Nanoparticle-based Liquid Marbles as Bioreactors with Novel Properties for Spheroid Co/culture and DNA Amplification

基於納米顆粒的液體彈珠作為具有優異性能的生物反應器用於細胞球培養或共培養以及脫氧核糖核酸擴增

Student thesis: Doctoral Thesis

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Award date4 Mar 2019

Abstract

The resistance to anticancer drugs is the principal cause of chemotherapy failure. However, in vitro drug sensitivity tests are usually performed in monolayer culture system, which presents a largely artificial cellular environment with little relevance to in vivo conditions and has limited value in predicting clinical efficacy of chemotherapeutic drugs. Three-dimensional (3D) cell culture methods have great potential for cell therapy, tissue engineering and drug sensitivity test because cells cultured as spheroid rather than monolayer could better mimic in vivo microenvironment. For example, spheroids have been used to reveal resistance mechanism to anticancer drugs, and to identify novel therapeutics for lung cancer, hepatocellular carcinoma, and breast cancer. However, the commonly used methods for culturing spheroid such as hanging drop and spinner flask techniques are typically not compatible with in situ imaging and lack of mobility control, which are essential for downstream drug screening. Considerable efforts have been made in recent years to improve tumor spheroid culture for studying fundamental cancer cell biology and pre-clinical research. Although dense hanging drop arrays have been developed for high-throughput drug screening, it remains a challenge to integrate real-time monitoring and drug test in the current system. Therefore, it is highly desirable to develop novel approaches for tumor spheroid culture with easy preparation, in situ monitoring and characterization.

Liquid marbles (LM), which are liquid droplets coated by hydrophobic particles at liquid-gas interfaces, have attracted growing interest for their ease of fabrication and manipulation. Liquid marble has been applied for cardiogenesis of embryonic stem cells, mammalian cell cryopreservation, cancer cell growth assay, and generation of olfactory ensheating cell spheroids. However, real-time in situ monitoring was not possible in these applications because of the opacity of these liquid marbles. Although several attempts had been made to realize real-time observation for reaction tracking in liquid marbles, they required tedious effert and involved the use of either powerful magnet to open the liquid marble or expensive infra-red (IR) detector. We hypothesized that the size of hydrophobic particles used in coating liquid marble was essential for the optical property of liquid marbles. We further envisage transparent and gas-permeable liquid marbles could be formed by using silica nanoparticles and be used for culturing and drug sensitivity testing of tumor spheroids.

In this study, transparent nanoparticle-based liquid marbles with high gas-permeability were prepared to culture tumor spheroids in three dimensions without the need of supplementary growth factor. The culturing process of spheroids from a population of cancer cells or a single cell in the transparent liquid marbles could be optically recorded continuously. Compared to monolayer cells and spheroids generated in multi-well plate, cancer spheroids cultured in the liquid marbles showed enhanced viability under the treatment of chemotherapeutic drugs and small interfering RNA (siRNAs). Further research on molecular biology reveals that the E-cadherin gene is down-regulated while N-cadherin gene is up-regulated in spheroids comparing to clusters.

The 3D culture system is successfully applied in 13 cancer cell lines and 2 normal cell lines, which proves that it is a reliable 3D culture system. Besides bulk cell seeding, spheroids cultured from single cells are also succeeded in 3 cancer cell lines, while as a control, nonadherent plates failed in culture spheroids in these cell lines. Coculture is also carried out in the liquid marbles systems. Cancer cells cocultured with immunes cell or whole blood revealed that only after a certain dilution, spheroids would grow in the coculture systems.

Liquid marbles as precisely controlled system still lacks heating mechanism. While previous magnetic liquid marbles only focus on manipulation, we firstly put forward a new idea to use alternating magnetic field (AMF) to activate the magnetic nanoparticles coated on the liquid marbles to heat up the liquid marbles and the content without any direct contact. With this unique heating mechanism, we can modulate the chemical reaction rate in the liquid marbles and test then develop magnetothermal therapy to induce cancer cell apoptosis. This heatable liquid marbles, as bioreactor, are also suitable for LAMP assay and DNA/RNA detection.

This dissertation studies the multifunction of liquid marbles in cell culture and diagnosis assays. The studies in each chapter are described below:

Chapter 1: This chapter reviews the development of liquid marbles and their multiple applications. More specifically, the key properties of liquid marbles are introduced. Next, the previous application of liquid marbles in chemistry, material and engineering are summarized. Furthermore, the cutting-edge applications of liquid marbles in biomedical sciences are overviewed and discussed.

Chapter 2: This chapter introduces transparent liquid marbles for 3-dimension culture. Firstly, the properties of 3D model are introduced. Then, the development and characterization of transparent liquid marbles are described. Furthermore, the spheroid culture is carried out in liquid marbles and is applied in corresponding drug sensitivity test.

Chapter 3: This chapter describes application of liquid marbles in culturing spheroids from single cells to select cancer stem cells and reveal heterogeneity of cancer cell. Furthermore, transparent liquid marbles are applied in coculture cancer cells with fresh blood to mimic in vivo microenvironment to reveal immune resistance of different cancer cell lines.

Chapter 4: This chapter describes the fabrication of novel iron oxide nanocube coated liquid marbles (iNLM) and their application as magnetic heatable bioreactor for reaction kinetics modulation and DNA amplification. This research highlights that with AMF, we firstly realize controllable and contactless heating of iron oxide coated liquid marbles (iNLM). Through select proper AMF power, iNLM can be heated to required temperature from 25ºC to 86ºC. Homogeneous temperature distribution is observed iNLM during heating and cooling. Multicentric heating is firstly realized in liquid marbles and iNLM are flexible in position for heating. Controlled pulse heating and cooling are realized in iNLM. Importantly, through using LAMP assay, DNA amplification is firstly realized in liquid marbles. This is the first application of liquid marble in molecular biological reaction.

Chapter 5: Conclusions and Future Prospectives
The work presented in this dissertation widen our sight for develop of transparent and gas-permeable liquid marbles for spheroids culture and iron oxide nanocubes coated liquid marble for magnetic responsive reactor for controllable and contactless heating. Further work is required to further investigate the mechanism of spheroids growth in liquid marbles and translate all these exciting findings to clinical applications. The field will provide opportunities for interdisciplinary collaborations from physics, chemistry, biology, pharmacy, and clinical medicine, and may open the door for bring multifunctional liquid marbles into clinical trials.

    Research areas

  • Liquid marble, Cancer spheroid, 3D culture, bioreactor, DNA amplification