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Review
, 153, 85-101

High-throughput Approaches for Screening and Analysis of Cell Behaviors

Affiliations
Review

High-throughput Approaches for Screening and Analysis of Cell Behaviors

Jungmok Seo et al. Biomaterials.

Abstract

The rapid development of new biomaterials and techniques to modify them challenge our capability to characterize them using conventional methods. In response, numerous high-throughput (HT) strategies are being developed to analyze biomaterials and their interactions with cells using combinatorial approaches. Moreover, these systematic analyses have the power to uncover effects of delivered soluble bioactive molecules on cell responses. In this review, we describe the recent developments in HT approaches that help identify cellular microenvironments affecting cell behaviors and highlight HT screening of biochemical libraries for gene delivery, drug discovery, and toxicological studies. We also discuss HT techniques for the analyses of cell secreted biomolecules and provide perspectives on the future utility of HT approaches in biomedical engineering.

Keywords: Biomaterial screening; Biomolecule delivery; Cell-biomaterial interactions; Cellular microenvironments; High-throughput biosensor; High-throughput system.

Figures

Figure 1
Figure 1
Schematic illustration of HT approaches for modulating and monitoring cell-material interactions. (a) HT platforms for the screenings of 2D and 3D cellular behaviors with different microenvironments. 2D environments can be engineered by using cell-specific binding peptides, wettability patterning technique, and ECM coatings. Cell spheroids and 3D cell-laden hydrogels can be used to monitor the cell behaviors in 3D environments. In specific purpose, cells can be externally stimulated via electrical or mechanical stimulation. (b) HT can provide the region-specific cell inoculation and delivery of bioactive molecules including genes, proteins, and chemical drugs to the cells via microarray and microwell methods. (c) The HT technique can also be utilized for the analysis of multiple cell-secreted factors and cellular products including proteins, genes, and enzymes.
Figure 2
Figure 2
HT approaches for the investigation of 2D stem cell behaviors. (a) HT screening platform to study the effects of cell-biomaterial interactions on human ESC behaviors, (b) which allowed for the comparison of human ESC colony formation efficiency by varying the composition of biomaterials and cell culture media. Combinatorial screening of three different cell culture substrate compositions (TCPS, hit-polymer 9, and hit-polymer 15A), four different protein coatings (matrigel, bovine serum (bovSerum), human serum (HuSerum), and human vitronectin (HuVitronectin)), and two different culture media (mouse embryonic fibroblast-conditioned media (MEF-CM) and chemically defined media (mTeSR1)) was conducted using the developed platform. (c) Fabrication of microarrays with engineered ECM allowed for investigating (d) stem cell behaviors as a function of hydrogel stiffness; and (e) extracellular matrix proteins. Figures adapted and reprinted with permission from (a, b) [34] and (c–e) [35].
Figure 3
Figure 3
Superhydrophobic-superhydrophilic patterning for 2D HT screening platform. (a) Schematic illustration of the workflow of HT screening platform for the study of cellular behaviors with conventional image-based analytical methods. (b) Homogenous seeding of diverse cell types on the superhydrophobic-superhydrophilic patterned substrate for screening cell behaviors with delivered biomolecules. Figures adapted and reprinted with permission from [44].
Figure 4
Figure 4
HT approaches to investigate 3D cellular behaviors. (a) Cell-laden gelatin methacryloyl (GE) hydrogel microarrays were developed for the identification of the compositional effects of ECM proteins (laminin (LN, 40 μg/mL), fibronectin (FN, 40 μg/mL), osteocalcin (OCN, 20 and 40 μg/mL)), culture media (normal media (NM) and osteogenic inducing media (IM)) supplement with different concentrations of bone morphogenic proteins (BMP-2 and BMP-5, 50 ng/mL). (b) High-throughput screening of mouse ESC behaviors within PEG 3D hydrogel microarrays in response to varying hydrogel stiffness and degradability, cell density, extracellular matrix components, cell-cell interactions, and soluble biomolecules. Figures adapted and reprinted with permission from (a) [39] and (b) [62].
Figure 5
Figure 5
