Patterning hypoxic multicellular spheroids in a 3D …ir.sia.cn/bitstream/173321/17615/1/Patterning hypoxic...plate cultures [22], hanging drops [23] and cell-hydrogel mixed spheroid

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Biotechnol. J. 2016, 11, 127–134 DOI 10.1002/biot.201500183

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1 Introduction

The preclinical validation process in cancer drug dis-covery generally involves a series of biochemical and cell-based assays followed by costly and time-consuming animal testing. However, the current system often leads to failure of drug compounds late in their development and following clinical trials. The federal Food and Drug Administration (FDA) approves less than 10% of new drugs [1]. Traditional two-dimensional (2D) cell cultures

made for testing anti-cancer drugs are simple and easy to operate. However, it is still difficult to reproduce the real and complex tumor microenvironment of the human body [2]. There is overwhelming evidence that in vitro three-dimensional (3D) culture models may more accu-rately reproduce the complexity and pathophysiology of in vivo tumor microenvironments in terms of their gene expression profiles, signaling pathway activity and drug sensitivity [3–5], and may thus be ideal tools for testing anti-cancer drugs.

Various techniques have been developed for 3D cul-turing [6], such as cell cultures in gel materials [7], cell aggregation to produce solid structures [8] and mixed cultures on porous scaffolds [9, 10]. Such techniques are likely to recapitulate the in vivo tumor microenviron-ment in a physiologically relevant manner [11–13]. Tumor

Research Article

Patterning hypoxic multicellular spheroids in a 3D matrix – a promising method for anti-tumor drug screening

Jingyun Ma1,2,3,*, Xu Zhang1,*, Yang Liu1, Haibo Yu3, Lianqing Liu3, Yang Shi1, Yanfeng Li1 and Jianhua Qin1

1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China2 The First Affiliated Hospital of Dalian Medical University, Dalian, China3 State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang, China

3D multicellular spheroid models are of great value in the investigation of tumor biology and tumor responses to chemotherapy and radiation. To establish a mimicking tumor microenvi-ronment in vitro, we developed a straightforward method by patterning hypoxic multicellular spheroids in a 3D matrix. The efficacy of this approach was evaluated by characterizing spheroid formation, invasive capability and phenotypic transition in aggressive human glioma cells. We observed enhanced cell proliferation, spheroid formation and invasive capability in U87 glioma cells transfected with hypoxia-inducible factors (HIFs) compared with non-treated cells. We also demonstrated that the overexpression of HIFs in hypoxic glioma cells may promote cell migration by epithelial-mesenchymal transition within the 3D matrix. Compared with conventional 3D cul-turing techniques, the simple operation, rapid prototyping, low cost and high throughput format of the micro-patterning method facilitates the characterization of cell proliferation, migration, phenotypic function and drug evaluation in physiologically relevant 3D microenvironments. This in vitro 3D system can recapitulate the physiologically relevant tumor microenvironment and is a promising method for 3D anti-tumor drug screening and the identification of novel targets for tumor invasion and angiogenesis.

Keywords: Drug screening · Hypoxia-inducible factors (HIFs) · Micro-patterning · Multicellular spheroids · Phenotype transition

Correspondence: Dr. Jianhua Qin, Dalian Institute of Chemical Physics, 457 Zhongshan Road, 116023, Dalian, China E-mail: [email protected]

Abbreviations: ECM, extracellular cell matrix; EMT, epithelial-mesenchymal transition; GFP, green fluorescent protein; HIFs, hypoxia-inducible factors; PDMS, poly-dimethylsiloxane; U87-HA-HIF1α, U87 cell lines transfected with HIF1α; U87-HA-HIF2α, U87 cell lines transfected with HIF2α

Received 12 APR 2015Revised 03 NOV 2015Accepted 07 DEC 2015Accepted article online 08 DEC 2015

* These authors contributed equally to this work.

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spheroids are heterogeneous cellular aggregates that are often characterized by multicellular components within a 3D extracellular matrix and hypoxic or necrotic regions. These multicellular tumor spheroids adopt similar pheno-types and respond to stimuli analogous to in vivo biologi-cal systems [14–17]. Thus, 3D spheroids are considered to be valid models for reproducing the properties of tumor microregions, intervascular regions and micrometastases [18, 19]. Since the first study on spheroid-based approach-

es in cancer research proposed by Sutherland et al. [20], much effort has been made to produce tumor spheroids via spontaneous aggregation [21], non-adherent well plate cultures [22], hanging drops [23] and cell-hydrogel mixed spheroid culturing systems [24]. A simple and high throughput approach for rapid and effective in vitro drug screening within a 3D extracellular cell matrix (ECM) has not been established. Such an approach would have to fulfill the requirements of the anti-cancer drug evaluation

Figure 1. (A) Schematic diagram of the glioma microenvironment and hypoxia-induced behavior in glioma cells. The microenvironment of malignant glioma is characterized by diffusely invasive-ness, hypoxia, and vascular proliferation. (B) Schematic diagram of the procedure for patterning multicellular spheroids in the 3D matrix. Steps 1–4: The process of fabricating the concave microwell structure. Steps 5–9: The procedure for producing and patterning multicellular spheroids in a 3D matrix.

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model to address the malignant phenotypes of dissemi-nated human tumors.

Malignant glioma is the most aggressive brain tumor type and has the highest fatality rate. Clinically, the inabil-ity to completely isolate or treat this high-grade glioma, or to inhibit the dispersion of cells from the primary tumor mass into the surrounding normal brain tissue, has result-ed in poor life prognosis among brain cancer patients. Malignant glioma is characterized by diffused invasive-ness, hypoxia, vascular proliferation, radioresistance and chemoresistance [25]. The tumor microenvironment of glioma cells is shown in Fig. 1A. Histopathological stud-ies confirm that invasion and thus a highly infiltrative phenotype is a major factor responsible for the ineffective treatment of malignant gliomas. It is well known that hypoxia is an independent prognostic factor of malignant tumors. Under low oxygen stress, hypoxia-inducible fac-tors (HIFs) function as master transcription factors that orchestrate the cellular response to hypoxia by mediating hypoxia-responsive genes involved in proliferation, sur-vival, invasion, angiogenesis, metastasis and resistance to chemo-radiation [26]. In malignant gliomas, hypoxia and HIF signaling pathways are recognized for the pivotal roles they play in the regulation of glioma aggressiveness. Some work has reported that HIF1α and HIF2α, two HIFα subunits, demonstrate competitive effects on the inva-sive behavior of gliomas [27, 28]. However, research on hypoxia-relevant glioma spheroid invasion in 3D micro-environments is still in its initial stages.

We propose a new and straightforward approach for patterning multicellular spheroids in 3D ECMs using a high throughput format that allows the evaluation of spheroid formation, growth and invasive ability in U87 glioma and hypoxic glioma cells exhibiting mesenchymal phenotype transition. Two varieties of transfected U87 cells were used to mimic in vivo hypoxia by transfecting HIF1α or HIF2α into the cells. The hypoxic tumor cells exhibited enhanced spheroid formation and invasive ability with mesenchymal phenotype transition compared with non-hypoxic cells. This approach demonstrated obvious advan-tages for quantitatively characterizing the dynamics of 3D tumor invasion by patterning spheroids in parallel within a natural extracellular matrix, thus facilitating the study of interactions between multicellular spheroids. This in vitro 3D system can recapitulate the physiologically relevant tumor microenvironment and is a promising method for 3D anti-tumor drug screening and the identification of novel targets of tumor invasion and angiogenesis.

2 Materials and methods

2.1 Design and fabrication of the microdevice

For multicellular spheroid formation, a hemispheric microwell device with a uniform diameter of 600 μm was

designed and fabricated. An SU-8 negative photoresist (3035, Micro Chem) was spun-coated on a clear glass, pre-baked for 60 min, exposed to a UV source for 60 s, immersed in ethyl lactate for incomplete development for 5 min, heated at 85°C for 5 min to change the structure into a hemispheric shape and exposed again to the UV source to fix the concave configuration. The depth and curvature of the microwell was adjusted by changing the developing and melting times of the SU-8. The final depth and width of the microwell were approximately 400 μm and 600 μm, respectively. Poly-dimethylsiloxane (PDMS, Sylgard 184, Dow Corning, USA) was chosen to construct the device due to its hydrophobicity and gas permeabil-ity. The PDMS hemispheric microwell device was com-pleted after two replications of the SU-8 template.

2.2 Generation of glioma spheroids

Human astrocytoma cell line U87 (Cell Bank of the Chi-nese Academy of Science, Shanghai, China) and two sub-lines stably transfected with the mutational form of hemagglutinin-tagged HIFs (U87-HA-HIF1α and U87-HA-HIF2α) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) basic (Life Technologies, Carlsbad, CA), which was supplemented with 10% fetal bovine serum at 37°C, 5% CO2, 21% O2 and constant humidity.

Before cell seeding in the PDMS hemispheric microw-ell, the surface of the device was immersed in 2% Pluron-icF-127 (Sigma-Aldrich, St Louis, USA) solution for 4 h and then washed twice with sterile water. The polymer adsorbed to the surface of the PDMS device, preventing cell attachment. Three types of glioma cell suspensions (U87, U87-HA-HIF1α and U87-HA-HIF2α) at optimized densities (1 × 105 cells/mL) were dropped onto the non-adhesive PDMS substrate replicated from the SU-8 mold with an array of microwell concave structure. After gentle shaking and standing for 30 min, the cells were trapped within the hemispheric microwells. A flow of culture medium was gently added to remove suspended cells from the microwells. The microchip was placed in humidified air with 5% CO2 at 37°C for three days to allow for cell sinking and multicellular spheroid formation.

To investigate cell proliferation and spheroid forma-tion in glioma cells, the different cell lines were seeded, at equal density (103 cells/mL), on the device at the same starting point. Cultures were maintained by replacing the medium on days 2, 4, 6, 8 and 10. Images were taken at intervals and growth curves were rapidly generated. The spheroid diameter as a function of culture time was used to calculate the cells’ growth curves.

2.3 Embedding glioma spheroids into the extracellular matrix

Multicellular spheroids in the microdevice were dropped with collagen type I (BD Biosciences, Bedford, MA, USA)




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solution at a diluted working concentration of 3 mg/mL. After crosslinking and solidifying the collagen mixture at 37°C for 30 min, once the spheroids were half-embedded with the first collagen layer, the collagen was overturned together with the PDMS microdevice and separated from the PDMS mold. By repeatedly adding and solidifying the collagen solution, the whole spheroids became embedded with collagen block. The tumor spheroids were fixed in the collagen matrix to investigate invasiveness by tak-ing photographs using an inverted phase contrast light microscope (Olympus IX-71, Japan) at indicated time points. By changing the position of the concave micro-wells in the microdevice, a controllable pattern of glioma spheroids in the collagen matrix was achieved.

2.4 Inhibitor treatment assay

The culture medium was treated with 40 μmol/L of pharmacologic HIF inhibitors following spheroid embed-ment to inhibit HIF1α-mediated transcription (Methyl-3-[[2-[4-(2-adamantyl)phenoxy]acetyl]amino]-4-hydroxy-benzoate, Santa Cruz Biotechnology, Santa Cruz, CA) and HIF2α translation (Methyl-3-(2-(cyano(methylsulfonyl)methylene)hydrazino)thiophene-2-carboxylate, Merck Millipore, Darmstadt, Germany). Bright images were taken by microscopy 24 h after treatment with the HIF inhibitors.

2.5 Fluorescence staining and imaging

The spheroids in the collagen matrix were treated with 4% paraformaldehyde (Sigma, USA), 0.1% Triton-X100 (Sigma) and normal goat serum, and incubated overnight with primary antibody against vimentin (Boster, Wuhan, CA) (1:100) at 4°C. After rinsing with PBS, the cells were incubated with TRITC-labeled anti-rabbit IgG for 45 min at room temperature. DAPI staining solution (Sigma) was used to stain nuclei and thus indicate cell positions. Fluorescent photographs were taken using a fluores-cence microscope (Olympus IX-71, Japan) and a confocal laser scanning biological microscope (Olympus FV1000, Japan). In the invasion assay, the spheroid specimen was divided into 40 layers for image analysis. Quantitative statistics were calculated by taking 40 individual stacks before z-axis projection for cell counting. To avoid repeat counting, each cell position was recorded using Image-Pro Plus software (Media Cybernetics, Rockville, MD). To characterize the 3D spheroid patterning method, U87 cells labeled with green fluorescent protein (GFP) were used for spheroid imaging.

3 Results

3.1 Facile and high throughput generation and patterning of glioma spheroids in a 3D matrix

We produced the high throughput multicellular spheroids on a microdevice fabricated with an array of concave con-figurations. As shown in Fig. 1B, the PDMS microdevice was generated following UV exposure, incomplete devel-opment and hot melting to SU-8 (the bottom structure of the sunken part changed from square to round due to incomplete development), and two PDMS replications from the SU-8 template. The high throughput concave array contained 3600 wells (60 × 60) over a total area of 25 cm2. After cell seeding onto the biocompatible and anti-adhesive PDMS microdevice, the glioma cells were physically aggregated and gradually experienced self-assembly. The mono-dispersed cells could then form 3D multi-cellular spheroids with uniform diameter. Following the formation of an array of tumor spheroids, complete packaging and patterning of glioma spheroids into the gels were achieved by covering and solidifying the col-lagen solution on the opposite sides of the spheroids.

As shown in Fig. 2A (i), with uniform and saturated seeding density, the dimensions of the cellular spheroids increased with the diameters of the microwell arrays. To obtain uniformly sized spheroids, we optimized the experimental conditions by culturing the cells for three days within the concave array with a diameter of 600 μm and a cell seeding density of 105 cells/mL. The average diameter of the spheroids was 300 μm, with a standard deviation of 1.56 (n = 50). The patterned spheroid configu-rations within the 3D matrix at the same seeding density are shown in Fig. 2A (ii).

3.2 Hypoxia enhanced proliferation and spheroid formation capability in glioma cells

Two varieties of HIF-transfected U87 cells were used to mimic in vivo hypoxia. Three glioma cell lines were tested: the U87 cell line, the U87 cell line transfected with HIF1α (U87-HA-HIF1α) and the U87 cell line transfected with HIF2α (U87-HA-HIF2α). Based on the spheroid gen-eration method, we first evaluated cell proliferation and spheroid formation in U87 cell lines and hypoxic glioma cells transfected with HIFs on the concave microdevice. We found that diameter significantly increased up to 10 days following cell seeding in U87 cells and HIFs-trans-fected cell lines, as shown in Fig. 2B (i). The cell growth assay showed that the average spheroid diameters of the three glioma cell lines increased over 10 days of culturing. However, U87-HA-HIF1α and U87-HA-HIF2α spheroids expanded progressively after four days of culturing com-pared with the U87 spheroids, indicating greater growth and spheroid formation ability in HIF-transfected glioma cells. We also observed that U87-HA-HIF1α spheroids




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expanded faster than U87-HA-HIF2α spheroids after six days of culturing, suggesting a potential overlay role of the HIFs involved in spheroid formation and growth.

Successive culture images of the three cell lines in the concave microwell device for up to 10 days are shown in Fig. 2B (ii). With similar diameters at day 2, the three types of U87 spheroids experienced different growth processes in the following days. The U87-HA-HIF1α spheroids expanded progressively and exhibited compact content and distinct profiles. The expansion rate of the U87-HA-HIF2α spheroids increased in the first six days and then declined, whereas that of U87 spheroids increased slowly with irregular profiles and loose internal structures.

3.3 Hypoxia enhanced invasive capability and phenotypic transition in glioma cells

In addition to cell proliferation and spheroid formation, we evaluated the invasion ability of hypoxic glioma cells in the 3D microenvironment using the approach described above. Fig. 3A shows real-time images of the invasive spheroids within the 3D matrix in the three glioma cell lines over 12 h. The U87 spheroids transfected with HA-HIF1α and HA-HIF2α exhibited strong invasion abil-ity in the 3D gels compared to U87 spheroids alone. The hypoxic glioma cells displayed more migrating cells with a mesenchyme-like shape in the front end.

