Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three dimensional tissue and organ models in vitro: their application in basic and practical research

Good and reliable three dimensional biomodel in vitro should mimic the in vivo structure and function of investigated tissue or organ. This can be achieved through the interaction of various cell types and the environment (extracellular matrix, as well as spatial cell contacts). While designing such a model it is necessary to find best nutritive and adhesive factors, to allow access to components of optimal matrix and to enable cell contacts in three dimensions. The response and function of such models reminds physiological function of tissue of origin. Over several years there has been an increasing number of experiments and publications reporting research involving spatial cell models. This model and its structural and molecular analysis clearly showed, that in comparison with conventional cultures, mainly monocultures grown as monolayers, spatial cultures resemble the in vivo situation with regard to cell shape and biological behaviour. Spatial arrangement of cells and the environment can direct tissue differentiation. Interesting example of the importance of the environment for the latter, is the finding that the ectopic implantation of embryonic cells transforms them into malignant tissue while the same cells located in the uterus undergo normal embryogenesis. The present review describes several three-dimensional models in vitro and their application in basic and practical studies. It is especially interesting and important in the light of developments of tissue modeling to obtain in vitro substitutes of damaged or impaired tissues and organs. Other demands come from animal protectionists who suggest replacement of experimental animals with cultures and tissue models in bio-, pharmacological, and toxicological assays. In response, European Union legislation introduces increasing restrictions on animal experiments. At last, researchers seek in vitro models which would enable avoiding discrepancies between in vitro and in vivo results.     

Effect of different factors on proliferation of antler cells, cultured in vitro

Antlers as a potential model for bone growth and development have become an object of rising interest. To elucidate processes explaining how antler growth is regulated, in vitro cultures have been established. However, until now, there has been no standard method to cultivate antler cells and in vitro results are often opposite to those reported in vivo. In addition, many factors which are often not taken into account under in vitro conditions may play an important role in the development of antler cells. In this study we investigated the effects of the antler growth stage, the male individuality, passaged versus primary cultures and the effect of foetal calf serum concentrations on proliferative potential of mixed antler cell cultures in vitro, derived from regenerating antlers of red deer males (Cervus elaphus). The proliferation potential of antler cells was measured by incorporation of (3)H thymidine. Our results demonstrate that there is no significant effect of the antler growth stage, whereas male individuality and all other examined factors significantly affected antler cell proliferation. Furthermore, our results suggest that primary cultures may better represent in vivo conditions and processes occurring in regenerating antlers. In conclusion, before all main factors affecting antler cell proliferation in vitro will be satisfactorily investigated, results of in vitro studies focused on hormonal regulation of antler growth should be taken with extreme caution.     

Mechanisms of T cell receptor antagonism: implications in the treatment of disease

The adaptive immune response is often required for the successful clearing of infectious pathogens. Antigen presenting cells (APC) present peptide antigens derived from pathogens to T cells via major histocompatibility complex (MHC) molecules. T cells then become activated and differentiate into effector cells with the capacity to kill infected cells or to induce an anti-pathogen antibody response. In autoimmunity, this T cell response is directed against self-antigens and often leads to deleterious effects on specific tissues. Likewise, T cell responses to allogeneic MHC molecules in transplants also leads to pathology. By introducing subtle changes in the antigenic peptide amino acid content, T cell activation can be inhibited, thereby preventing T cell effector functions. This strategy of TCR antagonism has been used successfully in vitro and in vivo to inhibit models of autoimmunity and allorecognition. In addition, a variety of pathogens that often result in chronic disease following infection, also have seemingly evolved natural mechanisms to inhibit T cell responses by antagonism. These microorganisms express natural variants of certain proteins, that when presented to T cells have the capacity to specifically inhibit T cell responses by functioning as antagonists or by modulating the nature of the T cell response. The understanding of how pathogens mediate this inhibition in vivo will be beneficial to ongoing studies in both autoimmunity and transplantation aimed at suppressing the harmful immune response, thereby controlling disease. TCR antagonism seems to have the potential to be used therapeutically to prevent or inhibit an undesired T cell response that will ultimately lead to disease.     

Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell- surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.     

An ultrastructural analysis of electromotor cell death in Torpedo marmorata and its counterpart in vitro

Summary Naturally occurring neuronal cell death has been investigated in the electromotor system of Torpedo marmorata and compared with neuronal death seen in expiant cultures of tissues from T. marmorata electric lobe. One objective of the study was to determine whether cell death in vitro was morphologically the same as cell death in vivo and to substantiate the validity of using in vitro models for studying naturally occurring cell death. Sequences of degeneration in vitro and in vivo have been established and compared: a single morphological sequence best represents this form of cell death in both conditions. In vivo, dying neurons are seen at all depths of the electric lobe indicating the involvement of different generations. Retrograde degeneration appears to be the first sign of cell death.     

