Cell lines as in vitro models for drug screening and toxicity studies

Cell culture is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. The use of tissue culture is a valuable tool to study problems of clinical relevance, especially those related to diseases, screening, and studies of cell toxicity mechanisms. Ready access to the cells provides the possibility for easy studies of cellular mechanisms that may suggest new potential drug targets and, in the case of pathological-derived tissue, it has an interesting application in the evaluation of therapeutic agents that potentially may treat the dysfunction. However, special considerations must be addressed to establish stable in vitro function. In primary culture, these factors are primarily linked to greater demands of tissue to adequately survive and develop differentiated conditions in vitro. Additional requirements include the use of special substrates (collagen, laminin, extracellular matrix preparations, etc.), growth factors and soluble media supplements, some of which can be quite complex in their composition. These demands, along with difficulties in obtaining adequate tissue amounts, have prompted interest in developing immortalized cell lines which can provide unlimited tissue amounts. However, cell lines tend to exhibit problems in stability and/or viability, though they serve as a feasible alternative, especially regarding new potential applications in cell transplant therapy. In this regard, stem cells may also be a source for the generation of various cell types in vitro. This review will address aspects of cell culture system application, with focus on immortalized cell lines, in studying cell function and dysfunction with the primary aim being to identify cell targets for drug screening.     

Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications.     

Biocompatible Smart Microcapsules Based on Chitosan‐Poly(vinyl alcohol) Copolymers for Cultivation of Animal Cells

In this study, two novel chitosan‐graft‐poly(vinyl alcohol) copolymers are synthesized and used as water‐soluble at physiological conditions polycations for preparation of smart microcapsules. The microcapsules provide growth and proliferation of eight mammalian cell lines, including hybridoma and tumor cells, at long‐term cell cultivation in vitro. The microcapsules are stable in cell culture medium but can be dissolved by changing pH value of the medium (up to 8.0–8.2), thus making possible a simple release of the entrapped cells. Monoclonal antibody production by encapsulated hybridoma cells is demonstrated. Cultivation of tumor cells within the microcapsules allows the formation of 3D multicellular spheroids, which can be proposed as an in vitro model for anticancer drug screening.     

Preferential killing of cancer cells using silicon carbide quantum dots

Silicon carbide quantum dots are highly luminescent biocompatible nanoparticles whose properties might be of particular interest for biomedical applications. In this study we investigated Silicon Carbide Quantum Dots (3C-SiC QDs) cellular localisation and influence on viability and proliferation on oral squamous carcinoma (AT-84 and HSC) and immortalized cell lines (S-G). They clearly localize into the nuclei, but the presence of 3C-SiC QDs in culture medium provoke morphological changes in cultured cells. We demonstrate that 3C-SiC QDs display dose- and time-dependent selective cytotoxicity on cancer versus immortalized cells in vitro. Since one of the limitations of classical antineoplastic drugs is their lack of selectivity, these results open a new way in the search for antiproliferative drugs.     

Design and Evaluation of Soluble Ocular Drug Insert for Controlled Release of Ciprofloxacin Hydrochloride

ABSTRACT

Purpose: Soluble ocular inserts of ciprofloxacin hydrochloride were prepared with the aim of achieving once a day administration. Design: Drug reservoir was prepared using natural hydrophilic polymer viz. gelatin while rate-controlling membrane was prepared using hydrophobic ethyl cellulose. Ocular inserts were evaluated for their physicochemical parameters like thickness, weight uniformity, drug content, percent moisture loss, and percent moisture absorption. The in vitro drug release studies were carried out using Bi-chambered donar receiver compartment model. Since targeted prolong release was observed in formulation CF2 and CF5, these formulations were further subjected to in vivo drug release study using rabbits as an animal model. In vitro drug release kinetic data was treated according to Zero, First, and Higuchi kinetics to access the mechanism of drug release. Results: Correlation between in vitro and in vivo drug release was found to be strong revealing the efficacy of the formulation. Conclusion: Formulation CF5 has achieved target of present study such as increase residence time, prolong drug release, reduction in frequency of administration, and, thus may improve the patient compliance.     

