Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2002
Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that ... more Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. The objective of this study was to examine the effect that the composition of intra- and extracellular trehalose solutions has on the survival of dried 3T3 fibroblasts. Trehalose was introduced into 3T3 fibroblasts using a genetically-engineered
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tol... more Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.
Circulating tumor cells (CTCs) are known to be a harbinger of cancer metastasis. The CTCs are kno... more Circulating tumor cells (CTCs) are known to be a harbinger of cancer metastasis. The CTCs are known to circulate as individual cells or as a group of interconnected cells called CTC clusters. Since both single CTCs and CTC clusters have been detected in venous blood samples of cancer patients, they needed to traverse at least one capillary bed when crossing from arterial to venous circulation. The diameter of a typical capillary is about 7µm, whereas the size of an individual CTC or CTC clusters can be greater than 20µm and thus size exclusion is believed to be an important factor in the capillary arrest of CTCs - a key early event in metastasis. To examine the biophysical conditions needed for capillary arrest, we have developed a custom-built viscoelastic solid-fluid 3D computational model that enables us to calculate, under physiological conditions, the maximal CTC diameter that will pass through the capillary. We show that large CTCs and CTC clusters can successfully cross capillaries if their stiffness is relatively small. Specifically, under physiological conditions, a 13µm diameter CTC passes through a 7µm capillary only if its stiffness is less than 500Pa and conversely, for a stiffness of 10Pa the maximal passing diameter can be as high as 140µm, such as for a cluster of CTCs. By exploring the parameter space, a relationship between the capillary blood pressure gradient and the CTC mechanical properties (size and stiffness) was determined. The presented computational platform and the resulting pressure-size-stiffness relationship can be employed as a tool to help study the biomechanical conditions needed for capillary arrest of CTCs and CTC clusters, provide predictive capabilities in disease progression based on biophysical CTC parameters, and aid in the rational design of size-based CTC isolation technologies where CTCs can experience large deformations due to high pressure gradients.
Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cel... more Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.
Long-term preservation of live cells is critical for a broad range of clinical and research appli... more Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g. oocytes, stem and other primary cells, genetically modified cells), careful optimization of preservation protocols becomes tedious and poses significant limitations for all but the most expert users. To address the challenge of long-term storage of critical, heterogeneous cell types, we propose a universal protocol for cell vitrification that is independent of cell phenotype and uses only low concentrations of cryoprotectant (1.5 M PROH and 0.5 M trehalose). We employed industrial grade microcapillaries made of highly conductive fused silica, which are commonly used for analytical chemistry applications. The minimal mass and thermal inertia of the microcapillaries enabled us to achieve ultrafast cooling rates up to 4,000 K/s. Using the same low, non-toxic concentration of cryoprotectant, we demonstrate high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines.
Abstract ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted repro... more Abstract ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted reproductive technology (ART) in humans and animals. However, conventional ICSI has proven to be difficult and inefficient, especially in mice. Fortunately, the application of piezo-electric technology to assist the injection process has made ICSI more effective and efficient in recent years. Despite this advance, the pipette tip is unstable in response to the force exerted by the piezoelectric pulse during injection. To some extent, this instability can be ...
