Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy

Three-dimensional (3D) cell culture has developed rapidly over the past 5–10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.     

Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling

Three-dimensional(3 D) culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3 D cell models. Thus, the 3 D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3 D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting. In addition, the progress of employing 3 D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3 D cell culture systems were discussed.     

Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate)-based biopolymer systems

Biodegradable biopolymers attract much attention in biology and medicine due to its wide application. The present review considers a biodegradable and biocompatible polymer of bacterial origin, poly(3-hydroxybutyrate), which has wide perspectives in medicine and pharmaceutics. It highlights basic properties of biopolymer (biodegradability and biocompatibility) and also biopolymer systems: various materials, devices and compositions based on the biopolymer. Application of poly(3-hydroxybutyrate)-based biopolymer systems in medicine as surgical implants, in bioengineering as cell culture scaffolds, and in pharmacy as novel drug dosage forms and drug systems are also considered.     

Scaffold-free cell-based systems

Cover illustration Scaffold-free cell-based systems. This issue of BTJ, edited by Umut Gurkan (Harvard Medical School, MA, USA) and Feng Xu (Xi' an Jiaotong University, China) highlights several applications of scaffold-free systems such as cell therapies for tissue regeneration, in vitro functional tissue and disease models for drug sensitivity testing, cell-based biosensors, and drug delivery. Cover image: Confocal image of a microtissue generated from human colorectal carcinoma cell line, HCT-116, stained for integrin, 1 (green), cytoskeleton (red) and nuclei (blue). Image: Irina Agarkova (InSphero AG, Zurich, Switzerland). See the article by Drewitz et al., http://dx.doi.org/10.1002/biot.201100290     

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure–activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.     

Synthetic 3D multicellular systems for drug development

Since the 1970s, the limitations of two dimensional (2D) cell culture and the relevance of appropriate three dimensional (3D) cell systems have become increasingly evident. Extensive effort has thus been made to move cells from a flat world to a 3D environment. While 3D cell culture technologies are meanwhile widely used in academia, 2D culture technologies are still entrenched in the (pharmaceutical) industry for most kind of cell-based efficacy and toxicology tests. However, 3D cell culture technologies will certainly become more applicable if biological relevance, reproducibility and high throughput can be assured at acceptable costs. Most recent innovations and developments clearly indicate that the transition from 2D to 3D cell culture for industrial purposes, for example, drug development is simply a question of time.     

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

Although the recent advances in stem cell engineering have gained a great deal of attention due to their high potential in clinical research, the applicability of stem cells for preclinical screening in the drug discovery process is still challenging due to difficulties in controlling the stem cell microenvironment and the limited availability of high-throughput systems. Recently, researchers have been actively developing and evaluating three-dimensional (3D) cell culture-based platforms using microfluidic technologies, such as organ-on-a-chip and organoid-on-a-chip platforms, and they have achieved promising breakthroughs in stem cell engineering. In this review, we start with a comprehensive discussion on the importance of microfluidic 3D cell culture techniques in stem cell research and their technical strategies in the field of drug discovery. In a subsequent section, we discuss microfluidic 3D cell culture techniques for high-throughput analysis for use in stem cell research. In addition, some potential and practical applications of organ-on-a-chip or organoid-on-a-chip platforms using stem cells as drug screening and disease models are highlighted.     

High mutagenic activity of 3-azido-1,2-propanediol (azidoglycerol, AG) in strain D7 of Saccharomyces cerevisiae

3-Azido-1,2-propanediol (azidoglycerol, AG) showed a high mutagenicity in strain D7 of Saccharomyces cerevisiae. At 5 mM it increased the spontaneous frequency of isoleucine revertants 3500 times and the frequency of gene convertants 3000 times during 24 h of growth, reducing the growth rate to 30%. In non-growth conditions, treatment with 150 mM of AG for 3 h reduced cell survival to 60% and enhanced the frequency of isoleucine revertants 490 times and tryptophan-independent convertants 50 times. At equal survival levels, AG was found to be 3000-fold more mutagenic and 200-fold more convertogenic than sodium azide.     

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived‐flavonoid monomers using a three‐dimensional stem cell‐derived neural model

An in vitro three‐dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell‐derived neural co‐culture model. Rat neural stem cells were differentiated into co‐culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two‐dimensional (2D) model using a two‐step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase‐3 activity in a dose‐dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co‐cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735–744, 2016     

Living in three dimensions

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.     

Recent and advanced therapy for oral cancer

Oral cancer is a common and deadly kind of tissue invasion, has a high death rate, and may induce metastasis that mostly affects adults over the age of 40. Most in vitro traditional methods for studying cancer have included the use of monolayer cell cultures and several animal models. There is a worldwide effort underway to reduce the excessive use of laboratory animals since, although being physiologically adequate, animal models rarely succeed in exactly mimicking human models. 3D culture models have gained great attention in the area of biomedicine because of their capacity to replicate parent tissue. There are many benefits to using a drug delivery approach based on nanoparticles in cancer treatment. Because of this, in vitro test methodologies are crucial for evaluating the efficacy of prospective novel nanoparticle drug delivery systems. This review discusses current advances in the utility of 3D cell culture models including multicellular spheroids, patient-derived explant cultures, organoids, xenografts, 3D bioprinting, and organoid-on-a-chip models. Aspects of nanoparticle-based drug discovery that have utilized 2D and 3D cultures for a better understanding of genes implicated in oral cancers are also included in this review.     

