In-Plate Cryopreservation of 2D and 3D Cell Models: Innovative Tools for Biomedical Research and Preclinical Drug Discovery.

Cell-based assays performed in multiwell plates are utilized in basic and translational research in a variety of cell models. The assembly of these multiwell platforms and their use is often laboratory specific, preventing the standardization of methods and the comparison of outputs across different analytical sites. Moreover, when cell models are based on primary cells with specialized culture requirements, including three-dimensional (3D) cell culture, their complexity and the need for manipulation by experienced operators can add significant cost and introduce long lead times to analysis, both of which are undesirable in any preclinical situation. To address this issue, we explored adaptations of cryopreservation technology that allow cells to be cryopreserved in-plate, ready for use in analysis, and have developed a method applicable to cells from different origins and different culture formats. Here we describe the application of this technology to conventional two-dimensional (2D) monolayers of human mesenchymal stem cells (MSCs) and human macrophages derived from primary monocytes, and to 3D cultures of hepatic organoids, colon organoids, and colon tumor organoids, each presented for cryopreservation in their obligate extracellular matrix. We demonstrated that cell viability, cell physiology, and cytotoxic sensitivity were maintained after cryopreservation, such that the models offer the means to uncouple model assembly from analytical use and to standardize cell models in product form for distribution to end users.

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species.

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

3D gastrointestinal models and organoids to study metabolism in human colon cancer.

Recent advances in the field of cancer metabolism raised awareness for the importance of the tumour microenvironment in tumour growth and progression. The initial theory by Heinrich Warburg was that cancer cells had a deficient oxidative respiration and thus had to perform aerobic glycolysis to produce energy. However, further research suggested that there is a metabolic reprogramming within the tumour microenvironment, controlled by communication between tumour and stromal cells. The importance of this communication exposes the need to use complex models in cancer research. Until recently, classic cell models included immortalized 2D cell lines or patient-derived tumour xenografts. Despite having contributed to many discoveries, these models present many limitations. Improved models are now being developed using 3D cell culture technology. These models are more physiologically relevant allowing the co-culture of different cells types and establishing a gradient concentration of solutes. Recent developments in organoid technology contributed largely to the expansion of 3D cell technology. Organoids can be developed from different tissues including tumours, representing the cell population and spatial organization of the tissue of origin. In the field of cancer metabolism, the interaction of different cell types, the stroma, and the different solutes and oxygen concentrations are crucial parameters. Current models to study metabolism either include only one cell population or are unable to represent solute/oxygen gradients or to collect samples in a proficient manner. The characteristics of organoid culture thus makes them a potent model to use in metabolic studies, drug development, disease model or even personalized medicine.

Bovine colon organoids: From 3D bioprinting to cryopreserved multi-well screening platforms.

Three-dimensional (3D) colon organoids, termed "colonoids", derived from adult stem cells represent a powerful tool in in vitro pharmaceutical and toxicological research. Murine and human colonoid models exist. Here we describe the establishment of bovine colonoids for agri-biotechnological applications, and extend the repertoire of colonoid culture options through proof-of-principle for bioprinting and novel in-plate cryopreservation technology. As a first step, we differentiated established long-term bovine colonoid cultures into mature colonoids. Tissue-specific differentiation was demonstrated by gene expression. Second, we investigated cryopreservation of colonoids in situ within an extracellular matrix in multi-well plates. Upon controlled thawing, cryopreserved 3D cultures grew at similar rates to unfrozen colonoids. Cytotoxic sensitivity to staurosporine was not significantly different between in situ freeze-thawed and unfrozen control cultures. Third, scalability of colonoid culture assembly by extrusion bioprinting into multi-well plates using GelMA bioink was assessed. With optimised bioprinting and crosslinking parameters, colonoids in GelMA were printed into 96 well culture plates and remained viable and proliferative post-print. For tissue-relevant in vitro studies we furthermore established differentiated colonoid-derived monolayer cultures on permeable membranes. Taken together, we outline novel in vitro approaches to study the ruminant colonic epithelium and introduce in-plate cryopreservation as convenient alternative to conventional in-vial cryopreservation.

Towards the Design of 3D Fiber-Deposited Poly(ε-caprolactone)/lron-Doped Hydroxyapatite Nanocomposite Magnetic Scaffolds for Bone Regeneration.

In the past few years, researchers have focused on the design and development of three-dimensional (3D) advanced scaffolds, which offer significant advantages in terms of cell performance. The introduction of magnetic features into scaffold technology could offer innovative opportunities to control cell populations within 3D microenvironments, with the potential to enhance their use in tissue regeneration or in cell-based analysis. In the present study, 3D fully biodegradable and magnetic nanocomposite scaffolds for bone tissue engineering, consisting of a poly(ε-caprolactone) (PCL) matrix reinforced with iron-doped hydroxyapatite (FeHA) nanoparticles, were designed and manufactured using a rapid prototyping technique. The performances of these novel 3D PCL/FeHA scaffolds were assessed through a combination of theoretical evaluation, experimental in vitro analyses and in vivo testing in a rabbit animal model. The results from mechanical com- pression tests were consistent with FEM simulations. The in vitro results showed that the cell growth in the magnetized scaffolds was 2.2-fold greater than that in non-magnetized ones. In vivo experiments further suggested that, after only 4 weeks, the PCL/FeHA scaffolds were completely filled with newly formed bone, proving a good level of histocompatibility. All of the results suggest that the introduction of magnetic features into biocompatible materials may confer significant advantages in terms of 3D cell assembly.