In this study, a stable, differentiated culture format (primary hepatocytes cultured between two ... more In this study, a stable, differentiated culture format (primary hepatocytes cultured between two layers of collagen gel) was used to study the effect of the inducer 3,3',4,4'-tetrachlorobiphenyl (TCB) on the activity of cytochrome P4501Al. P450IAI (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After I4 days of induction with 10mh M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 + 46.8 pmol R/fig DNA/hr (3.6 + 0.9 nmol R/IO6 cells/hr) compared with 34.9 + 3.8 pmol R/pg DNA/hr for cells cultured on a single layer of gel. At a TCB level of IOms M, the P450IAI activity in the double-gel configuration peaked at 220.8 + 37.0 pmol R/pg DNA/hr. Cessation of 10e6 M TCB induction produced a decrease in activity to 25.8 + 4.1 pmol R/pg DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 k-1 I. I pmol R/pg DNA/hr within 6 hr. reaching a maximal value of 131.0 + 38.6 nmol R/u~ DNAihr within I2 hr. Since TCB is ranidlv metabolized bv hepatocytes, a continuous perfusion cul&re system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10-s or IO-'M TCB, resulted in activities significantly higher than those of cultures induced by daily application of induction medium. A mechanistic model of TCB-dependent induction of P4501Al was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IAl activity in static culture, and the steady-state activity level of perfusion cultures. This work describes procedures for exposing stable hepatocyte cultures to either continuous or declining levels of consumable inducers and for measuring the activity of cytochrome P450IAl in cultured hepatocytes by a non-invasive method.
Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but... more Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but express low levels of liver-specific functions. We hypothesized that the microenvironment of adult hepatocytes (AHEPs) may upregulate these functions. Primary FHEPs were seeded on top of collagen-sandwiched AHEPs directly or separated by a porous transwell membrane insert. In direct co-cultures, albumin (ALB) secretion, urea synthesis, and cytochrome P450 (CytP450) activity were all approximately 2 times as high as the sum of the corresponding monocultures. Using a transwell porous insert led to similar results, suggesting a major role for soluble factors. When AHEPs and FHEPs were separated after co-culture, they both initially showed significantly higher ALB secretion than control monocultures, whereas urea synthesis was significantly lower for the FHEPs only. Functions of previously co-cultured FHEPs normalized over the course of a week, but AHEP function remained high even after separation. In conclusion, co-culturing AHEPs with FHEPs increases expression of liver-specific functions in both cell types. The effect on FHEPs, but not AHEPs, was reversible. Unraveling the underlying mechanisms and optimizing this phenomenon will be useful in making fetal liver cells a potential cell source for hepatic tissue-engineering applications.
Glioblastoma (GBM) is a highly aggressive brain cancer characterized by local invasion and angiog... more Glioblastoma (GBM) is a highly aggressive brain cancer characterized by local invasion and angiogenic recruitment, yet metastatic dissemination is extremely rare. Here, we adapted a microfluidic device to deplete hematopoietic cells from blood specimens of patients with GBM, uncovering evidence of circulating brain tumor cells (CTC). Staining and scoring criteria for GBM CTCs were first established using orthotopic patient-derived xenografts (PDX), and then applied clinically: CTCs were identified in at least one blood specimen from 13 of 33 patients (39%; 26 of 87 samples). Single GBM CTCs isolated from both patients and mouse PDX models demonstrated enrichment for mesenchymal over neural differentiation markers compared with primary GBMs. Within primary GBMs, RNA in situ hybridization identified a subpopulation of highly migratory mesenchymal tumor cells, and in a rare patient with disseminated GBM, systemic lesions were exclusively mesenchymal. Thus, a mesenchymal subset of GBM c...
Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interacti... more Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumorstromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumor-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumor-stromal interfacial interactions using ER+
During cancer progression, malignant cells in the tumour invade surrounding tissues. This transfo... more During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial-mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells. Individual cells with few neighbours dispersed with fast, straight trajectories, whereas cells that encountered many neighbours migrated collectively with epithelial biomarkers. We modelled these emergent dynamics using a physical analogy to phase transitions during binary-mixture solidification, and validated it using drug perturbations, which revealed that individually migrating cells exhibit diminished chemosensitivity. Our measurements also indicate a degree of phenotypic plasticity as cells interconvert between individual and collective migration. The study of multicellular behaviours with single-cell resolution should enable further quantitative insights into heterogeneous tumour invasion.
Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying pa... more Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b+ cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques
The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular vari... more The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan® reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.
