Cell marbles: A novel cell encapsulation technology by wrapping cell suspension droplets using electrospun nanofibers for developmental engineering

Highlights

• Developmental Engineering aims to imitate natural tissue regeneration processes using modular building blocks/materials.

• A technology to fabricate cell marbles (CMs) by wrapping cell suspensions with electrospun nanofibers is developed.

• CMs are mechanically stable to be handled as soft solids, thus accurately delivered using forceps to three distinct culture systems.

• Release of cells, culture media and nanofibers is achieved via controlled rupture of CMs.

• The prominent traits of the skin cells are well preserved during cell encapsulation and delivery processes.

• Our research demonstrates the great potential of CMs for developmental tissue engineering.

Abstract

Developmental Engineering aims to imitate natural tissue regeneration processes via an additive manufacturing approach. This research developes a technology to fabricate ready-made cell marbles (CMs) by wrapping cell suspension droplets of (3−15 μl) with electrospun hydrophobic nanofibers, as modular building blocks for developmental engineering. Human dermal fibroblasts and/or immortalised keratinocytes were suspended in the culture media cores of the CMs. The encapsulated cells were observed to precipitate at bottoms or up-inclined inner surfaces of the fibrous shells within 10 min. The CMs were mechanically strong enough to be handled as soft solids, thus easily and accurately delivered using forceps into three distinct culture systems, including tissue culture plastics, cellulosic scaffolds and in vitro fibrin wound models. The release of the cells, culture media and nanofibers into specific delivery points within the investigated culture systems was achieved via the controlled rupture of the CMs triggered by the simple hydrophobic-hydrophilic interaction between the nanofibers and the aqueous surroundings. Further cell and tissue culture studies indicated that the prominent traits of the skin cells were well preserved during cell encapsulation and delivery processes, suggesting the great potential of the CMs for additive tissue manufacturing in developmental engineering.

Introduction

Developmental Engineering has been proposed to imitate natural mechanisms that govern tissue formation and regeneration via in vitro tissue engineering approaches (Freeman and McNamara, 2017; Ingber et al., 2006; Marcucio et al., 2017), and “off the shelf” cells and mini tissue modular building blocks are essential for the additive manufacture of fully functional tissues using this novel strategy (Biggemann et al., 2018; Schon and Hooper, 2017). This work discusses an innovative, robust and cost-effective method to develop modular building blocks for Developmental Engineering using cells, culture media and electrospun polymeric nanofibres.

The technology here proposed is based on the concept of liquid marbles (LMs), namely non-stick droplets (normally aqueous) wrapped with micro- or nano-scale hydrophobic particles or powders. LMs are emerging as miniaturized platforms to prepare and manipulate liquids at microscale (Ireland et al., 2018; Kido et al., 2018; McHale and Newton, 2011). Due to their several advantages, such as reduced amounts of reagents, shortened reaction rate, enhanced efficiency and flexibility, LMs have been proposed for various applications including gas and liquid sensing (Tian et al., 2010), microreactors (Arbatan et al., 2012), water pollution detection (Bormashenko and Musin, 2009) and medical applications (Avrămescu et al., 2018). There is also a growing interest to further improve their mechanical properties (Chin et al., 2013) or develop analogous systems with higher robustness (Ueno et al., 2015). In our previous research, electrospun nanofibres with engineered wetting properties and morphologies have been optimised and then used to encapsulate water and oil drops (Mele et al., 2014). These fibre-coated drops exhibit mechanical stability and stress resistance higher than conventional liquid marbles due to the nanofibrous network formed in the surrounding cloaks. Due to the possibility of further modifying the nanofibers by incorporating functional particles, active molecules and drugs (Davis et al., 2015), and carrying out mixing processes inside the liquid cores, these robust, stable fibre-coated droplets are suitable as miniaturized bioreactors, sensors, micro-actuators and drug delivery systems (Mele et al., 2014).

Here, we show that cell culture medium droplets containing different types of human cells can be wrapped with nanofibrous cloaks to create cell marbles (CMs) for Developmental Engineering. A scalable process was firstly established in a Class II biological safety cabinet (BSC) to encapsulate human dermal fibroblasts (HDFs) and/or immortalised keratinocytes (HaCat cells) suspended in cell culture media with electrospun nanofibres of a fluoroacrylic copolymer; the distribution of these cells within the CMs was analysed via Phase contrast microscopy (PCM) and fluorescent microscopy (FM). The controlled rupture of the CMs and subsequent accurate delivery of the encapsulated cells and nanofibers was first investigated using tissue culture plastics (TCPs). The CMs were then used as an alternative strategy to seed and culture cells in three-dimensional (3D) porous cellulosic scaffolds and fibrin clot to further evaluate the efficacy of cell delivery and its potential as the ready-made modular building blocks for Developmental Engineering.