Biological models for phytochemical research: from cell to human organism.

Nutrigenomics represents a shift of nutrition research from epidemiology and physiology to molecular biology and genetics. Nutrigenomics seeks to understand nutrition influences on homeostasis, the mechanism of genetic predispositions for diseases, to identify the genes influencing risk of diet related diseases. This review presents some in vitro models applicable in nutrigenomic studies, and discusses the use of animal models, their advantages and limitations and relevance for human situation. In vitro and in vivo models are suitable for performance of DNA microarrays, proteomic and transcriptomic analyses. In vitro models (intracellular organelles and suborganellar compartments, cell cultures, or tissue samples/cultures) give insight in metabolic pathways and responses to test stimuli on cellular and molecular levels. Animal models allow evaluation of the biological significance of the effects recorded in vitro and testing of the hypothesis on how a specific factor affects specific species under specific circumstances. Therefore, the evaluation of the data in relation to human organism should be done carefully, considering the species differences. The use of in vitro and in vivo models is likely to continue as the effects of nutrition on health and disease cannot be fully explained without understanding of nutrients action at nuclear level and their role in the intra- and intercellular signal transduction. Through advances in cell and molecular biology (including genomic and proteomic), the use of these models should become more predictively accurate. However, this predictive value relies on an underpinning knowledge of the advantages and limitations of the model in nutrigenomic research as in other fields of biomedical research.

Functional expression of bcl-2 protein family and AIF in bovine mammary tissue in early lactation.

Cell proliferation and apoptosis were measured in bovine mammary tissue around the time of peak milk production in three heifers, and compared with changes in the expression of bcl-2-related intracellular signals and apoptosis-inducing factor (AIF). Cell proliferation and apoptosis were relatively constant during the study period with no significant change in the incidence of either event. However, the ratio of apoptotic to proliferating cells tended to change from 0.99 on day 45 of lactation to 1.82 on day 63 (P=0.064), suggesting that in the course of the study, a dynamic balance may have been succeeded by net cell loss. Average milk production recorded 6 d after biopsy was correlated with the estimated number of cells (r=0.898; P<0.01) but not with the apoptosis to proliferation ratio (r=-0.224, P>0.05). Turnover of the cell population was associated with relatively constant expression of anti-apoptotic bcl-2. Competitive PCR also indicated expression of bax, in contrast to observations in lactating rodent mammary tissue. Bax expression was relatively low compared with that of bcl-2, but immunohistochemical staining for bax protein, which was not detectable on day 45, was observed on day 53 and, more intensely, on day 60 when the protein appeared to be membrane-associated. A partial coding sequence for bovine AIF was identified and AIF expression was evaluated by in situ hybridization. The results indicated that AIF was expressed in luminal alveolar cells and that, in concert with a change in bax to bcl-2 ratio, they might contribute to signalling of a change in the dynamic balance of the cell population as lactation progresses.

Basement membrane integrity and keratinization in healthy and ulcerated bovine hoof tissue.

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal-epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.

Mammary apoptosis and lactation persistency in dairy animals.

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissue's ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

Transcription factor NFIC undergoes N-glycosylation during early mammary gland involution.

Expression of a 74-kDa nuclear factor I (NFI) protein is triggered in early involution in the mouse mammary gland, and its expression correlates with enhanced occupation of a twin (NFI) binding element in the clusterin promoter, a gene whose transcription is induced at this time (Furlong, E. E., Keon, N. K., Thornton, F. D., Rein, T., and Martin, F. (1996) J. Biol. Chem. 271, 29688-29697). We now identify this 74-kDa NFI as an NFIC isoform based on its interaction in Western analysis with two NFIC-specific antibodies. A transition from the expression of a 49-kDa NFIC in lactation to the expression of the 74-kDa NFIC in early involution is demonstrated. We show that the 74-kDa NFIC binds specifically to concanavalin A (ConA) and that this binding can be reversed by the specific ConA ligands, methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside. In addition, its apparent molecular size was reduced to approximately 63 kDa by treatment with the peptide N-glycosidase. The 49-kDa lactation-associated NFIC did not bind ConA nor was it affected by peptide N-glycosidase. Tunicamycin, a specific inhibitor of N-glycosylation, blocked formation of the 74-kDa NFI in involuting mouse mammary gland in vivo when delivered from implanted Elvax depot pellets. Finally, the production of the ConA binding activity could be reiterated in "mammospheres" formed from primary mouse mammary epithelial cells associated with a laminin-rich extracellular matrix. Synthesis of the 74-kDa NFIC was also inhibited in this setting by tunicamycin. Thus, involution triggers the production of an NFIC isoform that is post-translationally modified by N-glycosylation. We further show, by using quantitative competitive reverse transcriptase-PCR, that there is increased expression of the major mouse mammary NFIC mRNA transcript, mNFIC2, in early involution, suggesting that the involution-associated change in NFIC expression also has a transcriptional contribution.