The nature of the extracellular matrix profoundly influences the morphology and function of epith... more The nature of the extracellular matrix profoundly influences the morphology and function of epithelial cells, such as hepatocytes, in tissue culture. For Engelbroth-Holm-Swarm (EHS) sarcoma-derived gels (eg, Matrigel), rat hepatocytes exhibit limited spreading, and within 1-2 days they ...
Page 1. TISSUE ENGINEERING Volume 3, Number 1, 1997 Mary Ann Liebert, Inc. Role of j81 Integrin D... more Page 1. TISSUE ENGINEERING Volume 3, Number 1, 1997 Mary Ann Liebert, Inc. Role of j81 Integrin Distribution in Morphology and Function of Collagen-Sandwiched Hepatocytes PRABHAS V. MOGHE, Ph.D.,* ROBERT ...
In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and a... more In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and approximately 50 ongoing pregnancies being reported. While the ability to freeze human embryos has become a standard of practice in assisted reproductive technologies, obtaining reliable techniques for oocyte cryopreservation has been more difficult. The unique properties of the mature oocyte, such as the meiotic stage with sensitive spindle structure as well as the large cell volume, are responsible for the limited success obtained to date. There have been two approaches to cryopreserving the oocyte: (i) slow freeze-rapid thaw, and (ii) vitrification protocols with rapid cooling-rapid warming. Both methods have incorporated sugars (sucrose) as a beneficial non-permeating extracellular cryoprotectant. Studies of organisms that survive extreme conditions of freezing/dehydration have demonstrated the ability to accumulate intracellular sugars to afford protection and survival. A novel technique using microinjection of sugars into the oocyte for cryopreservation has been developed as an alternative approach to external addition of sugars. Freezing the human oocyte has been a challenging goal; however, developing research and efforts will, in the near future, provide women with an important option for their reproductive health.
The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits... more The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000°C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l propane-1,2-diol and 0.5 mol/l trehalose and achieve survival rates of 92.5%. Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.
Proceedings of the National Academy of Sciences, 2010
Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It ... more Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANO-VA" and submitted to the Comprehensive R Archive Network at http://www.r-project.org/. It is also available for download at http://gluegrant1.stanford.edu/TANOVA/. factorial design | longitudinal data | analysis of variance T ime-course microarray experiments are able to capture the temporal profiles for thousands of genes simultaneously to provide information on the dynamic changes of gene expression. A popular approach for analyzing time-course microarray data is to use clustering methods to group genes with similar temporal patterns (1-4). Cluster analysis may find functionally related genes, but the biological significance of the resulting gene clusters is often unclear. This is especially so when the time-course experiments are performed under different conditions that are defined by a set of experimental factors. Such experiments are called factorial time-course experiments.
Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2002
Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that ... more Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. The objective of this study was to examine the effect that the composition of intra- and extracellular trehalose solutions has on the survival of dried 3T3 fibroblasts. Trehalose was introduced into 3T3 fibroblasts using a genetically-engineered
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tol... more Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.
Circulating tumor cells (CTCs) are known to be a harbinger of cancer metastasis. The CTCs are kno... more Circulating tumor cells (CTCs) are known to be a harbinger of cancer metastasis. The CTCs are known to circulate as individual cells or as a group of interconnected cells called CTC clusters. Since both single CTCs and CTC clusters have been detected in venous blood samples of cancer patients, they needed to traverse at least one capillary bed when crossing from arterial to venous circulation. The diameter of a typical capillary is about 7µm, whereas the size of an individual CTC or CTC clusters can be greater than 20µm and thus size exclusion is believed to be an important factor in the capillary arrest of CTCs - a key early event in metastasis. To examine the biophysical conditions needed for capillary arrest, we have developed a custom-built viscoelastic solid-fluid 3D computational model that enables us to calculate, under physiological conditions, the maximal CTC diameter that will pass through the capillary. We show that large CTCs and CTC clusters can successfully cross capillaries if their stiffness is relatively small. Specifically, under physiological conditions, a 13µm diameter CTC passes through a 7µm capillary only if its stiffness is less than 500Pa and conversely, for a stiffness of 10Pa the maximal passing diameter can be as high as 140µm, such as for a cluster of CTCs. By exploring the parameter space, a relationship between the capillary blood pressure gradient and the CTC mechanical properties (size and stiffness) was determined. The presented computational platform and the resulting pressure-size-stiffness relationship can be employed as a tool to help study the biomechanical conditions needed for capillary arrest of CTCs and CTC clusters, provide predictive capabilities in disease progression based on biophysical CTC parameters, and aid in the rational design of size-based CTC isolation technologies where CTCs can experience large deformations due to high pressure gradients.
Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cel... more Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low-shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30-40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.
Long-term preservation of live cells is critical for a broad range of clinical and research appli... more Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g. oocytes, stem and other primary cells, genetically modified cells), careful optimization of preservation protocols becomes tedious and poses significant limitations for all but the most expert users. To address the challenge of long-term storage of critical, heterogeneous cell types, we propose a universal protocol for cell vitrification that is independent of cell phenotype and uses only low concentrations of cryoprotectant (1.5 M PROH and 0.5 M trehalose). We employed industrial grade microcapillaries made of highly conductive fused silica, which are commonly used for analytical chemistry applications. The minimal mass and thermal inertia of the microcapillaries enabled us to achieve ultrafast cooling rates up to 4,000 K/s. Using the same low, non-toxic concentration of cryoprotectant, we demonstrate high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines.
Abstract ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted repro... more Abstract ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted reproductive technology (ART) in humans and animals. However, conventional ICSI has proven to be difficult and inefficient, especially in mice. Fortunately, the application of piezo-electric technology to assist the injection process has made ICSI more effective and efficient in recent years. Despite this advance, the pipette tip is unstable in response to the force exerted by the piezoelectric pulse during injection. To some extent, this instability can be ...
In this study, a stable, differentiated culture format (primary hepatocytes cultured between two ... more In this study, a stable, differentiated culture format (primary hepatocytes cultured between two layers of collagen gel) was used to study the effect of the inducer 3,3',4,4'-tetrachlorobiphenyl (TCB) on the activity of cytochrome P4501Al. P450IAI (ethoxyresorufin O-deethylase) enzymatic activity was measured using the rate of conversion of ethoxyresorufin (ER) to resorufin (R). After I4 days of induction with 10mh M TCB, hepatocytes in the double collagen gel configuration exhibited a maximum activity of 180.3 + 46.8 pmol R/fig DNA/hr (3.6 + 0.9 nmol R/IO6 cells/hr) compared with 34.9 + 3.8 pmol R/pg DNA/hr for cells cultured on a single layer of gel. At a TCB level of IOms M, the P450IAI activity in the double-gel configuration peaked at 220.8 + 37.0 pmol R/pg DNA/hr. Cessation of 10e6 M TCB induction produced a decrease in activity to 25.8 + 4.1 pmol R/pg DNA/hr within 4 hr. Subsequent re-application of the inducer caused an increase in activity to 76.5 k-1 I. I pmol R/pg DNA/hr within 6 hr. reaching a maximal value of 131.0 + 38.6 nmol R/u~ DNAihr within I2 hr. Since TCB is ranidlv metabolized bv hepatocytes, a continuous perfusion cul&re system was developed to examine the effect of exposure to a constant level of TCB. Continuous perfusion of the cells with 10-s or IO-'M TCB, resulted in activities significantly higher than those of cultures induced by daily application of induction medium. A mechanistic model of TCB-dependent induction of P4501Al was developed using kinetic parameters estimated from static culture data. The model accurately predicted cyclic variations in P450IAl activity in static culture, and the steady-state activity level of perfusion cultures. This work describes procedures for exposing stable hepatocyte cultures to either continuous or declining levels of consumable inducers and for measuring the activity of cytochrome P450IAl in cultured hepatocytes by a non-invasive method.
Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but... more Fetal hepatocytes (FHEPs) are a potential source of highly proliferative transplantable cells but express low levels of liver-specific functions. We hypothesized that the microenvironment of adult hepatocytes (AHEPs) may upregulate these functions. Primary FHEPs were seeded on top of collagen-sandwiched AHEPs directly or separated by a porous transwell membrane insert. In direct co-cultures, albumin (ALB) secretion, urea synthesis, and cytochrome P450 (CytP450) activity were all approximately 2 times as high as the sum of the corresponding monocultures. Using a transwell porous insert led to similar results, suggesting a major role for soluble factors. When AHEPs and FHEPs were separated after co-culture, they both initially showed significantly higher ALB secretion than control monocultures, whereas urea synthesis was significantly lower for the FHEPs only. Functions of previously co-cultured FHEPs normalized over the course of a week, but AHEP function remained high even after separation. In conclusion, co-culturing AHEPs with FHEPs increases expression of liver-specific functions in both cell types. The effect on FHEPs, but not AHEPs, was reversible. Unraveling the underlying mechanisms and optimizing this phenomenon will be useful in making fetal liver cells a potential cell source for hepatic tissue-engineering applications.
Glioblastoma (GBM) is a highly aggressive brain cancer characterized by local invasion and angiog... more Glioblastoma (GBM) is a highly aggressive brain cancer characterized by local invasion and angiogenic recruitment, yet metastatic dissemination is extremely rare. Here, we adapted a microfluidic device to deplete hematopoietic cells from blood specimens of patients with GBM, uncovering evidence of circulating brain tumor cells (CTC). Staining and scoring criteria for GBM CTCs were first established using orthotopic patient-derived xenografts (PDX), and then applied clinically: CTCs were identified in at least one blood specimen from 13 of 33 patients (39%; 26 of 87 samples). Single GBM CTCs isolated from both patients and mouse PDX models demonstrated enrichment for mesenchymal over neural differentiation markers compared with primary GBMs. Within primary GBMs, RNA in situ hybridization identified a subpopulation of highly migratory mesenchymal tumor cells, and in a rare patient with disseminated GBM, systemic lesions were exclusively mesenchymal. Thus, a mesenchymal subset of GBM c...
Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interacti... more Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumorstromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumor-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumor-stromal interfacial interactions using ER+
During cancer progression, malignant cells in the tumour invade surrounding tissues. This transfo... more During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial-mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells. Individual cells with few neighbours dispersed with fast, straight trajectories, whereas cells that encountered many neighbours migrated collectively with epithelial biomarkers. We modelled these emergent dynamics using a physical analogy to phase transitions during binary-mixture solidification, and validated it using drug perturbations, which revealed that individually migrating cells exhibit diminished chemosensitivity. Our measurements also indicate a degree of phenotypic plasticity as cells interconvert between individual and collective migration. The study of multicellular behaviours with single-cell resolution should enable further quantitative insights into heterogeneous tumour invasion.
Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying pa... more Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b+ cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques
The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular vari... more The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan® reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.
The nature of the extracellular matrix profoundly influences the morphology and function of epith... more The nature of the extracellular matrix profoundly influences the morphology and function of epithelial cells, such as hepatocytes, in tissue culture. For Engelbroth-Holm-Swarm (EHS) sarcoma-derived gels (eg, Matrigel), rat hepatocytes exhibit limited spreading, and within 1-2 days they ...
Page 1. TISSUE ENGINEERING Volume 3, Number 1, 1997 Mary Ann Liebert, Inc. Role of j81 Integrin D... more Page 1. TISSUE ENGINEERING Volume 3, Number 1, 1997 Mary Ann Liebert, Inc. Role of j81 Integrin Distribution in Morphology and Function of Collagen-Sandwiched Hepatocytes PRABHAS V. MOGHE, Ph.D.,* ROBERT ...
