Organoid technologies meet genome engineering

Three‐dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two‐dimensional (2D) cell culture models. Organoids, the 3D self‐organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease‐associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.     

Electromagnetic exposure of scaffold-free three-dimensional cell culture systems

In recent years, a number of in vitro studies have reported on the possible athermal effects of electromagnetic exposure on biological tissue. Typically, this kind of study is performed on monolayers of primary cells or cell lines. However, two‐dimensional cell layer systems lack physiological relevance since cells in vivo are organized in a three‐dimensional (3D) architecture. In monolayer studies, cell‐cell and cell‐ECM interactions obviously differ from live tissue and scale‐ups of experimental results to in vivo systems should be considered carefully. To overcome this problem, we used a scaffold‐free 3D cell culture system, suitable for the exploration of electrophysiological effects due to electromagnetic fields (EMF) at 900 MHz. Dissociated cardiac myocytes were reaggregated into cellular spheres by constant rotation, and non‐invasive extracellular recordings of these so‐called spheroids were performed with microelectrode arrays (MEA). In this study, 3D cell culture systems were exposed to pulsed EMFs in a stripline setup. We found that inhomogeneities in the EMF due to electrodes and conducting lines of the MEA chip had only a minor influence on the field distribution in the spheroid if the exposure parameters were chosen carefully. Bioelectromagnetics 32:351–359, 2011. © 2011 Wiley‐Liss, Inc.     

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior¹. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system^2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D⁴. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved³. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D^6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.     

Raman spectroscopy investigation of biochemical changes in tumor spheroids with aging and after treatment with staurosporine

There has been increasing use of in vitro cell culture models that more realistically replicate the three‐dimensional (3D) environment found in vivo. Multicellular tumor spheroids (MTS) using cell lines or patient‐derived organoids have become an important in vitro drug development tool, where cells are grown in a 3D “sphere” that exhibits many of the characteristics found in vivo. Significantly, MTS develop gradients in nutrients and oxygen, commonly found in tumors that contribute to therapy resistance. While MTS show promise as a more realistic in vitro culture model, there is a massive need to improve imaging technologies for assessing biochemical characteristics and drug response in such models to maximize their translation into useful applications such as high throughput screening (HTS). In this study, we investigate the potential for Raman spectroscopy to unveil biochemical information in MTS and have investigated how spheroid age influences drug response, shedding light on increased therapy resistance in developing tumors. The wealth of molecular level information delivered by Raman spectroscopy in a noninvasive manner, could aid translation of these 3D models into HTS applications.      

Simulation and evaluation of 3D traction force microscopy

Measuring cell-generated forces by Traction Force Microscopy (TFM) has become a standard tool in cell mechanobiology. Although widely used in two dimensional (2D) experiments, only a few methods exist to measure traction in three-dimensional (3D) cell culture, since 3D volumetric high-resolution microscopy and more demanding computational approaches are required. Although it is commonly known that the selected experimental and computational setup highly influence the quality and accuracy of the results, no existing methods can adequately assess the errors involved in this process. We present a fully integrated simulation and evaluation platform that allows one to simulate TFM images and quantify errors of an applied approach for traction stress reconstruction, in order to improve experiments that attempt to measure mechanical interaction in cellular systems. In this context, we show that a careful parameter selection can decrease the reconstructed traction error by up to 40%.     

The Effects of Mycoplasma Contamination upon the Ability to Form Bioengineered 3D Kidney Cysts

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.     

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     

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.     

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening

Three‐dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative antibody staining, high‐content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model‐based prediction of spatially resolved affinities of proteins. As a proof‐of‐concept, we mapped twelve epitopes in 3D‐cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell culture combined with our automated high‐content assay will inspire future functional protein expression and drug assays.     

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.     

Three-dimensional perfused cell culture

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.     

Glutathione-Triggered Formation of a Fmoc-Protected Short Peptide-Based Supramolecular Hydrogel

A biocompatible method of glutathione (GSH) catalyzed disulfide bond reduction was used to form Fmoc-short peptide-based supramolecular hydrogels. The hydrogels could form in both buffer solution and cell culture medium containing 10% of Fetal Bovine Serum (FBS) within minutes. The hydrogel was characterized by rheology, transmission electron microscopy, and fluorescence emission spectra. Their potential in three dimensional (3D) cell culture was evaluated and the results indicated that the gel with a low concentration of the peptide (0.1 wt%) was suitable for 3D cell culture of 3T3 cells. This study provides an alternative candidate of supramolecular hydrogel for 3D cell culture and cell delivery.     