HT biomechanical stimulation systems. (a) Microarray platform for applying dynamic mechanical compressive strain across biomaterials such as PEG hydrogels. (b) Finite element simulations presenting the deformation of cell and hydrogel matrix under the compressive stress. According to the relative stiffness of cells and hydrogel matrix (E *matrix), the degree of cell deformations can be varied. (c) Photograph of bulging membranes with integrated strain sensor for the real-time monitoring of strain onto biomaterials. (d) Strain sensing of cyclic membrane bulging by using a carbon nanotube-based strain sensor. A time-dependent resistive strain of the sensors (ΔR/R0) demonstrated a good correlation with input pressure (P), although there is a hysteresis at loading and unloading strain in low input pressure regime. Figures adapted and reprinted with permission from (a, b) from [70] and (c, d) [73].
Figure 6
Figure 6
HT platforms for 3D microtissue behaviors. (a) HT 3D spheroids were formed in microwells to function as microbuilding blocks to generate an artificial osteochondral tissue construct. Culture conditions are defined by the media type (chondrogenic media (C) or osteogenic media (O)) and culture duration for the first 8 days and following 6 days. For instance, CO indicates cell culture with chondrogenic media for 8 days followed by 6 days culture with osteogenic media. Osteochondral-like tissue structure was obtained by co-culture of pre-osteogenic and pre-chondrogenic differentiated hMSC spheroids. (b) Microfluidic approach for monitoring of 3D microtissue behaviors. A suspension of fluorescent human colorectal carcinoma cells was flowed through microchannels to form 3D spheroids in hanging-drop microwells. The effect of FBS concentration on spheroid formation was explored simultaneously. Figures adapted and reprinted with permission from (a) [88], (b) [95].
Figure 7
Figure 7
Cell spot microarray methods for HT investigation of gene transfection. (a) Principle and workflow of the cell spot microarray method. (b) TurboGFP silencing of U-2OS sarcoma cells on cell spot microarray. Silencing efficacies of different siRNA concentrations were identified using a siRNA (siCTRL) as a negative group and a siRNA for CDC2 (siCDC2) as a control group. TuGFP = green, DNA = blue, scale bar = 2 mm. (c) Illustration of the patterning of a transfected cell microarray. (d) Average cell death rates (black columns) and the reverse transfection efficiency (gray columns) per one spot (n = 8). Figures adapted and reprinted with permission from (a,b) [105] and (c, d) [103].
Figure 8
Figure 8
HT systems for screening the effect of controlled delivery of drugs. (a) Schematic of the device fabrication and use: from fabricating the microwell array and chemical library printing to sandwiching the device for toxicological analysis. To validate the device, different concentrations of fluorescein isothiocyanate-dextran (FITC-Dextran) and Rhodamine B (0.02 to 200 ng/mL) were printed on PDMS posts and fluorescent intensities were evaluated after sandwiching. (b) A microarray system for released chemical screening (left) and resulting apoptosis and necrosis of MCF-7 cancer cells with various chemicals with different concentrations: staurosporine (STS), ethanol (EtOH), and hydrogen peroxide (H2O2). Apoptosis and necrosis were evaluated by annexin V-allophycocyanin and SYTOX orange, respectively. Figures adapted and reprinted with permission from (a) [113] and (b) [114].
Figure 9
Figure 9
HT analysis of gene expression. A droplet-based DNA barcoding platform for RNA sequencing. UMI = unique molecular identifier. Figures adapted and reprinted with permission from [126].
Figure 10
Figure 10
HT immunoassays. (a) In-channel antibody patterning of the microfluidic device for the highly sensitive immunoassay platform. (b) Fluorescence-based detection of carcinoembryonic antigen from a human serum sample of 6 patients using the microfluidic device. (c) Drug hepatotoxicity evaluation using a coupled 3D immunoChip with a 3D cell culture chip. The detection capability of the 3D immunoChip platform is comparable to conventional ELISA analysis. (d) Schematic diagram of SERS active nanoparticles encoding onto polymer bead with protein specific binder and scanning electron microscopic images at each step (i-iii). (e) Label-free detection of four different bio-ligands (HPQIG, IHPQG, IQHPQ, and biotin) by combining the SERS barcodes and streptavidin binding assay. Figures adapted and reprinted with permission from (a, b) [153], (c) [155], and (d, e) [159].

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