Biomedical advances from tissue culture

The demonstration that the “dedifferentiation” of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that cells in culture accurately represent cells in vivo but without the complex in vivo environment. This concept has been strengthened with the development of hormonally defined culture media in combination with functionally differentiated clonal cell lines, which have augmented the potential of tissue culture studies. The use of hormonally defined media in place of serum-supplemented media demonstrates that hormonal responses and dependencies can be discovered in culture. Discoveries of hormonal dependencies of cancer cells has led to therapies targeting intracellular signaling pathways while discoveries of hormonal responses of pluripotent cells are helping to identify the potential application of stem cells. In these and other ways tissue culture technology will continue to contribute to solving problems of human health.     

Tissue scaffold surface patterning for clinical applications

Patterned scaffold surfaces provide a platform for highly defined cellular interactions, and have recently taken precedence in tissue engineering. Despite advances in patterning techniques and improved tissue growth, no clinical studies have been conducted for implantation of patterned biomaterials. Four major clinical application fields where patterned materials hold great promise are antimicrobial surfaces, cardiac constructs, neurite outgrowth, and stem cell differentiation. Specific examples include applications of patterned materials to (i) counter infection by antibiotic resistant bacteria, (ii) establish proper alignment and contractile force of regrown cardiac cells for repairing tissue damaged by cardiac infarction, (iii) increase neurite outgrowth for central nervous system wound repair, and (iv) host differentiated stem cells while preventing reversion to a pluripotent state. Moreover, patterned materials offer unique advantages for artificial implants which other constructs cannot. For example, by inducing selective cell adhesion using topographical cues, patterned surfaces present cellular orientation signals that lead to functional tissue architectures. Mechanical stimuli such as modulus, tension, and material roughness are known to influence tissue growth, as are chemical stimuli for cell adhesion. Scaffold surface patterns allow for control of these mechanical and chemical factors. This review identifies research advances in scaffold surface patterning, in light of pressing clinical needs requiring organization of cellular interactions.     

Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.     

Neurons-on-a-Chip: In Vitro NeuroTools

Neurons-on-a-Chip technology has been developed to provide diverse in vitro neuro-tools to study neuritogenesis, synaptogensis, axon guidance, and network dynamics. The two core enabling technologies are soft-lithography and microelectrode array technology. Soft lithography technology made it possible to fabricate microstamps and microfluidic channel devices with a simple replica molding method in a biological laboratory and innovatively reduced the turn-around time from assay design to chip fabrication, facilitating various experimental designs. To control nerve cell behaviors at the single cell level via chemical cues, surface biofunctionalization methods and micropatterning techniques were developed. Microelectrode chip technology, which provides a functional readout by measuring the electrophysiological signals from individual neurons, has become a popular platform to investigate neural information processing in networks. Due to these key advances, it is possible to study the relationship between the network structure and functions, and they have opened a new era of neurobiology and will become standard tools in the near future.     

Cell patterning using magnetite nanoparticles and magnetic force

Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithography have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the number of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodology, which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.     

Improving the integrity of three-dimensional vascular patterns by poly(ethylene glycol) conjugation

Development of functional tissue-engineering constructs may require that multiple cell types be organized in controlled three-dimensional (3-D) microarchitectures with proper nutrient diffusion and vascularization. In the past few years, a variety of microscale techniques have demonstrated the ability to control protein and cell attachment in defined patterns. Nevertheless, maintenance of these patterns over time has been a significant challenge due to nonspecific protein adsorption and cell migration. To this end, we have investigated the effectiveness of poly(ethylene glycol) (PEG) thin films in maintaining the integrity of 3-D cellular patterns, using human umbilical vein endothelial cells (HUVEC) as a model system. These HUVEC constructs were created using extracellular matrix (ECM)-based microfluidic patterning. Our results indicated that PEG-conjugated substrates improve cell pattern integrity as compared to control silicon. The compliance multifactor (a measure of pattern integrity; higher value means lower pattern integrity) was about 3.66 +/- 0.29 on day 5 for PEG-conjugated surfaces, compared with 8.23 +/- 0.42 for control surfaces ECM-based microfluidic patterning coupled with stable PEG-conjugated surfaces may serve as a vital tool for vascularized tissue engineering.     

Nanotopographical control of human osteoprogenitor differentiation

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.     

Activity of mesenchymal stem cells in therapies for chronic skin wound healing

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care, particularly as the number of patients increases while technology aimed at stimulating wound healing in these cases remains inefficient. Mesenchymal stem cells (MSCs) have proved to be an attractive cell type for various cell therapies due to their ability to differentiate into various cell lineages, multiple donor tissue types, and relative resilience in ex-vivo expansion, as well as immunomodulatory effects during transplants. More recently, these cells have been targeted for use in strategies to improve chronic wound healing in patients with diabetic ulcers or other stasis wounds. Here, we outline several mechanisms by which MSCs can improve healing outcomes in these cases, including reducing tissue inflammation, inducing angiogenesis in the wound bed, and reducing scarring following the repair process. Approaches to extend MSC life span in implant sites are also examined.     

