A multicellular spheroid formation and extraction chip using removable cell trapping barriers

This paper presents a multicellular spheroid chip capable of forming and extracting three-dimensional (3D) spheroids using removable cell trapping barriers. Compared to the conventional macro-scale spheroid formation methods, including spinning, hanging-drop, and liquid-overlay methods, the recent micro-scale spheroid chips have the advantage of forming smaller spheroids with better uniformity. The recent micro spheroid chips, however, have difficulties in extracting the spheroids due to fixed cell trapping barriers. The present spheroid chip, having two PDMS layers, uses removable cell trapping barriers, thereby making it easy to form and extract uniform and small-sized spheroids. We have designed, fabricated and characterized a 4 × 1 spheroid chip, where membrane cell trapping barriers are inflated at a pressure of 50 kPa for spheroid formation and are deflated at zero gauge pressure for simple and safe extraction of the spheroids formed. In this experimental study, the cell suspension of non-small lung cancer cells, H1650, is supplied to the fabricated spheroid chip in the pressure range 145-155 Pa. The fabricated spheroid chips collect the cancer cells in the cell trapping regions from the cell suspension at a concentration of 2 × 10(6) ml(-1), thus forming uniform 3D spheroids with a diameter of 197.2 ± 11.7 μm, after 24 h incubation at 5% CO(2) and 37°C environment. After the removal of the cell trapping barriers, the spheroids formed were extracted through the outlet ports at a cell inlet pressure of 5 kPa. The cells in the extracted spheroids showed a viability of 80.3 ± 7.7%. The present spheroid chip offers a simple and effective method of obtaining uniform and small-sized 3D spheroids for the next stage of cell-based biomedical research, such as gene expression analysis and spheroid inoculation in animal models.     

Microfluidic platform for studying the anti-cancer effect of ursolic acid on tumor spheroid

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.     

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.     

Comparison of protein expression profiles between monolayer and spheroid cell culture of HT-29 cells revealed fragmentation of CK18 in three-dimensional cell culture

The use of three-dimensional cell culture models, so-called multicellular tumor spheroids, is a special approach in experimental cancer research, because spheroids are similar to in vivo tumors in structural as well as functional sense. Cells grown in spheroids exhibit alterations of cell cycle regulation, induction of apoptosis and differentiation and can acquire multidrug resistance. In this study we investigated the protein expression in human colorectal cancer cells grown in monolayer and in spheroid cultures using proteomics. Evaluation by computer-assisted image analysis revealed overexpression of three cytokeratin 18 fragments that were generated in vivo. Cytokeratin 18 has previously been described as a target for caspase-mediated cleavage during apoptosis and our results indicate that apoptosis may take place in spheroids. Other proteins upregulated in spheroids include calreticulin precursor, a rho GDP dissociation inhibitor variant, several cytokeratins and peroxiredoxin 4. Some of these proteins have already been linked to chemoresistance and apoptotic phenomena.     

On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.     

High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.     

Diffusion-mediated in situ alginate encapsulation of cell spheroids using microscale concave well and nanoporous membrane

Here, we present a novel and simple process of spheroid formation and in situ encapsulation of the formed spheroid without intervention. A hemispherical polydimethylsiloxane (PDMS) micromold was employed for the formation of uniform sized spheroids and two types of nano-porous membrane were used for the control of the crosslinking agent. We characterized the transport properties of the membrane, and the selection of alginate hydrogel as a function of gelation time, alginate concentration, and membrane type. Using the developed process and micromold, HepG2 cell spheroids were successfully formed and encapsulated in alginate without replating. This method allows spheroid encapsulation with minimal damage to the spheroid while maintaining high cell viability. We demonstrate the feasibility of this method in developing a bio-artificial liver (BAL) chip by evaluating viability and function of encapsulated HepG2 spheroids. This method may be applied to the encapsulation of several aggregating cell types, such as β-cells for islet formation and stem cells for embryonic body preservation, or as a model for tumor cell growth and proliferation in a 3D hydrogel environment.     

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 μm. The diffusive limit of competition for media occurred with a pitch of ≥1250 μm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

Poly(N‐isopropylacrylamide)‐Based Polymers as Additive for Rapid Generation of Spheroid via Hanging Drop Method

Multicellular tumor spheroid (MCTS) mimics microenvironment for tumor formation and provides predictive insight for in vivo tests. The hanging drop (HD) method of spheroid generation is cost effective, but it is limited by a long time duration for spheroid development and a low rate of formation of larger spheroids. Toward addressing those limitations, thermoresponsive copolymers with poly(N‐isopropylacrylamide) (p(NIPA)) backbone are developed, to be used as additives in the MCTS formation via HD method. Upon investigation it is found that in the presence of the polymer, robust and compact spheroids are formed in a short duration of 48 h. Larger spheroids (350–600 µm) can be formed by increasing the number of cells. Spheroids are characterized for their 3D shape and different cellular layers, and drug uptake study is done to prove the efficacy of the spheroids generated in drug screening.     

