Mélanie Grondin

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.

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 (β1-integrin, E-cadherin, and β-catenin). Immunoblot analyses revealed that the levels of β1-integrin, E-cadherin, and β-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 β1-integrin (62–74%) > β-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 β1-integrin, E-cadherin, and β-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. Biotechnol. Bioeng. 2009;103: 582–591.

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.

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.

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.

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.