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.     

Overview of Cell Models: From Organs Cultured in a Petri Dish to Organs-on-Chips

In this review, we tried to elucidate the origin and development of different animal and human cell culture methodologies used to evaluate the effects of various factors and substances in vitro. Organ cultures and conventional two-dimensional cultures of dissociated cells of various types, such as primary, tumor, induced pluripotent, stem, etc., have their advantages and drawbacks but usually do not represent accurate models for studying biological processes that take place in living organisms. Nowadays, high-throughput cell assays on the basis of various methods of signal detection (optical utilizing colorimetric, luminescent and fluorescent methods of detection, and electrochemical) are widely used at early stages of drug development for selection of the most active compounds and evaluation of their cytotoxic effects. The use of animals as models for drug testing is being criticized because of the lack of correlation between the results obtained in studies on them and on humans, and also because of the high cost and ethical issues. Therefore, much effort is put to create models based on human cells. This is how cultures emerged that utilize a three-dimensional network to simulate the architecture of tissues in vivo, and then so-called organs-on-chips—microfluidic microfabricated devices combining several types of cells—that replicate physical and chemical parameters of the microenvironment of cells in living organisms. In summary, experimental cell models have come a long way from the whole organs cultivated in a growth medium to almost complete reconstruction of organs in vitro based on the cutting-edge engineering approach with the use of different cell types. This currently enables one to replicate complex biological processes and study the influence of different substances and factors on them more successfully.     

Cell replication and aging: in vitro and in vivo studies.

The relationship between human aging and cell replication has been investigated using two complementary approaches: in vitro studies of human fibroblasts derived from young and old volunteer members of the Baltimore Longitudinal Study and in vivo examinations of bone marrow cell populations from young and old mice and rats. Total proliferative capacity measured as either the onset of cell culture senescence or as in vitro life span was significantly diminished in cell cultures derived from old human donors when compared to parallel cultures established from young donors. Acute replicative abilities as measured by percent replicating cells, cell pupulation doubling time, cell number at confluency, and colony size distribution were also significantly decreased in human old cell populations. An in vivo cytogenetic technique for measuring cell replication was developed utilizing the differential staining properties of metaphase chromosomes of cells that have replicated in the presence of bromodeoxyuridine. With this technique, cell cycle times have been derived in vivo as well as in vitro. Preliminary in vivo results in both mice and rats indicate that cell replication is slowed in old animal cell populations. Further research will be directed both in vitro and in vivo at discerning the mechanisms for this impairment of cellular replication with aging.     

3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers

Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.     

Interaction of chlamydiae and host cells in vitro.

The obligately intracellular bacteria of the genus Chlamydia, which is only remotely related to other eubacterial genera, cause many diseases of humans, nonhuman mammals, and birds. Interaction of chlamydiae with host cells in vitro has been studied as a model of infection in natural hosts and as an example of the adaptation of an organism to an unusual environment, the inside of another living cell. Among the novel adaptations made by chlamydiae have been the substitution of disulfide-bond-cross-linked polypeptides for peptidoglycans and the use of host-generated nucleotide triphosphates as sources of metabolic energy. The effect of contact between chlamydiae and host cells in culture varies from no effect at all to rapid destruction of either chlamydiae or host cells. When successful infection occurs, it is usually followed by production of large numbers of progeny and destruction of host cells. However, host cells containing chlamydiae sometimes continue to divide, with or without overt signs of infection, and chlamydiae may persist indefinitely in cell cultures. Some of the many factors that influence the outcome of chlamydia-host cell interaction are kind of chlamydiae, kind of host cells, mode of chlamydial entry, nutritional adequacy of the culture medium, presence of antimicrobial agents, and presence of immune cells and soluble immune factors. General characteristics of chlamydial multiplication in cells of their natural hosts are reproduced in established cell lines, but reproduction in vitro of the subtle differences in chlamydial behavior responsible for the individuality of the different chlamydial diseases will require better in vitro models.     

