involved in tri-n-butyltin-induced apoptotic signaling in rat hepatocytes.

Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes.

Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent.