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.     

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.     

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.     

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.     

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.     

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.     

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.     

Targeted and Oxygen-Enriched Nanoplatform for Enhanced Photodynamic Therapy: In Vitro 2D Cell and 3D Spheroid Model Evaluation

Hypoxic microenvironment and limited penetration of photosensitizers within solid tumors are two crucial factors that restrict photodynamic therapy (PDT) efficacy. Herein, a new fluorinated mixed micelle ( M60@PFC-Ce6 ) is developed as a tumor-penetrating and oxygen-enriching nanoplatform, which consists of chlorin e6 (Ce6) and perfluorocarbons (PFCs) co-loaded into fluorinated micelles to relieve hypoxia conditions as well as folate as targeting ligand that facilitates the selective biodistribution within tumor solids. The incorporation of fluorinated copolymers into mixed micelles exhibits not only a great increase in the oxygen-loading capacity, but also improves the stability of liquid PFCs emulsion within micelles without leakage. M60@PFC-Ce6 shows excellent oxygen delivery capability, good intracellular reactive oxygen species (ROS) generation, and superior phototoxicity in vitro for both 2D monolayer of cells and 3D multicellular spheroid model. These results indicate the enriched oxygen delivery and increased cellular uptake resulting from folate-targeted ability to enhance ROS production and PDT efficacy. The penetration study of M60@PFC-Ce6 into a 3D spheroid confirms that small micellar size and folate-conjugation are beneficial for micelles to penetrate and accumulate within spheroids. Thus, a new nanoplatform with enriched oxygen-carrying amounts, better drug penetration, and stable micellar properties that relieve tumor hypoxia and improve PDT efficacy is provided.     

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.     

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.     

Highly robust protein production by co-culture of CHO spheroids layered on feeder cells in serum-free medium

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.     

扫描电化学显微镜在生物医学领域的应用研究进展

研究病变细胞和组织的异常表现可为理解重大疾病发生发展的病理机理和新型药物筛选提供重要参考.扫描电化学显微镜(Scanning electrochemical microscopy,SECM)是一种基于电化学原理的扫描探针显微镜,通过记录探针在样品表面扫描时的电流或电位等信息,对活细胞的形态和多种化学信息进行原位、实时、精准表征.近10年来,SECM在重大疾病相关的细胞、细胞球和微组织层次的应用研究得到快速发展.本文从与疾病相关的SECM研究角度入手,分别从单细胞、细胞球和微组织层次小结SECM近10年来在生物医学领域的应用研究进展.首先介绍SECM的仪器组成、探针种类和工作模式,其次分别介绍SECM在神经细胞、心肌细胞和肿瘤细胞的应用进展,之后介绍SECM近期在细胞球和微组织的最新应用,最后提出并展望SECM技术在生物医学领域进一步应用所面临的挑战和发展方向.     

Quantitative bioimaging of platinum group elements in tumor spheroids

Limited drug penetration into tumor tissue is a significant factor to the effectiveness of cancer therapy. Tumor spheroids, a 3D cell culture model system, can be used to study drug penetration for pharmaceutical development. In this study, a method for quantitative bioimaging of platinum group elements by laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS) is presented. Different matrix-matched standards were used to develop a quantitative LA-ICP-MS method with high spatial resolution. To investigate drug penetration, tumor spheroids were incubated with platinum complexes (Pt(II)acetylacetonate, cisplatin) and the palladium tagged photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). Distribution and accumulation of the pharmaceuticals were determined with the developed method.     

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.     

A Rho Kinase (ROCK) Inhibitor,Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.     

GROWTH DELAY STUDIES OF THE RESPONSE OF V-79 MULTICELL SPHEROIDS EXPOSED TO DHE and RED LIGHT

Abstract Multicell tumour spheroids (MTS) of V-79 Chinese hamster cells have been used to study the role of a number of treatment and microenvironmental parameters in the modification of tumour response to Photodynamic Therapy (PDT) using visible light in combination with the photosensitizing compound dihematoporphyrin ether (DHE). The kinetics of DHE uptake into MTS, determined by fluorimetry of extracted porphyrins, indicate that after extended incubation (i.e. 24 h) the mean cellular DHE content in larger (˜300 μ.m and 400 u.m) MTS is significantly less than that for smaller (˜200 μm) MTS, consistent with a hypothesis that DHE uptake into the internal regions of spheroids is diffusion-limited. The response of spheroids to PDT, as assessed by the endpoint of growth delay, indicates that the kinetics of spheroid volume alteration and cell loss, as well as the potential for regfrrwth, are markedly dependent on both the drug and light exposure levels used. The oxygen dependence of this response has been investigated after light irradiation of spheroid cultures equilibrated with either 21% O₂ (i.e. air) or 0% 0₂ (i.e. N₂). While treatment in air results in significant growth delay, the growth kinetics of DHE-treated spheroids irradiated under N₂ were essentially unchanged from those of untreated spheroids. These observations clearly demonstrate an important role for oxygen, at the time of irradiation, in determining the response of spheroids to PDT.     

Instructed Assembly as Context‐Dependent Signaling for the Death and Morphogenesis of Cells

Context‐dependent signaling is a ubiquitous phenomenon in nature, but ways to mimic the essence of these nano‐ and microscale dynamic molecular processes by noncovalent synthesis in the cellular environment have yet to be developed. Herein we present a dynamic continuum of noncovalent filaments formed by the instructed assembly (iA) of a supramolecular phosphoglycopeptide (sPGP) as context‐dependent signals for controlling the death and morphogenesis of cells. Specifically, ectophosphatase enzymes on cancer cells catalyze the formation of sPGP filaments to result in cell death; however, damping of the enzyme activity induces the formation 3D cell spheroids. Similarly, the ratio of stromal and cancer cells in a coculture can be used to modulate the expression of the ectophosphatase, so that the iA process leads to the formation of cell spheroids. The spheroids mimic the tumor microenvironment for drug screening.     

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.     

Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.     

Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.