To quantitatively evaluate the invasion ability of glioma cells, their spheroids were cultured for 24 h and measured with DAPI nuclei staining followed by confocal

imaging, as shown in Fig. 3B (i–iii). In a confocal micro-scope, a z-stack consists of thin sections of the spheroid culture at different layers, which makes it possible to follow the entire invasion process from all parts of the spheroid in the matrix. The 3D image of spheroids was then constructed based on the overlaying of all of the images from one z-stack. As shown in Fig. 3B (iv), the ability of the cells to migrate was evaluated quantitatively according to the mathematical model described earlier [29]. We defined the edge of the spheroid as the starting point and divided the cells’ migration regions into differ-ent and equal zones along the radial direction. We used directional motility to evaluate the invasive capability of glioma cell spheroids. By counting the number of nuclear staining cells within respective zones, we estimated the cell density as a function of radial distance from the spheroid edge. According to the statistical results, U87-HA-HIF2α spheroids showed the strongest directional motility, which is consistent with the results above.

To determine whether HIF1α or HIF2α overexpression is the fundamental factor of glioma invasion, the cells’ spheroids were treated with HIF1α or HIF2α inhibitor for 24 h and an invasion assay was conducted. Both inhibi-tors had a significant inhibitory effect on HIF-induced tumor invasion in the U87 cell lines, as shown in Fig. 4. The cells consistently lost their invasive mesenchyme-like phenotype after treatment with the HIF inhibitors. Thus, the results demonstrated that invasion capability was reduced by treatment with an HIF inhibitor. Mesenchy-mal transformation confers cells with enhanced migratory

Figure 2. (A) Characterization of the multicellular spheroid formation and patterning method. (i) Spheroid for-mation with controllable size (scale bars = 100 µm). (ii) Images of spheroid array patterning with different arrange-ments. Green represents glioma cells transfected with green fluorescent pro-tein (GFP) (scale bars = 500 µm). (B) Assays of the proliferation and spheroid forming capabilities of different glioma cells (U87, U87-HA-HIF1α, U87-HA-HIF2α), at seeding concentrations of 105 cells/mL for 10 days of culture in a cell non-adhesive PDMS concave microdevice. (i) Diameters of the spheroids formed by the three cell lines (n = 6). (ii) Bright field images of the spheroid array in the concave microde-vice (scale bars = 150 µm).




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ability. To investigate whether hypoxia-promoted mesen-chymal transformation of glioma cells results in a migra-tory phenotype, we observed the expression of the mes-

enchymal marker vimentin using immunofluorescence in different glioma cells. For this assay, the spheroids were allowed to migrate into the 3D collagen matrix, which more accurately simulated the microenvironment of the cell. As shown in Fig. 5A, the three cells lines experienced mesenchymal change during the invasion process. The mesenchymal degree of the cells was quantified by cal-culating the ratio of the length to width per cell in each of the three U87 cell lines [30, 31], as indicated in Fig. 5B. It was shown that U87-HA-HIF1α spheroids and U87-HA-HIF2α spheroids presented more aggressive phenotypic features than U87 spheroids.

4 Discussion

We showed that multicellular tumor spheroids embed-ded in 3D gels provide promising platforms for in vitro tumor mimics with aggressive phenotypes and respond to stimuli analogous to in vivo tumor microenvironments. This approach is characterized by the ability to produce uniform multicellular spheroids in parallel, simultaneously pattern an array of spheroids within a natural 3D ECM and incorporate hypoxic factors in the 3D tumor microenvi-ronment. We observed enhanced cell proliferation, spher-oid formation, tumor invasion and phenotype transition abilities in glioma cells transfected with HIF1α and HIF2α compared with non-treated glioma cells. The results demonstrated that hypoxia enhances tumor spheroid formation and cell migration in the 3D ECM in an HIF-dependent manner. Furthermore, the results showed that this process can be attenuated by a pharmacologi-cal blockade of HIFα. These data suggest that although HIF1α preferentially promotes cell migration, HIF2α is also necessary for it to occur.