In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and a... more In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and approximately 50 ongoing pregnancies being reported. While the ability to freeze human embryos has become a standard of practice in assisted reproductive technologies, obtaining reliable techniques for oocyte cryopreservation has been more difficult. The unique properties of the mature oocyte, such as the meiotic stage with sensitive spindle structure as well as the large cell volume, are responsible for the limited success obtained to date. There have been two approaches to cryopreserving the oocyte: (i) slow freeze-rapid thaw, and (ii) vitrification protocols with rapid cooling-rapid warming. Both methods have incorporated sugars (sucrose) as a beneficial non-permeating extracellular cryoprotectant. Studies of organisms that survive extreme conditions of freezing/dehydration have demonstrated the ability to accumulate intracellular sugars to afford protection and survival. A novel technique using microinjection of sugars into the oocyte for cryopreservation has been developed as an alternative approach to external addition of sugars. Freezing the human oocyte has been a challenging goal; however, developing research and efforts will, in the near future, provide women with an important option for their reproductive health.
The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits... more The ideal cryopreservation protocol would combine the benefits of slow freezing with the benefits of vitrification. This report describes a method for the ultra-rapid vitrification of oocytes using slush nitrogen in quartz capillaries. The approach minimizes the thermal mass of the vitrification vessel by using open microcapillaries made of highly conductive quartz and achieves cooling rates of 250,000°C/min. The process of vitrification can be optimized by maximizing the rate at which the sample is cooled, which allows for the use of lower cryoprotectant concentrations. Mouse oocytes can be successfully vitrified using 1.5 mol/l propane-1,2-diol and 0.5 mol/l trehalose and achieve survival rates of 92.5%. Fertilization and blastocyst formation rates of vitrified-warmed and fresh oocytes were not significantly different. A total of 120 blastocysts from each of the vitrified-warmed and fresh oocytes were transferred to surrogate mothers and 23 and 27 offspring were born respectively. All offspring in both groups were healthy, grew and bred normally and gave rise to a second generation of pups. Thus, an ultra-rapid vitrification technique has been developed for mouse oocytes that uses low concentrations of cryoprotectants and slush nitrogen in quartz capillaries, which combines the benefits of slow freezing and vitrification.
Proceedings of the National Academy of Sciences, 2010
Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It ... more Time-course microarray experiments are capable of capturing dynamic gene expression profiles. It is important to study how these dynamic profiles depend on the multiple factors that characterize the experimental condition under which the time course is observed. Analytic methods are needed to simultaneously handle the time course and factorial structure in the data. We developed a method to evaluate factor effects by pooling information across the time course while accounting for multiple testing and nonnormality of the microarray data. The method effectively extracts gene-specific response features and models their dependency on the experimental factors. Both longitudinal and cross-sectional time-course data can be handled by our approach. The method was used to analyze the impact of age on the temporal gene response to burn injury in a large-scale clinical study. Our analysis reveals that 21% of the genes responsive to burn are age-specific, among which expressions of mitochondria and immunoglobulin genes are differentially perturbed in pediatric and adult patients by burn injury. These new findings in the body's response to burn injury between children and adults support further investigations of therapeutic options targeting specific age groups. The methodology proposed here has been implemented in R package "TANO-VA" and submitted to the Comprehensive R Archive Network at http://www.r-project.org/. It is also available for download at http://gluegrant1.stanford.edu/TANOVA/. factorial design | longitudinal data | analysis of variance T ime-course microarray experiments are able to capture the temporal profiles for thousands of genes simultaneously to provide information on the dynamic changes of gene expression. A popular approach for analyzing time-course microarray data is to use clustering methods to group genes with similar temporal patterns (1-4). Cluster analysis may find functionally related genes, but the biological significance of the resulting gene clusters is often unclear. This is especially so when the time-course experiments are performed under different conditions that are defined by a set of experimental factors. Such experiments are called factorial time-course experiments.
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Papers by Mehmet Toner