MECHANISMS BEHIND THE RESISTANCE OF SPHEROIDS TO PHOTODYNAMIC TREATMENT: A FLOW CYTOMETRY STUDY

The influence of cell heterogeneity on response to photodynamic treatment (PDT) has been investigated using the human colon adenocarcinoma line WiDr, grown as spheroids and exposed to hematoporphyrin derivative. The spheroids show a marked spheroid size-dependent resistance to PDT. Using a flow cytometer, cell sub-populations have been separated, on the basis of drug fluorescence, from single cell suspensions prepared from 500 microm diameter spheroids. Cells low in fluorescence have been shown to be resistant to PDT, have a smaller median cell volume, and be enhanced in G1-type cells. These cells also show reduced low density lipoprotein uptake. The results suggest that spheroid size-dependent resistance to PDT is related to a decreasing growth fraction with increasing spheroid size. Heterogeneity of drug uptake could be a potential limitation to clinical PDT.     

Studies of anticancer drug cytotoxicity based on long‐term HepG2 spheroid culture in a microfluidic system

Cell‐on‐a‐chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high‐throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5‐fluorouracil, 5‐FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long‐term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5‐FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5‐FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5‐FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.     

Spheroid cultivation of HT-29 carcinoma cell line in liquid marbles

The ability to simulate the 3D structure of a human body is essential to increase the efficiency of drug development. In vivo conditions are significantly different in comparison to in vitro conditions. A standardly used cell monolayer on tissue culture plastic (2D cell culture) is not sufficient to simulate the transfer phenomena occurring in living organisms, therefore, cell growth in a 3D space is desired. Drug absorption, distribution, metabolism, excretion and toxicity could be tested on 3D cell aggregates called spheroids, decrease the use of animal models and accelerate the drug development. In this work, the formation of spheroids from HT-29 human colorectal adenocarcinoma cells was successfully achieved by means of the so-called liquid marbles, which are liquid droplets encapsulated by a hydrophobic powder. During the cultivation in the medium inside the liquid marbles, cells spontaneously formed spherical agglomerates (spheroids) without the need of any supporting scaffold. The study focused on the influence of different parameters—namely liquid marble volume, seeding cell density and time of cultivation—on the final yield and quality of spheroids. This work has shown that using liquid marbles as microbioreactors is a suitable method for the cultivation of HT-29 cells in the form of spheroids.     

Photodynamic Efficacy of Cercosporin in 3D Tumor Cell Cultures

In the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco-PDT, in contrast to the other two cell lines (T47D and T98G). While 300 μm diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 μm) start to regrow following blue-light PDT and exhibit high viability. Cerco-PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (~590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco-PDT. U87 spheroids also experienced a respiratory collapse following cerco-PDT, but retained half their glycolytic activity.     

Cell Spheroid Formation on the Surface of Multi-Block Copolymers Composed of Poly(2-methoxyethyl acrylate) and Polyethylene Glycol

3D structured cells have great drug screening potential because they mimic in vivo tissues better than 2D cultured cells. In this study, multi-block copolymers composed of poly(2-methoxyethyl acrylate) (PMEA) and polyethylene glycol (PEG) are developed as a new kind of biocompatible polymers. PEG imparts non-cell adhesion while PMEA acts as an anchoring segment to prepare the polymer coating surface. The multi-block copolymers show higher stability in water than PMEA. A specific micro-sized swelling structure composed of a PEG chain is observed in the multi-block copolymer film in water. A single NIH3T3-3-4 spheroid is formed in 3 h on the surface of the multi-block copolymers with 8.4 wt% PEG. However, at a PEG content of 0.7 wt%, spheroid formed after 4 days. The adenosine triphosphate (ATP) activity of cells and the internal necrotic state of the spheroid change depending on PEG loading in the multi-block copolymers. As the formation rate of cell spheroid on low-PEG-ratio multi-block copolymers is slow, internal necrosis of cell spheroid is less likely to occur. Consequently, the cell spheroid formation rate by changing the PEG chain content in multi-block copolymers is successfully controlled. These unique surfaces are suggested to be useful for 3D cell culture.     

