**1. Introduction**

Over the last century, two-dimensional (2D) in-vitro cell culture has been used in studying the responses to stimulation from biological and biochemical materials, such as drugs, toxic materials, and detoxification. Traditional in-vitro cell tests are based on 2D culture on a flat surface, and the 2D culture environment is completely different from the human body. Cells inside the human body are surrounded by extracellular matrix (ECM) and tissue fluid in a complex three-dimensional (3D) space. Thus, cells are difficult to activate in 2D environments to maintain differentiation and expression of tissue-specific physiological functions and physiological activity [1,2].

Tissue-on-a-chip is a recapitulation of the biological and mechanochemical environment of human body tissues using a small device chip [1]. This technique includes cells, chemical and physical environments, and the microenvironment. It is used in the development of in-vitro disease models, drug screening, toxicity testing, and disease research, by providing a cellular environment that better mimics human physiological conditions [3–5]. Typical tissue-on-a-chip consists of an integrated microscale engineering system, several types of cells, and culture medium. The microelectromechanical

system (MEMS) allows the use of microfluidic devices to reconstitute distinct features of the tissue–tissue interface, physiological movements, and biochemical environment similar to the human body. Marino et al. [6] presented a microfluidic system to mimic the blood–brain barrier (BBB). The system consisted of porous microtubes fabricated using two-photon lithography and enabled co-culturing brain microcapillary cells and functioning.

Recently, various 3D environmental features, such as microporous membrane [7,8], hydrogel [9–11], and nanofibers [12,13], have been introduced to tissue-on-a-chip applications to provide the similar structures and functions of the human body to cell culture. The hydrogel is highly permeable and an excellent biocompatible material. Annabi et al. coated microfluidic channels with synthesized photo-crosslinkable gelatin and tropoelastin-based hydrogel to improve cardiomyocyte culture in a polydimethylsiloxane (PDMS) surface [9]. Au et al. fabricated hepatic tissue models by encapsulating HepG2 and NIH-3T3 cells in a hydrogel. Their platform showed better results than 2D cell culture systems in drug screening [14]. Gumuscu et al. [15] proposed a microfluidic cell culture platform composed of 3D collagen hydrogel compartments, and they applied their system for co-culture of human intestinal cells and drug screening in preliminary level. Porous membranes are considered mimic basal membranes of barrier tissue, such as tissue–tissue, tissue–liquid, and tissue–air interfaces in organ-on-a-chip [16,17].

Nanofibers have diameters ranging from tens to hundreds of nanometers and similar morphology to the extracellular matrix of the human body. In particular, nanofibers are well suited for cell nutrient exchange, communication, and efficient cellular responses because of large surface areas, high porosity, and spatial interconnectivity [18,19]. As a result, nanofibers have been extensively applied to a variety of applications such as porous membrane and scaffold in biotechnology [20–22]. In addition, cells are easily attached and better proliferated in electrospun composite nanofiber than in a conventional 2D culture environment, such as a petri dish [23].

Here, we present a microfluidic chip with a nanofiber scaffold, which can provide a 3D human body ECM-like environment to cell culture and monitor cell status and activity using a conditioned culture medium. The nanofiber scaffold was electrospun so that it composed various diameter fibers, thereby providing highly porous morphology to cells under the well-defined microfluidic chip conditions. In particular, a perfusion method, which enables real-time monitoring cell status, was demonstrated. The developed chip was applied to 3D culture of HepG2 cells, which has various functions, such as cell growth and secretion of proteins. Our results demonstrated that HepG2 cells were cultured with 3D culture-specific morphology (i.e., large spheroids) [3,24], and their protein production was successfully monitored for 14 days.
