A Novel Scaffold-Based Hybrid Multicellular Model for Pancreatic Ductal Adenocarcinoma-Toward a Better Mimicry of the in vivo Tumor Microenvironment

With a very low survival rate, pancreatic ductal adenocarcinoma (PDAC) is a deadly disease. This has been primarily attributed to (i) its late diagnosis and (ii) its high resistance to current treatment methods. The latter specifically requires the development of robust, realistic in vitro models of PDAC, capable of accurately mimicking the in vivo tumor niche. Advancements in the field of tissue engineering (TE) have helped the development of such models for PDAC. Herein, we report for the first time a novel hybrid, polyurethane (PU) scaffold-based, long-term, multicellular (tri-culture) model of pancreatic cancer involving cancer cells, endothelial cells, and stellate cells. Recognizing the importance of ECM proteins for optimal growth of different cell types, the model consists of two different zones/compartments: an inner tumor compartment consisting of cancer cells [fibronectin (FN)-coated] and a surrounding stromal compartment consisting of stellate and endothelial cells [collagen I (COL)-coated]. Our developed novel hybrid, tri-culture model supports the proliferation of all different cell types for 35 days (5 weeks), which is the longest reported timeframe in vitro. Furthermore, the hybrid model showed extensive COL production by the cells, mimicking desmoplasia, one of PDAC's hallmark features. Fibril alignment of the stellate cells was observed, which attested to their activated state. All three cell types expressed various cell-specific markers within the scaffolds, throughout the culture period and showed cellular migration between the two zones of the hybrid scaffold. Our novel model has great potential as a low-cost tool for in vitro studies of PDAC, as well as for treatment screening

Chemotherapy Assessment in Advanced Multicellular 3D Models of Pancreatic Cancer: Unravelling the Importance of Spatiotemporal Mimicry of the Tumor Microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is a challenge for global health with very low survival rate and high therapeutic resistance. Hence, advanced preclinical models for treatment screening are of paramount importance. Herein, chemotherapeutic (gemcitabine) assessment on novel (polyurethane) scaffold-based spatially advanced 3D multicellular PDAC models is carried out. Through comprehensive image-based analysis at the protein level, and expression analysis at the mRNA level, the importance of stromal cells is confirmed, primarily activated stellate cells in the chemoresistance of PDAC cells within the models. Furthermore, it is demonstrated that, in addition to the presence of activated stellate cells, the spatial architecture of the scaffolds, i.e., segregation/compartmentalization of the cancer and stromal zones, affect the cellular evolution and is necessary for the development of chemoresistance. These results highlight that, further to multicellularity, mapping the tumor structure/architecture and zonal complexity in 3D cancer models is important for better mimicry of the in vivo therapeutic response

Role of growth morphology in the formulation of NaCl-based selective media for injury detection of Escherichia coli, Salmonella Typhimurium and Listeria innocua

Sublethal injury (SI) poses major public health concerns since injured cells are responsible for serious limitations in food diagnostics and are susceptible to recovery, often developing adaptive stress responses. Detection of SI is based on the difference in plate counts between non-selective media, which represent the total cell population, and selective media, to which injured cells become sensitive. Selective media for detection of sublethal membrane damage are often based on NaCl supplement, although there is a lack of consensus in the literature about appropriate levels. Planktonic cells are generally used to investigate SI mechanisms, although they often exhibit different stress tolerance than cell colonies in/on solid food (model) systems. In this work, the effect of growth morphology, colony size and concentration of the gelling agent in the growth media, on the maximum non-inhibitory NaCl concentration in the plating medium was assessed for Escherichia coli, Salmonella Typhimurium and Listeria innocua. Stationary phase cultures of planktonic cells and large and small colonies grown in either 1.5% (w/v) xanthan gum-based system or 2.5% (w/v) xanthan gum-based system exhibited significantly different viable counts and osmotolerance. The effect of cell arrangement and xanthan gum percentage in the growth media depended on the microorganism under investigation. Additionally, differences in the maximum non-inhibitory concentration were evident, with 5.0% (w/v) NaCl for the Gram-negative bacteria and 6.5% (w/v), for L. innocua. Different extent of colony shrinkage and morphological damage was observed as NaCl concentration in the plating medium increased. This information will contribute to define NaCl-based selective media for accurate SI detection under realistic scenarios. © 2014

Role of growth morphology in the formulation of NaCl-based selective media for injury detection of Escherichia coli, Salmonella Typhimurium and Listeria innocua

Sublethal injury (SI) poses major public health concerns since injured cells are responsible for serious limitations in food diagnostics and are susceptible to recovery, often developing adaptive stress responses. Detection of SI is based on the difference in plate counts between non-selective media, which represent the total cell population, and selective media, to which injured cells become sensitive. Selective media for detection of sublethal membrane damage are often based on NaCl supplement, although there is a lack of consensus in the literature about appropriate levels. Planktonic cells are generally used to investigate SI mechanisms, although they often exhibit different stress tolerance than cell colonies in/on solid food (model) systems. In this work, the effect of growth morphology, colony size and concentration of the gelling agent in the growth media, on the maximum non-inhibitory NaCl concentration in the plating medium was assessed for Escherichia coli, Salmonella Typhimurium and Listeria innocua. Stationary phase cultures of planktonic cells and large and small colonies grown in either 1.5% (w/v) xanthan gum-based system or 2.5% (w/v) xanthan gum-based system exhibited significantly different viable counts and osmotolerance. The effect of cell arrangement and xanthan gum percentage in the growth media depended on the microorganism under investigation. Additionally, differences in the maximum non-inhibitory concentration were evident, with 5.0% (w/v) NaCl for the Gram-negative bacteria and 6.5% (w/v), for L. innocua. Different extent of colony shrinkage and morphological damage was observed as NaCl concentration in the plating medium increased. This information will contribute to define NaCl-based selective media for accurate SI detection under realistic scenarios. © 2014

Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium

Aim: The effect of spent medium, obtained after different time-temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied. Methods and results: Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time-temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium - compared to the unmodified BHI broth - are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance. Conclusions: It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Significance and impact of study: This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry. © 2011

Heat inactivation of Escherichia coli K12 MG1655: effect of microbial metabolites and acids in spent medium

Aim: The effect of spent medium, obtained after different time-temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied. Methods and results: Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time-temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium - compared to the unmodified BHI broth - are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance. Conclusions: It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance. Significance and impact of study: This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry. © 2011