Amy Y. Hsiao

Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy

Multicellular spheroids are three dimensional in vitro microscale tissue analogs. The current article examines the suitability of spheroids as an in vitro platform for testing drug delivery systems. Spheroids model critical physiologic parameters present in vivo, including complex multicellular architecture, barriers to mass transport, and extracellular matrix deposition. Relative to two-dimensional cultures, spheroids also provide better target cells for drug testing and are appropriate in vitro models for studies of drug penetration. Key challenges associated with creation of uniformly sized spheroids, spheroids with small number of cells and co-culture spheroids are emphasized in the article. Moreover, the assay techniques required for the characterization of drug delivery and efficacy in spheroids and the challenges associated with such studies are discussed. Examples for the use of spheroids in drug delivery and testing are also emphasized. By addressing these challenges with possible solutions, multicellular spheroids are becoming an increasingly useful in vitro tool for drug screening and delivery to pathological tissues and organs.

Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids.

The niche microenvironment in which cancer cells reside plays a prominent role in the growth of cancer. It is therefore imperative to mimic the in vivo tumor niche in vitro to better understand cancer and enhance development of therapeutics. Here, we engineer a 3D metastatic prostate cancer model that includes the types of surrounding cells in the bone microenvironment that the metastatic prostate cancer cells reside in. Specifically, we used a two-layer microfluidic system to culture 3D multi-cell type spheroids of fluorescently labeled metastatic prostate cancer cells (PC-3 cell line), osteoblasts and endothelial cells. This method ensures uniform incorporation of all co-culture cell types into each spheroid and keeps the spheroids stationary for easy tracking of individual spheroids and the PC-3s residing inside them over the course of at least a week. This culture system greatly decreased the proliferation rate of PC-3 cells without reducing viability and may more faithfully recapitulate the in vivo growth behavior of malignant cancer cells within the bone metastatic prostate cancer microenvironment.

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.

We previously reported the development of a simple, user‐friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti‐cancer drug sensitivity testing. The 384 hanging drop array plate allows for high‐throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell‐based high‐throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence‐ and colorimetric‐based assays through Z‐factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Biotechnol. Bioeng. 2012; 109:1293–1304.

Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device.

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.

Multiplexed spectral signature detection for microfluidic color-coded bioparticle flow.

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.

High-speed tuning of visible laser wavelength using a nanoimprinted grating optical tunable filter

We report on a microelectromechanical tunable optical filter incorporating strain-tunable nanoimprinted elastomeric grating with a pitch varied by 18%. This device enables tuning of optical fiber-guided laser wavelength between lambda=473 and 532 nm within 0.5 ms by mechanically modulating the pitch with a silicon microactuator. We also demonstrate the use of the device for obtaining two-color images of livedead-stained cells with the color intensity ratio varied by the actuator voltage applied. The small structure of the device integrated on a silicon chip may be used in portable systems for optical switching and spectroscopy.

Abstract A72: Prostate cancer spheroid formation in a 384 hanging drop array plate.

Background: Bridging the gap between in vitro and in vivo studies is critical to therapeutic advancement. Achieving more physiological tumor models in vitro will attenuate dependence on preclinical animal testing and facilitate more accurate prediction of a compound9s antitumor behavior in vivo. Multicellular tumor spheroids are 3-dimensional, self-assembled clusters of cancer cells formed in an environment where cell-cell interactions dominate in the absence of cell-surface interactions. A spheroid composed of prostate cancer cells, or prostatosphere, not only provides a 3-dimensional tumor model, but also allows the formation of extracellular matrix as would occur in vivo. Thus, drug screening performed on spheroids should yield results of greater physiological significance than screening performed on 2-dimensional tissue cultures. Furthermore, the use of a 384 hanging drop array plate has previously been shown to enable the convenient formation of spheroids from COS7, mES, and A431.H9 cell lines[1]. Here we chronicle the compatibility of the 384 hanging drop array plate with four prostate cancer cell lines for prostatosphere formation. Methods: Prostate cancer cell suspensions were deposited into staggered access holes on the 384 hanging drop array plate, creating 15 to 20 L hanging drops with cell concentrations ranging from 500 to 5000 cells per drop. In order to arrest drop evaporation, the hanging drop array plate was sandwiched between a lid and 96-well plate filled with distilled water and was wrapped in Parafilm. Drops were observed using bright field microscopy and imaged daily (Olympus IX71). The image processing software ImageJ was used to obtain spheroid dimensions, and mean spheroid diameter was calculated using the following equation: d = (a × b)∘(1/2), where a and b are orthogonal diameters of the spheroid. Average size was reported as mean diameter ± standard deviation and was measured on the second day after spheroid formation was observed. Results: Spheroid formation was observed within 24 hours for LNCaP and LAPC4 cells, and within 48 hours for VCaP and DU145 cells. LNCaP, LAPC4, and VCaP spheroids characteristically formed one single spheroid per drop, with larger spheroids obtained at higher cell concentrations. These three cell lines exhibited round and oblong spheroid morphologies at low cell concentration, with spheroid shape becoming more irregular, branched, or partially hollow at higher cell concentrations. Hanging drops containing DU145 cells tended to contain multiple spheroids of smaller diameter. Thus, DU145 spheroids were usually limited to round or oblong morphologies. DU145 spheroids attained an average size of 72.1 ± 43.7 microns. Conclusion: These prostate cancer cell lines are shown to form prostatospheres when cultured in the 384 hanging drop array plate. Preliminary evidence suggests that additional prostate cancer cell lines are also capable of doing so. Our group intends to screen antitumor therapeutics on prostatospheres in comparison to prostate cancer cells under traditional 2-dimensional tissue culture conditions. Co-culture spheroid formation incorporating prostate cancer cells, endothelial cells, and osteoblasts to simulate the tumor microenvironment is expected, as is drug screening on such spheroids. References : 1. Y. Tung, A. Y. Hsiao, S. G. Allen, Y. Torisawa, M. Ho, and S. Takayama, Analyst, 2011, 136, 473–478. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A72.