Three‐dimensional cell culture model utilization in cancer stem cell research

Three‐dimensional (3D) cell culture models are becoming increasingly popular in contemporary cancer research and drug resistance studies. Recently, scientists have begun incorporating cancer stem cells (CSCs) into 3D models and modifying culture components in order to mimic in vivo conditions better. Currently, the global cell culture market is primarily focused on either 3D cancer cell cultures or stem cell cultures, with less focus on CSCs. This is evident in the low product availability officially indicated for 3D CSC model research. This review discusses the currently available commercial products for CSC 3D culture model research. Additionally, we discuss different culture media and components that result in higher levels of stem cell subpopulations while better recreating the tumor microenvironment. In summary, although progress has been made applying 3D technology to CSC research, this technology could be further utilized and a greater number of 3D kits dedicated specifically to CSCs should be implemented.     

Growth and survival signalling in B16F10 melanoma cells in 3D culture

The two‐way communication between the ECM (extracellular matrix) and the cytoplasm via the integrins has many functions in cancer cells, including the suppression of apoptosis. As cells in a 3D (three‐dimensional) architecture resemble the in vivo situation more closely than do cells in more conventional 2D cultures, we have employed a substratum that prevents cell adhesion and induces cell aggregation to determine why highly metastatic B16F10 melanoma cells resist anoikis. We compared the behaviour of B16F10 cells in 2D [on tPS (tissue culture polystyrene)] and 3D culture {on polyHEMA [poly(2‐hydroxyethylmethacrylate)]} configurations. For this, we analysed cell morphology, proliferation, apoptosis and the activation status of several proteins involved in cell proliferation and survival [RhoA, FAK (focal adhesion kinase), Akt, ERK1/2 (extracellular‐signal‐regulated kinase 1/2)]. B16F10 cells in 3D architecture were able to proliferate as cell aggregates for 3 days, after which the number of cells decreased. The normal Swiss 3T3 cells used as an anoikis‐sensitive control did not proliferate on the anti‐adhesive substratum. Rho A was activated in B16F10 aggregates throughout their time in culture, whereas it was not in Swiss 3T3 aggregates. An absence of apoptotic activity was correlated with the proliferation of B16F10 cells in aggregates: caspase 3 was significantly activated only after 3 days in culture on polyHEMA. FAK and Akt were transiently activated, and their inactivation was correlated with the induction of apoptosis. ERK1/2 were activated throughout the 3D culture. No survival protein was activated in Swiss 3T3 aggregates. Data obtained from cells in 3D culture suggest that B16F10 cells are resistant to anoikis through the activation of the FAK and Akt signalling pathways.     

Culture phases,cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures

Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.     

Fibrin scaffold enhances function of insulin producing cells differentiated from human umbilical cord matrix-derived stem cells

Tissue engineering is a new strategy which proposed to treat numerous human diseases nowadays. Three dimensional (3D) scaffolds fill the gap between two dimensional cell culture (2D) and animal tissues through mimicking the environmental behaviors surrounding the cells. In this study, hUCMs into insulin producing cells in fibrin scaffold were differentiated compare to conventional culture condition. Differentiation rate was estimated by real time PCR, immunocytochemistry (ICC) and the chemiluminesence (CLIA) and enzyme immunoassay (EIA). Real time PCR's results showed an increasing expression in NKX2.2, PDX1 and INS (producing the hormone insulin) genes in fibrin scaffold. Furthermore ICC analysis exhibited that insulin and pro-insulin proteins were more in fibrin scaffolds. CLIA and EIA on insulin and C peptide secretion indicated that both of groups were sensitive to the glucose challenge test but significant higher response was observed in fibrin scaffold (6.5 fold in 3D, 1.8 fold in 2D culture). It could be concluded that differentiation of hUCM cells into insulin producing cells in fibrin scaffold 3D culture system is much more efficient than 2D conventional culture system.     

Three‐dimensional culture systems for the expansion of pluripotent embryonic stem cells

Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two‐dimensional (2D) static culturing techniques are inadequate for large‐scale production. The culture of mammalian cells in three‐dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher‐S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher‐S out‐performed the Solohill microcarriers. When cultured for successive passages on Cultispher‐S microcarriers, mESCs maintained their pluripotency, demonstrated by self‐renewal, expression of pluripotency markers and the ability to undergo multi‐lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher‐S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra‐1–60, NANOG, and OCT‐4. Our study highlights the importance of optimization of initial seeding parameters and provides proof‐of‐concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695. © 2010 Wiley Periodicals, Inc.