In vitro and in vivo methods to determine the interactions of osteogenic cells with biomaterials

To assess new biomaterials for possible use as bone graft substitutes, a number of techniques allow interactions with osteoblastic cells to be studied, with respect to effects on proliferation and differentiation of osteoprogenitors. In vitro models include the use of bone explant cultures, fetal rat calvarial-derived osteoblast cells, primary stromal populations, transformed and non-transformed cell lines and immortalized osteoblast cell lines. However, these assessments are limited by the extent of osteogenic differentiation and bone formation that can be observed in vitro, species differences and phenotypic drift of cells cultured in vitro. The use of in vivo experimental systems such as the segmental/calvarial bone defect model, the subcutaneous implant model and the diffusion chamber implantation model circumvent some of these issues and, in the appropriate model, provide data on efficacy, biocompatibility and osteointegration of a biomaterial. The combination of in vitro and in vivo approaches together with the development of new cell labeling techniques, in particular the ability to genetically mark and select specific human bone cell populations provides new avenues for their potential evaluation in combination with appropriate biomaterials for clinical use. These in vitro and in vivo techniques are reviewed and those recently developed for assessment of human osteogenic cells should be applicable to many other cell systems where knowledge of specific human tissue or cell interactions with biomaterials is required. © 1999 Kluwer Academic Publishers     

Processing of nanolitre liquid plugs for microfluidic cell-based assays

Abstract

Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.     

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet‐Microarray Platform

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo “microenvironment” compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96‐ and 384‐well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic–superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single‐spheroid‐microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24–48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high‐throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.     

Subcellular‐Scale Drug Transport via Ultrasound‐Degradable Mesoporous Nanosilicon to Bypass Cancer Drug Resistance

Delivering and releasing anticancer agents directly to their subcellular targets of action in a controlled manner are almost the ultimate goal of pharmacology, but it is challenging. In recent decades, plenty of efforts have been made to send drugs to tumor tissue or even specifically to cancer cells; however, at the subcellular scale, cancer cells have multiple cunning ways to hinder drugs from reaching their final action targets. Here, we demonstrate a strategy to bypass the last defense of cancer drug resistance by contolling the drug transportation and release at subcellular scale. We developed a platform based on ultrasound‐degradable mesoporous nanosilicon, which allows drug delivery towards, ultrasound controlled drug release into the cell nucleus. This strategy altered the drug distribution within cells and remarkably enhanced the drug accumulation ratio at the action target, i.e. nucleus. In vitro and in vivo studies proved that this strategy reduced the drug dosage by an order of magnitude, prolonged drug retention and amplified therapeutic efficacy in tumor‐bearing mice. These results offer new insights into bypassing cancer drug resistance through transport and release drugs directly to their action targets in a controlled manner.     

The response of primary rat and human osteoblasts and an immortalized rat osteoblast cell line to orthopaedic materials: comparative sensitivity of several toxicity indices

When studying the biocompatibility of orthopaedic biomaterials it isoften necessary to discriminate between responses which show mild cytotoxicity.It is therefore essential to use a very sensitive index of toxicity. We havecompared the sensitivity of four well-established indices of toxicity: totalcell protein content, leakage of lactate dehydrogenase (LDH), reducedglutathione content and the MTT assay, with that of a novel index, alkalinephosphatase (ALP) activity. Comparisons were made by detecting nickel chloridetoxicity in osteoblasts. ALP activity, the novel method, proved the mostsensitive index of toxicity and it provides a convenient automated assay forassessing the interactions of materials with osteoblasts. The responses tonickel chloride and to aqueous extracts prepared from carbon fibre reinforcedepoxy and polyetheretherketone (peek), two candidate materials for orthopaedicimplants, were compared in primary and immortalized rat osteoblasts, and !in primary human osteoblasts. Although the immortalized rat osteoblast cell line,FFC, was consistently the most sensitive cell type, the responses of the humancells and the FFC cell line were similar in terms of ALP activity throughout therange of nickel concentrations studied. Neither peek nor epoxy material extractsshowed a significant decrease in the MTT or ALP responses in any of the threecell types. Our data suggest that immortalized rat osteoblasts may provide anin vitro model system for screening the biocompatibility of orthopaedicpolymers.     

Assessment of Transport Barriers Using Cell and Tissue Culture Systems

Abstract

The delivery of the next generation of drugs, particularly polar peptides and proteins, will represent a major challenge to the pharmaceutical scientists. To successfully deliver these potential drugs to specific targets, strategies will have to be developed to circumvent epithelial (e.g., intestinal, buccal, nasal, epidermal) and endothelial (e.g., vascular) cells, which represent both physical and metabolic barriers. This presentation will focus on the possible utility of cultured epithelial and endothelial cells to screen the permeability properties of potential drug candidates, to elucidate transcellular transport mechanisms, to evaluate the potential cellular site of metabolism, and to test strategies to prevent metabolism and/or to enhance the permeability properties of drug candidates. Topics to be discussed include: (a) advantages and disadvantages of cell culture systems; (b) factors important in selecting an appropriate cell culture system which will mimic the in vivo biological barrier; (c) characterization and validation of in vitro cell culture model systems; (d) factors important in selecting a porous membrane and a diffusion apparatus for transcellular transport studies; (e) the effect of cell culturing conditions on the transport characteristics of cultured cells; and (f) factors important in interpretation of in vitro cell culture data.     

Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

Abstract

Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (φ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.     

Cell‐Assembled Graphene Biocomposite for Enhanced Chondrogenic Differentiation

Graphene‐based nanomaterials are increasingly being explored for use as biomaterials for drug delivery and tissue engineering applications due to their exceptional physicochemical and mechanical properties. However, the two‐dimensional nature of graphene makes it difficult to extend its applications beyond planar tissue culture. Here, graphene–cell biocomposites are used to pre‐concentrate growth factors for chondrogenic differentiation. Bone marrow‐derived mesenchymal stem cells (MSCs) are assembled with graphene flakes in the solution to form graphene‐cell biocomposites. Increasing concentrations of graphene (G) and porous graphene oxide (pGO) are found to correlate positively with the extent of differentiation. However, beyond a certain concentration, especially in the case of graphene oxide, it will lead to decreased chondrogenesis due to increased diffusional barrier and cytotoxic effects. Nevertheless, these findings indicate that both G and pGO could serve as effective pre‐concentration platforms for the construction of tissue‐engineered cartilage and suspension‐based cultures in vitro.     

Nanomedicine-nanoemulsion formulation improves safety and efficacy of the anti-cancer drug paclitaxel according to preclinical assessment

Paclitaxel is an important anticancer drug and is currently used to treat a variety of cancers, including ovarian carcinomas, breast cancer, non-small cell lung cancer, and AIDS-related Kaposi's sarcoma. The objectives of the studies were to assess and compare the safety and efficacy of EmPAC (a newly developed nanoemulsion formulation of paclitaxel) versus Taxol (the injectable formulation of paclitaxel involving the use of polyethylated or polyoxyl castor oil currently used in the clinic). The objectives were also to investigate the mechanism for the improved safety and efficacy of EmPAC over Taxol. These results showed that EmPAC had better anti-tumor efficacy than Taxol, according to in vitro cell culture studies and studies in animal tumor models. EmPAC had improved anti-tumor efficacy even in tumor cell lines that are known to be multi-drug resistant. Part of the mechanism of action for the improved efficacy may be related to EmPAC inducing greater cellular uptake of paclitaxel into tumor cells than Taxol did, according to the in vitro cell culture radioactive-labeled studies and in vitro cell culture antibody studies. It may also partly be because EmPAC delivered more paclitaxel to the tumor mass than Taxol, while the delivery of paclitaxel to other tissues (e.g., blood, muscle, liver, spleen, kidney and lung) were similar between the two formulations of paclitaxel, according to studies in animals with tumor xenograft. EmPAC also had better safety than Taxol according to toxicology studies in rabbits. This may be because EmPAC does not contain the toxic ingredients used in formulating Taxol (such as polyethylated or polyoxyl castor oil). These results support the clinical development of the nanoemulsion formulation of paclitaxel.     

In vitro Toxicity Testing of Nanoparticles in 3D Cell Culture

Common 2D cell cultures do not adequately represent the functions of 3D tissues that have extensive cell–cell and cell–matrix interactions, as well as markedly different diffusion/transport conditions. Hence, testing cytotoxicity in 2D cultures may not accurately reflect the actual toxicity of nanoparticles (NPs) and other nanostructures in the body. To obtain more adequate and detailed information about NP–tissue interactions, we here introduce a 3D‐spheroid‐culture‐based NP toxicology testing system. Hydrogel inverted colloidal crystal (ICC) scaffolds are used to create a physiologically relevant and standardized 3D liver tissue spheroid model for in vitro assay application. Toxicity of CdTe and Au NPs are tested in both 2D and 3D spheroid cultures. The results reveal that NP toxic effects are significantly reduced in the spheroid culture when compared to the 2D culture data. Tissue‐like morphology and phenotypic change are identified to be the major factors in diminishing toxicity. Acting as an intermediate stage bridging in vitro 2D and in vivo, our in vitro 3D cell‐culture model would extend current cellular level cytotoxicity to the tissue level, thereby improving the predictive power of in vitro NP toxicology.     

