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Advanced biomedical applications of cell clusteroids based on aqueous twophase Pickering emulsion systems

Wang, Anheng


Anheng Wang


Vesselin Paunov


The development of in vitro models for advancing the research of cell biology and cell physiology is of great importance to the fields of biotechnology, cancer study, drug testing, toxicity study. The emerging field includes tissue engineering and regenerative medicine will benefit from the models in a great way. Traditional mammalian two-dimensional (2D) methods cells culture encountered its limitations, and it is recently agreed that the three-dimensional (3D) cell culture features the in vivo environment more similarly owing to the increased cell-cell interactions and complex architecture like natural organ and tissue. In Chapter 1, we review the methods of generating 3D multicellular cell models on their merits and disadvantages. The assays that were mostly utilized to characterize the function of spheroids were also discussed. The application of 3D cell models has advanced the basic cell sciences, especially in understanding tumour biology, cancer drug discovery and cancer metastasis. Another potential down-stream application of the 3D cell models is that they could be utilized as basic building blocks for tissue constructs. In brief, emerging technologies aiming to generate and assess spheroids are pushing their application and wider their utilizations in drug testing and tissue engineering.
Chapter 2 describes the design and optimization of methods for encapsulating and generating the clusteroids of Hep-G2. The discontinuous and separated phases were selected from bio-compatible PEO and DEX, and the stabilizer was made from foodgrade whey protein particles. It was due in part to the spontaneous partitioning of the cells to the DEX phase within the DEX droplets that the cells were easily captured. The experiment showed that a number of different parameters could be adjusted in order to ensure that the cell clusteroids were adequately encapsulated and generated by varying the ratio of DEX:PEO. With our method, we were able to show that a very large number of individual cell clusteroids could be produced. In addition, we utilized FDA assays to assess the viability of the clusteroids collected after the preparation procedures in order to demonstrate that they remain highly viable after the treatment.
In Chapter 3, we demonstrated how to apply our ATPS based 3D cell culture method to the co-culture level. As a result, these two types of cells were able to coexist in a single droplet and be compacted into clusteroids during coculture. Hep-G2/ECV 304 cells collected in co-culture were both carcinoma cells, which would require the presence of blood vessels as the clusteroids grew larger. A simple change in the initial cell ratio added to the DEX phase proved that the cell ratios of two types of cells within co-cultured clusteroids were variable. We found that using carcinoma cell lines in vitro to produce vascularized co-culture clusteroids could be a facile procedure.
Chapter 4 examined the feasibility of co-culturing human liver cells with primary endothelial lines that are capable of angiogenesis by using w/w Pickering emulsion. A primary endothelial cell line could provide a better simulation of angiogenesis because it emulates the in vivo environment. The primary cells showed no repulsion to the carcinoma cell lines. Angiogenesis proteins were less abundant in HUVECs than in ECVs (Chapter 3). As a VEGF pump, Hep-G2 cells were used in the co-culture model to stimulate the angiogenesis of HUVEC cells by releasing VEGF. This model could be an ideal for investigating drug toxicology and other applications related to
tissue engineering.
Chapter 5 aimed to find a suitable application for the massive amount of the clusteroids collected from w/w Pickering emulsion. We generated a dense layer of 3D keratinocytes clusteroids to simulate skin in vitro. The S. aureus and P. aeruginosa could form biofilm on the clusteroids layer without breaking the structure of the 3D cell interactions. We further designed a nanotherapeutics based on antibiotic encapsulated Carbopol NPs, surface functionalised with a protease- alcalase. In comparison with non-coated ciprofloxacin and alcalase, this nanocarrier demonstrated a significant increase in antibacterial activity. In the clusteroids model, such nanoparticles did not pose a significant threat, and the clusteroids could continue to proliferate despite the presence of such nanoparticles. Moreover, this work showed that 3D cell clusteroids could have the potential to be used in further biomedical applications in the future.
Chapter 6 aim to broaden the application of the clusteroids layer to simulate the urinary track infection, which is commonly seen and tricky to solve in nosocomial infection. The aim of chapter 6 was to develop a 3D urothelial cell clusteroids model that mimicked the inner cell wall of the bladder infected with C.albicans biofilm. Using Fluconazole-loaded shellac nanotherapeutics in conjunction with a cationic enzyme Lysozyme to functionalize the Fluconazole nanocarriers, we made it possible to remove fungal biofilms from 3D urothelial clusteroids. An extensive array of fungal biofilm infections, or bacterial infections, could be mimicked using a protocol such as this by changing the species of cellular type and pathogen type of the organism.


Wang, A. (2022). Advanced biomedical applications of cell clusteroids based on aqueous twophase Pickering emulsion systems. (Thesis). University of Hull. Retrieved from

Thesis Type Thesis
Publication Date 2022-09
Deposit Date Mar 24, 2023
Publicly Available Date Mar 24, 2023
Keywords Chemistry
Public URL
Additional Information Department of Chemistry, The University of Hull


Thesis (11.6 Mb)

Copyright Statement
© 2022 Anheng Wang. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

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