Epithelial-mesenchymal transition (EMT) is recog-nized as a critical process involved in cancer progression. It is known to be an important feature of epithelial tumors and is increasingly recognized as a key event in non-epi-thelial cancers, such as gliomas. Tumor aggressiveness is usually accompanied by EMT, which decreases the adhe-sion capacity of cells with a mesenchymal phenotype and increases cell migration and invasion [32]. The HIF-transfected spheroids exhibited more aggressive features and phenotype change than the U87 spheroids, which indicates that hypoxia activates mesenchymal transition in glioma cells. The presented approach facilitates the characterization of EMT features during tumor invasion in a 3D ECM and drug sensitivity testing in a dynamic manner.

Compared with previous 3D bioengineering methods of single spheroid invasion assays, such as non-adhesive 96-well plate [22], hanging droplets [23] and microin-jection [33], the simple operation, rapid prototyping, low cost and high throughput format of the proposed approach facilitates the characterization of cell prolifera-

Figure 3. Dynamic and quantitative analysis of metastatic glioma cells in a 3D matrix. (A) Time-course invasion of the different U87 cell lines using real time images: (i) U87, (ii) U87 transfected with HA-HIF1α and (iii) U87 transfected with HA-HIF2α. (B) Quantitative evaluation of inva-sive capability in the different U87 cell lines after 24 h of invasion. Confo-cal images of the spheroid invasion in (i) U87, (ii) U87 transfected with HA-HIF1α, (iii) U87 transfected with HA-HIF2α and (iv) cell counting schematic and statistical results (scale bars = 100 µm).




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tion, migration, phenotypic function and drug evaluation in physiologically relevant 3D microenvironments. The method has the ability to simultaneously pattern multiple spheroids into 3D ECMs in a controlled fashion, which avoids repeatedly seeding the single spheroid separately (as in the plate format) and enables the study of multiple invasive spheroids in a high throughput format. The patterned format enables further exploration of the inter-action between adjacent spheroids with a controllable distance and angle in 3D microenvironments, which may be useful for the study of angiogenesis and anti-angiogen-esis drug testing in tumor progression.

In summary, we successfully established a new in vitro 3D system to mimic tumor microenvironments by patterning multicellular spheroids within a 3D ECM in a controllable and high throughput format. This approach enables the quantitative analysis of tumor invasion and phenotypic EMT status in a dynamic manner. The find-ings may not only benefit the mechanistic study of tumor metastasis and pharmacological high throughput 3D drug evaluation for preclinical trials, but may also facilitate further study of the interactions between multicellular spheroids and angiogenesis in tumor biology.

This research was supported by International Sci-ence & Technology Cooperation Program of China (No. 2015DFA00740), Key Laboratory of Separation Science for Analytical Chemistry (Dalian Institute of Chemical Physics, Chinese Academy of Sciences), National Nature Science Foundation of China (No. 81201689, 31171149).

The authors declare no financial or commercial conflict of interest.

Figure 4. Effects of the HIF1α inhibitor and HIF2α inhibitor on the inva-sive capability of two types of U87 spheroids transfected with HIFs. (A) U87 transfected with HA-HIF1α. (B) U87 transfected with HA-HIF2α. (C) Quantification of the invasive area affected by HIF inhibitors. Error bars indicate the standard error of the mean relative to the control: ***p < 0.001 (scale bars = 500 µm).

Figure 5. Characterization of invasive glioma cells with phenotypic transition in the 3D matrix after 24 h of invasion. (A) Morphologies of the three lines of invasive cells from the patterned spheroids by immunofluorescent staining. (B) Quantitative evaluation of the mesenchymal degree by the length to width ratio in the different U87 cell lines (scale bars = 50 µm).