The Role of Lymphatic Transport in Enhancing Oral Protein and Peptide Drug Delivery

ABSTRACT

The gastrointestinal lymphatic system is a specific transport pathway through which dietary lipids, fat-soluble vitamins, and water-insoluble peptide-type molecules (e.g., cyclosporine A) can gain access to the systemic circulation. Drugs transported by way of the gastrointestinal lymphatic system bypass the liver and avoid potential hepatic first-pass metabolism. Lymphatic delivery of immunomodulatory and low therapeutic index protein and peptide drugs used in the treatment of cancer cell metastases and HIV presents an opportunity to maximize therapeutic benefit while minimizing general systemic drug exposure. Furthermore, lymphatic drug transport may promote drug incorporation into the body's lipid-handling system, thus offering the potential to manipulate drug distribution and residence time within the body. This review article will discuss the potential utilization of lymphatic transport in enhancing the oral absorption of protein- and peptide-like drugs.     

Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

Abstract

This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide) exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has developed cell sheet-based tissue engineering for therapeutic uses with single layer or multilayered cell sheets, which were recovered from the thermoresponsive cell culture dish. Using surface derivation techniques, we developed a new generation of thermoresponsive cell culture dishes to improve culture conditions. We also designed a new methodology for constructing well-defined organs using microfabrication techniques.     

Flexible Nanoparticles Reach Sterically Obscured Endothelial Targets Inaccessible to Rigid Nanoparticles

Molecular targeting of nanoparticle drug carriers promises maximized therapeutic impact to sites of disease or injury with minimized systemic effects. Precise targeting demands addressing to subcellular features. Caveolae, invaginations in cell membranes implicated in transcytosis and inflammatory signaling, are appealing subcellular targets. Caveolar geometry has been reported to impose a ≈50 nm size cutoff on nanocarrier access to plasmalemma vesicle associated protein (PLVAP), a marker found in caveolae in the lungs. The use of deformable nanocarriers to overcome that size cutoff is explored in this study. Lysozyme‐dextran nanogels (NGs) are synthesized with ≈150 or ≈300 nm mean diameter. Atomic force microscopy indicates the NGs deform on complementary surfaces. Quartz crystal microbalance data indicate that NGs form softer monolayers (≈60 kPa) than polystyrene particles (≈8 MPa). NGs deform during flow through microfluidic channels, and modeling of NG extrusion through porous filters yields sieving diameters less than 25 nm for NGs with 150 and 300 nm hydrodynamic diameters. NGs of 150 and 300 nm diameter target PLVAP in mouse lungs while counterpart rigid polystyrene particles do not. The data in this study indicate a role for mechanical deformability in targeting large high‐payload drug‐delivery vehicles to sterically obscured targets like PLVAP.     

Thyroid Cells Exposed to Simulated Microgravity Conditions – Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.     

Intracellular accumulation and cytotoxicity of doxorubicin with different pharmaceutical formulations in human cancer cell lines

The structure of both carrier and anticancer drug affects the intracellular fate of a transported drug. The study investigated in vitro intracellular accumulation and cytotoxic activity of doxorubicin-loaded solid lipid nanoparticles (SLN), doxorubicin in pegylated liposomes (Caelyx) and free doxorubicin. Intracellular doxorubicin levels and cytotoxic activity were determined by high performance liquid chromatography with fluorescence detection, and by the trypan blue dye exclusion assay, respectively. Doxorubicin-loaded SLN inhibited cell growth more strongly than either free or liposomal doxorubicin, in human colorectal adenocarcinoma, HT-29, retinoblastoma Y79, and glioblastoma U373 cell lines. The IC50 values for doxorubicin-loaded SLN were significantly lower after 24 h exposure than those for free doxorubicin in all cell lines; after 48 h exposure they were lower than those for liposomal doxorubicin in HT-29 and Y79 cells. The enhanced cytotoxic activity of doxorubicin-loaded SLN was associated with increased drug incorporation in cells: intracellular doxorubicin levels were significantly enhanced after exposure to drug-loaded SLN versus either free or liposomal drug. Rate of intracellular accumulation and cytotoxic activity also differed among different cell lines; in particular, cells of epithelial origin were found to be more sensitive to doxorubicin-loaded SLN. In conclusion, the greater sensitivity of HT-29, Y79, and U373 cells to doxorubicin-loaded SLN than to the other drug formulations may be due to the capability of the delivery system to enhance drug action, through a marked uptake and accumulation of SLN within the cell.