Drug testing on 3D in vitro tissues trapped on a microcavity chip

Close to realistic responses to anti-cancer drugs are not adequately provided in monolayer or single cells assays. 3-dimensional multicellular cultures (spheroids) mimicking in vivo-like conditions are established as cell biological models for microtumors/metastases. For a non-invasive real-time monitoring of the electrical parameters of such spheroid cultures we designed, fabricated and tested a 3D multifunctional electrode-based microcavity array. In a non-adherent assay acute tests with tumor spheroids were done maintaining their spherical shape and cellular arrangement. The sensor chip with 15 individual square microcavities containing four gold electrodes each was used for impedance spectroscopy to analyze the tissue models in terms of morphological and structural changes. Cell type specific differences in the spectra and varying responses to several anti-tumor drugs were found. Further development of the prototype will provide a promising tool for the use in pharmacological high-throughput studies.     

Self‐Assembling Peptide Gels for 3D Prostate Cancer Spheroid Culture

Progress in prostate cancer research is presently limited by a shortage of reliable in vitro model systems. The authors describe a novel self‐assembling peptide, bQ13, which forms nanofibers and gels useful for the 3D culture of prostate cancer spheroids, with improved cytocompatibility compared to related fibrillizing peptides. The mechanical properties of bQ13 gels can be controlled by adjusting peptide concentration, with storage moduli ranging between 1 and 10 kPa. bQ13's ability to remain soluble at mildly basic pH considerably improved the viability of encapsulated cells compared to other self‐assembling nanofiber‐forming peptides. LNCaP cells formed spheroids in bQ13 gels with similar morphologies and sizes to those formed in Matrigel or RADA16‐I. Moreover, prostate‐specific antigen (PSA) is produced by LNCaP cells in all matrices, and PSA production is more responsive to enzalutamide treatment in bQ13 gels than in other fibrillized peptide gels. bQ13 represents an attractive platform for further tailoring within 3D cell culture systems.     

Investigation of the Effective Action Distance Between Hematopoietic Stem/Progenitor Cells and Human Adipose-Derived Stem Cells During Their In Vitro Co-culture

The in vitro suitable action distance between umbilical cord blood-derived hematopoietic stem/progenitor cells and its feeder cell, human adipose-derived stem cells, during their co-culture, was investigated through a novel transwell co-culture protocol, in which the distance between the two culture chambers where each cell type is growing can be adjusted from 10 to 450 μm. The total cell number was determined with a hemacytometer, and the cell morphology was observed under an inverted microscope each day. After 7 days of co-culture, the fold-expansion, surface antigen expression of CD34(+) and CFU-GM assay of the hematopoietic mononuclear cells (MNCs) were analyzed. The results showed that there was an optimal communication distance at around 350 μm between both types of stem cells during their in vitro co-culture. By using this distance, the UCB-MNCs and CD34(+) cells were expanded by 15.1?±?0.2 and 5.0?±?0.1-fold, respectively. It can therefore be concluded that the optimal action distance between stem cells and their supportive cells, when cultured together for 7 days, is of around 350 μm.     

A mesoscale modelling study of nematic liquid crystals confined to ellipsoidal domains

Director configurations of nematic liquid crystalline molecules packed in ellipsoidal domains have been investigated using mesoscale modelling techniques. Interactions between the directors were described by the Lebwohl-Lasher potential. Four different ellipsoidal shapes (sphere, oblate spheroid, prolate spheroid, and ellipsoid) were studied under homogeneous and homeotropic surface anchoring conditions. The model has been characterized by computing thermodynamic and structural properties as a function of ellipsoidal shape (prolate and oblate) and size. The predicted director configuration in ellipsoids resulting from homeotropic surface anchoring is found to be very different from that in spherical domains. The bipolar configuration involving homogeneous surface anchoring is nearly identical in the four cases. The effect of an external electric field, applied at different orientations with respect to the major axis of the ellipsoid, has been probed as a function of the magnitude of the field and the ellipsoidal size and shape. The orientation of directors is most easily accomplished parallel and perpendicular to the major axis for the oblate and prolate spheroids, respectively, for homeotropic anchoring, and along the bipolar symmetry axis for homogeneous anchoring. In domains with homeotropic surface anchoring, the oblate spheroid and elongated ellipsoid are predicted to be the most efficient geometries for PDLC applications; for homogeneous anchoring conditions, the prolate spheroid and elongated ellipsoid are predicted to be the most efficient.