Cell-specific mechanisms and systemic signalling as emerging themes in light acclimation of C3 plants

Chloroplasts perform essential signalling functions in light acclimation and various stress responses in plants. Research on chloroplast signalling has provided fundamental information concerning the diversity of cellular responses to changing environmental conditions. Evidence has also accumulated indicating that different cell types possess specialized roles in regulation of leaf development and stress acclimation when challenged by environmental cues. Leaf veins are flanked by a layer of elongated chloroplast-containing bundle sheath cells, which due to their central position hold the potential to control the flux of information inside the leaves. Indeed, a specific role for bundle sheath cells in plant acclimation to various light regimes is currently emerging. Moreover, perception of light stress initiates systemic signals that spread through the vasculature to confer stress resistance in non-exposed parts of the plant. Such long-distance signalling functions are related to unique characteristics of reactive oxygen species and their detoxification in bundle sheath cells. Novel techniques for analysis of distinct tissue types, together with Arabidopsis thaliana mutants with vasculature-specific phenotypes, have proven instrumental in dissection of structural hierarchy among regulatory processes in leaves. This review emphasizes the current knowledge concerning the role of vascular bundle sheath cells in light-dependent acclimation processes of C3 plants.     

Bioreactors in tissue engineering—principles,applications and commercial constraints

Bioreactor technology is vital for tissue engineering. Usually, bioreactors are used to provide a tissue-specific physiological in vitro environment during tissue maturation. In addition to this most obvious application, bioreactors have the potential to improve the efficiency of the overall tissue-engineering concept. To date, a variety of bioreactor systems for tissue-specific applications have been developed. Of these, some systems are already commercially available. With bioreactor technology, various functional tissues of different types were generated and cultured in vitro. Nevertheless, these efforts and achievements alone have not yet led to many clinically successful tissue-engineered implants. We review possible applications for bioreactor systems within a tissue-engineering process and present basic principles and requirements for bioreactor development. Moreover, the use of bioreactor systems for the expansion of clinically relevant cell types is addressed. In contrast to cell expansion, for the generation of functional three-dimensional tissue equivalents, additional physical cues must be provided. Therefore, bioreactors for musculoskeletal tissue engineering are discussed. Finally, bioreactor technology is reviewed in the context of commercial constraints.     

Immunology

It has been known for the past 85 years that mucosal responses can be stimulated by local presentation of antigen and that the gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless dietary proteins. How these types of responses are induced and regulated remains unclear. In the gut attention has for some time been focused on Peyer's patches (PP) due to evidence that they play a vital role in the induction of humoral and cellular responses. Moreover, it has been established that MHC class II molecules are found in the gut mucosa on a variety of cell types outside PP, namely the lamina propria (LP). Fed antigens have also been detected in the LP and studies have shown that LP cells can stimulate allogeneic mixed lymphocyte responses and are capable of presenting soluble protein antigen to naïve T cells. This article reviews the present understanding of the possible roles of PP and LP in intestinal immunity in terms of induction, regulation, surveillance of immune responses and the antigen presenting cell types involved.     

Dynamic imaging of the immune system: progress, pitfalls and promise

Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.     

Development and applications of a model for cellular response to multiple chemotactic cues

The chemotactic response of a cell population to a single chemical species has been characterized experimentally for many cell types and has been extensively studied from a theoretical standpoint. However, cells frequently have multiple receptor types and can detect and respond chemotactically to more than one chemical. How these signals are integrated within the cell is not known, and we therefore adopt a macroscopic phenomenological approach to this problem. In this paper we derive and analyze chemotactic models based on partial differential (chemotaxis) equations for cell movement in response to multiple chemotactic cues. Our derivation generalizes the approach of Othmer and Stevens [29], who have recently developed a modeling framework for studying different chemotactic responses to a single chemical species. The importance of such a generalization is illustrated by the effect of multiple chemical cues on the chemotactic sensitivity and the spatial pattern of cell densities in several examples. We demonstrate that the model can generate the complex patterns observed on the skin of certain animal species and we indicate how the chemotactic response can be viewed as a form of positional indicator. Received: 15 February 1999 / Revised version: 1 February 2000 / Published online: 14 September 2000     

Responsive systems for cell sheet detachment

Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.     