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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com

EditorialBiotechnology Journal – we are looking forward to a new decade Jing Zhu

http://dx.doi.org/10.1002/biot.201500668

ReviewPolyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applicationsXiaohong Wang, Heinz C. Schröder and Werner E. G. Müller


ReviewQuantum dot as probe for disease diagnosis and monitoringAbhishek Mukherjee, Yumi Shim and Joon Myong Song


ReviewRegulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactorsSébastien Sart, Spiros N. Agathos, Yan Li and Teng Ma


Review Potential applications of keratinocytes derived from human embryonic stem cellsMohammad M. Movahednia, Fahad K. Kidwai, Doorgesh S. Jokhun, Christopher A. Squier, Wei Seong Toh and Tong Cao


Research ArticleSelecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cellsAtze T. Das, Xue Zhou, Stefan W. Metz, Monique A. Vink and Ben Berkhout


Research ArticleUse of transposase and ends of IS608 enables precise and scarless genome modification for modulating gene expression and metabolic engineering applications in Escherichia coliChandresh Thakker, Kevin Lin, Heidi Martini-Stoica and George N. Bennett


Research ArticleUltra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditionsMakoto Kurihara, Yuki Ohmuro-Matsuyama, Keiichi Ayabe, Takahiro Yamashita, Hideki Yamaji and Hiroshi Ueda


Research ArticleMonolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressureYu Isakari , Ales Podgornik, Noriko Yoshimoto and Shuichi Yamamoto


Research ArticleOvercoming low yields of plant-made antibodies by a protein engineering approachJulia Zischewski, Markus Sack and Rainer Fischer


Research ArticleHigh yield of recombinant human apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatographyVignesh N. Janakiraman, Abdelmajid Noubhani, Krishnan Venkataraman, Mookambeswaran Vijayalakshmi and Xavier Santarelli


Biotechnology Journal – list of articles published in the January 2016 issue.

Cover illustrationThis issue includes articles on an ultra-sensitive protein-protein interaction assay, an approach to overcome low yields of plant-made antibodies and a method to efficiently generate germline transgenic mice. The cover shows inorganic polyphosphates (polyP) that constitute an extracellular polymer for energy release, delivery and transport. PolyP use blood platelets as transport vehicles to reach their target cells, but have also been successfully encapsulated into microparticles allowing the development of novel therapeutics strategies. Image by Werner Müller.












Research ArticlePatterning hypoxic multicellular spheroids in a 3D matrix – a promising method for anti-tumor drug screeningJingyun Ma, Xu Zhang, Yang Liu, Haibo Yu, Lianqing Liu, Yang Shi, Yanfeng Li and Jianhua Qin


Research ArticleOptimal model-based design of the twin-column CaptureSMB process improves capacity utilization and productivity in protein A affinity captureDaniel Baur, Monica Angarita, Thomas Müller-Späth and Massimo Morbidelli


Research ArticleInclusion of mPRISM potential for polymer-induced protein interactions enables modeling of second osmotic virial coefficients in aqueous polymer-salt solutionsMarcel Herhut, Christoph Brandenbusch and Gabriele Sadowski


Research ArticleEnvironmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivationsSarah Lieder, Michael Jahn, Joachim Koepff, Susann Müller and Ralf Takors


Biotech MethodBridging the gap between PAT concepts and implementation: An integrated software platform for fermentationViki R Chopda, James Gomes and Anurag S. Rathore


Biotech MethodIn vivo amyloid aggregation kinetics tracked by time-lapse confocal microscopy in real-timeAnna Villar-Piqué, Alba Espargaró, Salvador Ventura and Raimon Sabate


Biotech MethodCytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic miceWiebke Garrels, Thirumala R. Talluri, Maren Ziegler, Ilka Most, Diego O. Forcato, Marco Schmeer, Martin Schleef, Zoltán Ivics and Wilfried A. Kues


Rapid CommunicationAsymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto acidsSam Mathew, Seong-Su Jeong, Taeowan Chung, Sang-Hyeup Lee and Hyungdon Yun


© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.biotechnology-journal.com









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Patterning hypoxic multicellular spheroids in a 3D …ir.sia.cn/bitstream/173321/17615/1/Patterning hypoxic...plate cultures [22], hanging drops [23] and cell-hydrogel mixed spheroid