3D tumour models: novel in vitro approaches to cancer studies

3D in vitro models have been used in cancer research as a compromise between 2-dimensional cultures of isolated cancer cells and the manufactured complexity of xenografts of human cancers in immunocompromised animal hosts. 3D models can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario in which they are found. These 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Approaches to create more biomimetic 3D models of cancer include, but are not limited to, (i) providing the appropriate matrix components in a 3D configuration found in vivo, (ii) co-culturing cancer cells, endothelial cells and other associated cells in a spatially relevant manner, (iii) monitoring and controlling hypoxia- to mimic levels found in native tumours and (iv) monitoring the release of angiogenic factors by cancer cells in response to hypoxia. This article aims to overview current 3D in vitro models of cancer and review strategies employed by researchers to tackle these aspects with special reference to recent promising developments, as well as the current limitations of 2D cultures and in vivo models. 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Here we review current strategies in the field of modelling cancer, with special reference to advances in complex 3D in vitro models.     

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.     

Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.     

Determination of the biological activity of Clostridium difficile toxins in in vivo and in vitro experiments

The biological activity of the filtrates of 29 C. difficile strains was studied in vivo (suckling white mice) and in vitro (cell cultures of different species and origin). The action of the filtrates on the experimental models in vivo was evaluated from the cytotoxic effect index, while in vitro the intensity of the cytotoxic effect was evaluated from the percentage of dead cells in the monolayer. The results of the comparative determination of toxicity characteristics in vivo and in vitro demonstrated that cell cultures were more sensitive experimental models than suckling white mice. The use of cell cultures permitted the quantitative evaluation of the cytotoxic activity of the filtrates under study, as well as the detection of their cell-directed action at minimal concentrations.     

Comparison of in vivo and in vitro subcellular localization of estrogen receptors alpha and beta in oligodendrocytes

The existence of estrogen receptors (ERs) in oligodendrocytes (OLGs) in vivo and in vitro is unresolved, as their presence has been reported in some studies and their absence in others. Using molecular and immunocytochemical techniques, we describe the subcellular localization of ERalpha and ERbeta in OLGs in vivo and in vitro. Both ERalpha and ERbeta are detected in an immortalized OLG cell line and in enriched OLG cultures by RT-PCR and western blot. Immunocytochemistry of OLGs from enriched cultures shows ERalpha receptors are nuclear, whereas ERbeta receptors are cytoplasmic. Confocal and deconvolution microscopy of enriched OLG cultures reveals ERbeta immunoreactivity is concentrated in perikarya and veins of OLG membrane sheets; lesser reactivity is present in their plasma membranes and nuclei. In vivo, we readily detect ERalpha in neurons but not in OLGs, even though we used different fixation procedures and different ERalpha antibodies. The presence of ERalpha in cultured OLGs may be due to culture media that contains factors stimulating ERalpha expression but are reduced in normal brain. In vivo, ERbeta immunoreactivity is readily detectable in OLG cytoplasm and in myelin sheaths. Incubation of glial cultures without or with increasing concentrations of 17beta-estradiol (E2) shows that E2 significantly accelerates OLG process formation.     

Response to challenging dose of X-rays as a predictive assay for molecular epidemiology

Human biomonitoring, as a tool to identify health risk from environmental exposures, has gained increasing interest especially in the areas of cancer risk assessment and response to therapy. Chromosome aberrations resulting from direct DNA breakage or from inhibition of DNA repair or synthesis, as measured in peripheral blood lymphocytes, have been used successfully in the assessment of environmental health. Susceptibility to the induction of genotoxicity has been evaluated by the use of an in vitro challenge dose of UV or X-rays. In this report, DNA damage was analyzed with the use of single cell gel electrophoresis (SCGE) assay in healthy donors and cancer patients. Studies have shown a good correlation between DNA damage induced in vivo or in vitro and cytogenetic measures. Results from studies on susceptibilities and repair competence in 475 controls, exposed workers and cancer patients are discussed. The possible effects of exposures and influence of the diet and other confounding factors are shown. The prospective use of a challenging dose of radiation combined with the SCGE assay as a predictive assay is suggested and the limitations are discussed.     

Expansion of human hematopoietic stem cells for transplantation: trends and perspectives

Umbilical cord blood transplantation is clinically limited by its low progenitor cell content. Ex vivo expansion has become an alternative to increase the cell dose available for transplants. Expansion has been evaluated in several ways such as static cultures combining growth factors or mimicking the natural microenvironment using co-culture systems. However, static cultures have a small volume capacity and therefore large-scale expansion has been addressed using bioreactors. These and other biotechnological approaches for the expansion of hematopoietic progenitors and their utility to study several aspects of hematopoietic stem cell biology are discussed here.