scholarly journals Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging

2021 ◽  
Vol 7 (17) ◽  
pp. eabc1323
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
C. Saavedra ◽  
Y. Kim ◽  
P. Le ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Drug Screening ◽  
Three Dimensional ◽  
3D Culture ◽  
Geometric Control ◽  
Hanging Drop ◽  
Design Elements ◽  
Trade Offs ◽  
On Chip

Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research.

scholarly journals Identification and evaluation of an appropriate housekeeping gene for real-time gene profiling of hepatocellular carcinoma cells cultured in three-dimensional scaffold

2021 ◽  
Author(s):  
Anjana Kaveri Badekila ◽  
Praveen Rai ◽  
Sudarshan Kini
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Culture ◽  
Real Time ◽  
Internal Control ◽  
18S Rrna ◽  
Three Dimensional ◽  
Melting Curve ◽  
3D Culture ◽  
Gene Profiling ◽  
3D Microenvironment

Abstract Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three-dimensional cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of recapitulation of in vivo tumors. In this aspect, expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture (two-dimensional), and alginate-carboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analyzed for hepatocellular carcinoma (Huh-7) cell line. The real-time gene expression using RT-qPCR of HCC spheroids in 3D culture were analyzed by determining the primer efficiency, melting curve and quantification cycle analysis of the selected candidate HKG’s. Further, RT-qPCR data were validated using analysis softwares i.e., geNorm and NormFinder for statistical significance. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates and considered them as potential internal controls during varying cell culture conditions.

scholarly journals Three-dimensional visualization and analysis of flowing droplets in microchannels using real-time quantitative phase microscopy

Lab on a Chip ◽  
2021 ◽  
Vol 21 (1) ◽  
pp. 75-82
Author(s):  
Yingdong Luo ◽  
Jinwu Yang ◽  
Xinqi Zheng ◽  
Jianjun Wang ◽  
Xin Tu ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Three Dimensional ◽  
Phase Variation ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
On Chip

We present real-time quantitative phase microscopy (RT-QPM) that can be used for on-chip three-dimensional visualization of droplets and high-throughput quantitative molecular measurement via real-time extraction of sample-induced phase variation.

Media additives to promote spheroid circularity and compactness in hanging drop platform

2015 ◽  
Vol 3 (2) ◽  
pp. 336-344 ◽  
Author(s):  
Brendan M. Leung ◽  
Sasha Cai Lesher-Perez ◽  
Toshiki Matsuoka ◽  
Christopher Moraes ◽  
Shuichi Takayama
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Three Dimensional ◽  
Hanging Drop

Three-dimensional spheroid cultures have become increasingly popular as drug screening platforms, especially with the advent of different high throughput spheroid forming technologies.

scholarly journals Multi-Organs-on-Chips: Towards Long-Term Biomedical Investigations

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 675 ◽  
Author(s):  
Yi Zhao ◽  
Ranjith Kankala ◽  
Shi-Bin Wang ◽  
Ai-Zheng Chen
Keyword(s):  
Real Time ◽  
Three Dimensional ◽  
Drug Efficacy ◽  
Dynamic Responses ◽  
Physical Parameters ◽  
Real Time Monitoring ◽  
Multiple Organs ◽  
On Chip

With advantageous features such as minimizing the cost, time, and sample size requirements, organ-on-a-chip (OOC) systems have garnered enormous interest from researchers for their ability for real-time monitoring of physical parameters by mimicking the in vivo microenvironment and the precise responses of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models. Recent advancements of OOC systems have evidenced the fabrication of ‘multi-organ-on-chip’ (MOC) models, which connect separated organ chambers together to resemble an ideal pharmacokinetic and pharmacodynamic (PK-PD) model for monitoring the complex interactions between multiple organs and the resultant dynamic responses of multiple organs to pharmaceutical compounds. Numerous varieties of MOC systems have been proposed, mainly focusing on the construction of these multi-organ models, while there are only few studies on how to realize continual, automated, and stable testing, which still remains a significant challenge in the development process of MOCs. Herein, this review emphasizes the recent advancements in realizing long-term testing of MOCs to promote their capability for real-time monitoring of multi-organ interactions and chronic cellular reactions more accurately and steadily over the available chip models. Efforts in this field are still ongoing for better performance in the assessment of preclinical attributes for a new chemical entity. Further, we give a brief overview on the various biomedical applications of long-term testing in MOCs, including several proposed applications and their potential utilization in the future. Finally, we summarize with perspectives.

scholarly journals Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Olga Schmal ◽  
Jan Seifert ◽  
Tilman E. Schäffer ◽  
Christina B. Walter ◽  
Wilhelm K. Aicher ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Three Dimensional ◽  
3D Culture ◽  
Hanging Drop ◽  
Mixed Cell ◽  
Hematopoietic Stem ◽  
Extracellular Matrix Components ◽  
Stem And Progenitor Cells

Efficientex vivoexpansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

scholarly journals Robust real-time 3D imaging of moving scenes through atmospheric obscurant using single-photon LiDAR

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rachael Tobin ◽  
Abderrahim Halimi ◽  
Aongus McCarthy ◽  
Philip J. Soan ◽  
Gerald S. Buller
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Moving Objects ◽  
Single Photon ◽  
Three Dimensional ◽  
Depth Profiling ◽  
High Sensitivity ◽  
Single Photon Detection ◽  
Photon Detection ◽  
Uncertainty Estimates

AbstractRecently, time-of-flight LiDAR using the single-photon detection approach has emerged as a potential solution for three-dimensional imaging in challenging measurement scenarios, such as over distances of many kilometres. The high sensitivity and picosecond timing resolution afforded by single-photon detection offers high-resolution depth profiling of remote, complex scenes while maintaining low power optical illumination. These properties are ideal for imaging in highly scattering environments such as through atmospheric obscurants, for example fog and smoke. In this paper we present the reconstruction of depth profiles of moving objects through high levels of obscurant equivalent to five attenuation lengths between transceiver and target at stand-off distances up to 150 m. We used a robust statistically based processing algorithm designed for the real time reconstruction of single-photon data obtained in the presence of atmospheric obscurant, including providing uncertainty estimates in the depth reconstruction. This demonstration of real-time 3D reconstruction of moving scenes points a way forward for high-resolution imaging from mobile platforms in degraded visual environments.

scholarly journals Binary defocusing technique based on complementary decoding with unconstrained dual projectors

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Xuexing Li ◽  
Wenhui Zhang
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Three Dimensional ◽  
Low Resolution ◽  
Fusion Technique ◽  
Space Time Coding ◽  
Depth Extraction ◽  
Core Idea ◽  
3D Scene ◽  
Binary Defocusing

AbstractBinary defocusing technique can effectively break the limitation of hardware speed, which has been widely used in the real-time three-dimensional (3D) reconstruction. In addition, fusion technique can reduce captured images count for a 3D scene, which helps to improve real-time performance. Unfortunately, it is difficult for binary defocusing technique and fusion technique working simultaneously. To this end, our research established a novel system framework consisting of dual projectors and a camera, where the position and posture of the dual projectors are not strictly required. And, the dual projectors can adjust defocusing level independently. Based on this, this paper proposed a complementary decoding method with unconstrained dual projectors. The core idea is that low-resolution information is employed for high-resolution phase unwrapping. For this purpose, we developed the low-resolution depth extraction strategy based on periodic space-time coding patterns and the method from the low-resolution order to high-resolution order of fringe. Finally, experimental results demonstrated the performance of our proposed method, and the proposed method only requires three images for a 3D scene, as well as has strong robustness, expansibility, and implementation.

scholarly journals AVATAR: AI Vision Analysis for Three-dimensional Action in Real-time

2022 ◽  
Author(s):  
Daesoo Kim ◽  
Dae-Gun Kim ◽  
Anna Shin ◽  
Yong-Cheol Jeong ◽  
Seahyung Park
Keyword(s):  
Artificial Intelligence ◽  
High Resolution ◽  
Real Time ◽  
Closed Loop ◽  
Three Dimensional ◽  
Body Parts ◽  
Behavioural Analysis ◽  
Action Sequences ◽  
Freely Moving ◽  
Behavioural Experiments

Artificial intelligence (AI) is an emerging tool for high-resolution behavioural analysis and conduction of human-free behavioural experiments. Here, we applied an AI-based system, AVATAR, which automatically virtualises 3D motions from the detection of 9 body parts. This allows quantification, classification and detection of specific action sequences in real-time and facilitates closed-loop manipulation, triggered by the onset of specific behaviours, in freely moving mice.

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