Advances in generation of three-dimensional skin equivalents: pre-clinical studies to clinical therapies

The inability of two-dimensional cell culture systems to adequately map the structure and function of complex organs like skin necessitates the development of three-dimensional (3D) skin models. A diverse range of 3D skin equivalents have been developed over the last few decades for studying complex properties of skin as well as for drug discovery and clinical applications for skin regeneration in chronic wounds, such as diabetic foot ulcers, where the normal mechanism of wound healing is compromised. These 3D skin substitutes also serve as a suitable alternative to animal models in industrial applications and fundamental research. With the emergence of tissue engineering, new scaffolds and matrices have been integrated into 3D cell culture systems, along with gene therapy approaches, to increase the efficacy of transplanted cells in skin regeneration. This review summarizes recent approaches to the development of skin equivalents as well as different models for studying skin diseases and properties and current therapeutic applications of skin substitutes.     

3D Cultures of Prostate Cancer Cells Cultured in a Novel High-Throughput Culture Platform Are More Resistant to Chemotherapeutics Compared to Cells Cultured in Monolayer

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

MS imaging of multicellular tumor spheroids and organoids as an emerging tool for personalized medicine and drug discovery

MS imaging (MSI) is a powerful tool in drug discovery because of its ability to interrogate a wide range of endogenous and exogenous molecules in a broad variety of samples. The impressive versatility of the approach, where almost any ionizable biomolecule can be analyzed, including peptides, proteins, lipids, carbohydrates, and nucleic acids, has been applied to numerous types of complex biological samples. While originally demonstrated with harvested organs from animal models and biopsies from humans, these models are time consuming and expensive, which makes it necessary to extend the approach to 3D cell culture systems. These systems, which include spheroid models, prepared from immortalized cell lines, and organoid cultures, grown from patient biopsies, can provide insight on the intersection of molecular information on a spatial scale. In particular, the investigation of drug compounds, their metabolism, and the subsequent distribution of their metabolites in 3D cell culture systems by MSI has been a promising area of study. This review summarizes the different ionization methods, sample preparation steps, and data analysis methods of MSI and focuses on several of the latest applications of MALDI-MSI for drug studies in spheroids and organoids. Finally, the application of this approach in patient-derived organoids to evaluate personalized medicine options is discussed.     

From 3D cell culture to organs-on-chips

3D cell-culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional 2D culture systems. We review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue interfaces, spatiotemporal chemical gradients, and mechanical microenvironments of living organs. These 'organs-on-chips' permit the study of human physiology in an organ-specific context, enable development of novel in vitro disease models, and could potentially serve as replacements for animals used in drug development and toxin testing.     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Lack of coordination between glucose-6-phosphatase and gamma glutamyltranspeptidase activities in rat hepatocytes maintained in primary culture

Summary Glucose-6-phosphatase activity decreases whereas gamma glutamyltranspeptidase activity increases during hepatocarcinogenesis and the maintenance of hepatocytes in primary culture. This report describes the effect of culture conditions that are known to preserve hepatic glucose-6-phosphatase activity on gamma glutamyltranspeptidase activity. The results indicate that the regulation of glucose-6-phosphatase and gamma glutamyltranspeptidase activities is not coordinated in primary cultures of hepatocytes. This work was supported by a grant from the USPHS, NIH grant AG00439 awarded to Dr. Christopher C. Widnell and a Category I Research Development Award from the University of Pittsburgh to Dr. Kathleen Dobrosielski-Vergona. Editor's Statement Information communicated in this article contributes to a greater understanding of the mechanisms regulating liver cell metabolism and provides some further insight concerning the complexity of the controls involved in vitro, and presumably in vivo. David W. Barnes     

Proteomic Analysis of Stromal and Epithelial Cell Communications in Human Endometrial Cancer Using a Unique 3D Co‐Culture Model

Epithelial and stromal communications are essential for normal uterine functions and their dysregulation contributes to the pathogenesis of many diseases including infertility, endometriosis, and cancer. Although many studies have highlighted the advantages of culturing cells in 3D compared to the conventional 2D culture system, one of the major limitations of these systems is the lack of incorporation of cells from non‐epithelial lineages. In an effort to develop a culture system incorporating both stromal and epithelial cells, 3D endometrial cancer spheroids are developed by co‐culturing endometrial stromal cells with cancerous epithelial cells. The spheroids developed by this method are phenotypically comparable to in vivo endometrial cancer tissue. Proteomic analysis of the co‐culture spheroids comparable to human endometrial tissue revealed 591 common proteins and canonical pathways that are closely related to endometrium biology. To determine the feasibility of using this model for drug screening, the efficacy of tamoxifen and everolimus is tested. In summary, a unique 3D model system of human endometrial cancer is developed that will serve as the foundation for the further development of 3D culture systems incorporating different cell types of the human uterus for deciphering the contributions of non‐epithelial cells present in cancer microenvironment.     

Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.