generation-of-3d-tumor-spheroids-for-drug-evaluation-studies

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Southeast University

Jiangsu Avatarget Biotechnology Co., Ltd.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility.

Two-photon lithography (TPL) is a powerful tool to construct small-scale objects with complex and precise 3D architectures. While the limited selection of chemical functionalities on the printed structures has restricted the application of this method in fabricating functional objects and devices, this study presents a facile, efficient, and extensively applicable method to functionalize the surfaces of the objects printed by TPL. TPL-printed objects, regardless of their compositions, can be efficiently functionalized by combining trichlorovinylsilane treatment and thiol-ene chemistry. Various functionalities can be introduced on the printed objects, without affecting their micro-nano topographies. Hence, microstructures with diverse functions can be generated using non-functional photoresists. Compared to existed strategies, this method is fast, highly efficient, and non photoresist-dependent. In addition, this method can be applied to various materials, such as metals, metal oxides, and plastics that can be potentially utilized in TPL or other 3D printing technologies. The applications of this method on the biofunctionalization of microrobots and cell scaffolds are also demonstrated.

Facile Surface Functionalization Strategy for Two-Photon Lithography Microstructures.

3D skin equivalents have been increasingly used in the pharmaceutical and cosmetic industries, but the troublesome operation procedure and low throughput restricted their applications as  safety evaluation models. Organ-on-a-chip, an emerging powerful tool in tissue/organ modeling, could be utilized to improve the function of the skin model compared with that of traditional static skin models, as well as innovate an automatic and modular way for construction or detection. In this research, we grew and differentiated human keratinocytes within a microfluidic chip to construct an integrated epidermis-on-a-chip (iEOC) system, which is specially designed to integrate multi-culture units with integrated bubble removal structures as well as trans-epithelial electrical resistance (TEER) electrodes for barrier function detection . After 14 days of culture at the air-liquid interface (ALI), the constructed epidermis-on-a-chip demonstrated histological features similar to those observed in normal human epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers, especially the TEER value reached 3 kΩ cm and prevented more than 99% of Cascade Blue-607 Da permeation owing to the enhanced barrier function. Further immunofluorescence analysis also indicated typical keratin expression including keratin-14, keratin-10, loricrin, involucrin, and filaggrin. With the TEER monitoring integration in the chip, it could be convenient for scale-up high-quality epidermis-on-chip fabrication and correlated investigation. Additionally, the iEOC can distinguish all the 10 known toxins and non-toxins in irritation measurement by MTT assay, which is consistent with animal testing according to the OECD. Preliminarily detection of irritation responses like inflammatory cytokines also predicted different irritation reactions. This high fidelity epidermis-on-a-chip could be a potential alternative in  skin irritation evaluation. This microchip and automated microfluidic systems also pave the way for scalable testing in multidisciplinary industrial applications.

Construction of a high fidelity epidermis-on-a-chip for scalable  irritation evaluation.

A 3D tumor spheroid has been increasingly applied in pharmaceutical development for its simulation of the tumor structure and microenvironment. The embedded-culture of a tumor spheroid within a hydrogel microenvironment could help to improve the mimicking of in vivo cell growth and the development of 3D models for tumor invasiveness evaluation, which could enhance its drug efficiency prediction together with cell viability detection. NCI-H23 spheroids and CT-26 spheroids, from a non-small cell lung cancer and colorectal cancer cell line, respectively, together with extracellular matrix were generated for evaluating their sensitivity to AMG510 (a  inhibitor) under normoxia and hypoxia conditions, which were created by an on-stage environmental chamber. Results demonstrated that NCI-H23, the  moderate expression cell line, only mildly responded to AMG510 treatment in normal 2D and 3D cultures and could be clearly evaluated by our system in hypoxia conditions, while the negative control CT-26 (-mutant) spheroid exhibited no significant response to AMG510 treatment. In summary, our system, together with a controlled microenvironment and imaging methodology, provided an easily assessable and effective methodology for 3D in vitro drug efficiency testing and screenings.

Evaluation of AMG510 Therapy on -Mutant Non-Small Cell Lung Cancer and Colorectal Cancer Cell Using a 3D Invasive Tumor Spheroid System under Normoxia and Hypoxia.

Article	Year
Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility. American journal of human genetics\| PubMed ID: 32502391	2020
Facile Surface Functionalization Strategy for Two-Photon Lithography Microstructures. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 34269514	2021
Construction of a high fidelity epidermis-on-a-chip for scalable irritation evaluation. Lab on a chip\| PubMed ID: 34581381	2021
Evaluation of AMG510 Therapy on -Mutant Non-Small Cell Lung Cancer and Colorectal Cancer Cell Using a 3D Invasive Tumor Spheroid System under Normoxia and Hypoxia. Bioengineering (Basel, Switzerland)\| PubMed ID: 36550998	2022

Xiaoran Li

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