How Do Medical Students Respond to Emotional Cues and Concerns Expressed by Simulated Patients during OSCE Consultations? – A Multilevel Study

Objectives

How medical students handle negative emotions expressed by simulated patients during Objective Structured Clinical Examinations (OSCE) has not been fully investigated. We aim to explore (i) whether medical students respond differently to different types of patients’ emotional cues; and (2) possible effects of patients’ progressive disclosure of emotional cues on students’ responses.

Methods

Forty OSCE consultations were video recorded and coded for patients’ expressions of emotional distress and students’ responses using a validated behavioural coding scheme (the Verona Coding Definitions of Emotional Sequence). Logistic multilevel regression was adopted to model the probability of the occurrence of student reduce space response behaviour as a function of the number of patients’ expressions of emotional cues.

Results

We found that medical students offered responses that differed to emotional cue types expressed by simulated patients. Students appeared to provide space to emotional cues when expressed in vague and unspecific words and reduce space to cues emphasizing physiological or cognitive correlates. We also found that medical students were less likely to explore patients’ emotional distress nearer the end of the consultation and when the duration of a patient speech turn got larger. Cumulative frequency of patients’ emotional cues also predicted students’ reduce space behaviour.

Practical Implications

Understanding how medical students manage negative emotions has significant implications for training programme development focusing on emotion recognition skills and patient-centred communication approach. In addition, the statistical approaches adopted by this study will encourage researchers in healthcare communication to search for appropriate analytical techniques to test theoretical propositions.     

Tissue culture: the unlimited potential

Lack of tissue-specific differentiated functions of cells in tissue culture, once thought to be due to “dedifferentiation”, was shown to be due to selective overgrowth of fibroblasts by a series of simple experiments that challenged the prevailing dogma. Following this insight, enrichment culture techniques (alternate animal and culture passage) were designed to give functionally differentiated tumor cells selective advantage over the fibroblasts. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types, providing the final point of destruction of the dogma of “dedifferentiation.” Instead, the hypothesis was proposed that cells in culture accurately represent cells in vivo, but without the complex in vivo environment. With the development of hormonally defined media and its combination with functionally differentiated clonal cell lines, this concept has been strengthened and the potential of tissue culture studies has been greatly augmented. Hormonally defined media allow the culture of cell types that cannot be grown in conventional, serum-supplemented media. These approaches demonstrate that hormonal responses and dependencies can be discovered in culture. Following this thinking and the discovery of hormonal dependencies of cancer cells has led to a new rationale for therapy. Tissue culture and cell technology continue to play an important role in solving human health problems.     

Adhesion dynamics: mechanisms and measurements

Adhesion to the extracellular matrix (ECM) is a fundamental requirement for survival, differentiation and migration of numerous cell types during both embryonic development and adult homeostasis. Different types of adhesion structures have been classified in different cell types or tissue environments. The best studied of these are focal adhesions which are found on a wide variety of cell types and will be the main focus of this review. Many years of research into the control of adhesion has yielded a wealth of information regarding the complexity of protein composition of these critical points of cell:ECM contact. Moreover, it has emerged that adhesions are not only highly ordered, but also dynamic structures under tight spatial control at the subcellular level to enable localised responses to extracellular cues. However, it is only in the last decade that the relative dynamics of these adhesion proteins have been closely studied. Here we provide an overview of the imaging strategies that have been developed and implemented to study the intricacies and hierarchy of protein turnover within focal adhesions. The caveats of employing these imaging techniques, as well as future directions will also be discussed.     

The protective effect of specific medium additives with respect to bubble rupture

A significant degree of cell damage is observed during suspension cell culture with air sparging. Protective agents can be added to the culture medium to protect the cells from damage. It has been observed that cells tend to adhere to air-medium interfaces and cell damage is mainly due to this cell-bubble interaction; protective additives have been found to prevent this cell adhesion to the bubble surfaces. In this article, it is demonstrated that the interfacial tension between the air and medium is related to the effectiveness of the protective additives to prevent adhesion of cells to this interface. Five different types of additives (Pluronic F-68, Methocels, dextran, Polyvinyl alcohol, and polyethylene glycols) were studied in an effort to determine their protective characteristics. Liquid-vapor interfacial tensions of the culture medium, with and without the additives, were measured by two different techniques (maximum bubble pressure method and Wilhelmy plate method). In addition, visualization techniques showed that in the presence of certain protective additives cells do not adhere to the bubble surface. Results obtained from these experiments indicate that the additives which rapidly lower the liquid-vapor interfacial tension of the culture medium also prevent adhesion of cells to the bubble surface. Experiments have also been conducted to determine the number of cells killed due to bubble rupture, and it was observed that this number is related to the amount of cells adhering to the bubble surface. (c) 1995 John Wiley & Sons, Inc.This article is a US Government Work and, as such, is in the public domain in the United States of America.     

The tail of integrin activation

Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation.