Novel versatile 3D bio-scaffold made of natural biocompatible hagfish exudate for tissue growth and organoid modeling

Highlights

• Hagfish exudate was utilized as a novel biomaterial for 3D bio-scaffolding.

• Successful tissue growth and organoid formation achieved using hagfish exudate

• Natural hagfish exudate was ~100% biocompatible non-cytotoxic substrate for cell culture.

• Chloroformization was used as a simple process for sterilization of hagfish exudate.

• Simple sonication regulated fibrocity and beaded structure for simulated nerve system.

Abstract

Hagfish exudate is a natural biological macromolecule made of keratin intermediate filament protein skeins and mucin vesicles. Here, we successfully examined this remarkable biomaterial as a substrate for three-dimensional (3D) cell culturing purposes. After the sterilization with chloroform vapor, Dulbecco's modified eagle medium was mixed with the exudate to rupture the vesicles and skeins; a highly soft, adherent, fibrous and biocompatible hydrogel was formed. A variety of cells, including Hela-FUCCI, NMuMG-FUCCI, 10T1/2 and C2C12, was cultured on the hagfish exudate. A remarkable 3D growth by ~2.5 folds after day 3, ~5 folds after day 5, ~10 folds after day 7 and ~15 folds after day 14 were seen compared to day one of culturing in the hagfish exudate scaffold. In addition, the phase contrast, fluorescent and confocal microscopy observations confirmed the organoid shape formation within the three-week culture. The viability of cells was almost 100% indicating the great in vitro and in vivo potential of this exceptional biomaterial with no cytotoxic effect.

Introduction

In order to mimic a natural niche-base environment, a proper 3D substrate is necessitated for cell growth, tissue alteration and organ development both in-vivo and in-vitro [1]. This setup is crucial for drug screening and understanding the mechanisms of diseases when new medicines are utilized [2,3]. Nevertheless, an appropriate scaffold with multiple usages for 3D cell culture has yet remained an ongoing challenge in tissue engineering. Since a couple of decades ago after the emergence of modern tissue engineering and organ implanting, fully biocompatible scaffolds which mimic the natural extracellular matrix of tissues have remained either very costly, e.g., dentistry, or a challenge for regular uses of biomedical sectors [4]. In addition, their production process may include unprecedented cytotoxic elements [5]. Here, we process and examine hagfish exudate for the first time to utilize it as a 3D-scaffold biomaterial for cell culture without considerable modification or blending with other biomaterials, no crosslinking with macromeres, and no polymerization with other monomers.

Hydrogels are one of the most important bio-substances for cell-culturing [6,7]. They are utilized as suitable scaffolds for cell retention to gradually produce and hold the growth factors including oxygen, nutrients, and proteins in their water-swollen network [[8], [9], [10]]. Compared to synthetic hydrogels, natural bio-substrates have the advantage of providing bioactive components and facilitating the adhesion, growth and maturation of cells cultured on them [11,12]. Among natural hydrogels, protein-based biogels are highly advantageous for cell culture due to their inherent sequence-dependent ability to mediate cell signaling, adhesion and controlling tissue developments [13]. In addition, they have the capability of resembling the natural viscoelastic and strain-stiffening characteristics of extracellular matrix of native tissues [14,15]. Unfortunately, during an isolating process of protein biomaterials from their origin tissues using mechanical, chemical or biochemical methods, the microstructure of proteins may change and turn into a relatively new biomaterial with new cellular and biological characteristics [16].

Keratin is an abundant insoluble intermediate filament (IF) protein with ~10 nm thickness [17]. It belongs to one of the most important super-families of self-assembled fibrous proteins in the cytoskeleton cells of most animals [18,19]. Keratin is stable in natural conditions with retained innate characteristics to maintain cell integrity, cell adhesion, growth, proliferation, migration, tissue regeneration, and to protect the cells from apoptosis [20]. Keratin is an in-vitro and in-vivo biodegradable material, though its degradation is slower than other biologically degradable polymers like poly(lactic acid) and collagen (type I); it does not leave any abnormal scar in the tissue sites after transplantation [21]. These characteristics make keratin as a suitable cellular scaffold for tissue and nerve regeneration, wound healing, bone repair, and controlled drug-release [22,23]. In spite of incredible characteristics and unique advantages of keratin, the process of extraction and purification of this protein is difficult, time-consuming and cost-ineffective compared to the ones from other natural proteins, e.g., collagen [24]. On the other hand, available procedures to extract and purify keratin damage its biomechanical properties, hence, they need complementary stages, including blending with different natural and synthetic polymers or bio-glasses to rebuild the strength, flexibility and porosity [25,26].

In contrast to more highly evolved vertebrates where keratin intracellularly remain within the epithelial cells or become epidermal appendages (such as wool and feather), a hagfish IF deposits in the extracellular space as a fine exudate through the gland pores out of the body of a hagfish [27]. Therefore, the necessity of extracting the keratinous biomaterial from the complex tissues or coarse structures is eliminated. In fact, the hagfish keratin polypeptide possesses homologous regions to human epidermal keratin, a characteristic that suits it as a highly bio- and immuno-compatible material for culturing human cells and similar transplantation organs [36]. So, systematic investigations are required to find the potential of hagfish exudate scaffold for the expansion of human cells. These unique features encouraged us to examine the natural fibrous and beaded protein structure of the exudate for cell culturing.

Some groups have engineered native hagfish thread and rendered it as a biomimetic foundation [28]. Kim et al. blended the keratin, which was extracted from hagfish slime threads, with polylactic acid (PLA) polymer solution to construct a nanofibrous scaffold using electrospinning technique [29]. From their results, hagfish keratin had better properties than wool keratin which improved the cellular attachment, proliferation and acceleration for the differentiation of stem cells. The procedures of extracting and blending, however, eliminated the incredible natural bioactivity of nanofibrous as well as the bead structure of pure hagfish protein threads and skeins. In another study, two types of semi-crystalline nanocomposite films of hagfish slime IF proteins were produced through solubilizing processes. Although both films were immediately hydrated and softened in water [30], their natural bioactive characteristics changed noticeably.

Hagfishes (Craniata: Myxini) are cosmopolitan ancient species of chordates living deep in oceans. Their place on the evolutionary tree specifies an evolutionary transition from rudimentary to true vertebrates [31]. Each live fish can release thick glandular exudate which is enough to form ~20 L of slime as a defense mechanism, the release of exudate till fully formation of slime takes only 100 ms [32]. The viscose hagfish slime is one of the softest biomaterials with the elastic modulus of 0.02 Pa in an aqueous environment [33]. This highly soft biomaterial can be used to augment the healing process of wound by covering the injured area of the skin [34]. Hagfish exudate is highly biodegradable because of its natural keratin-base composition [35]. It contains organic osmolytes, inorganic ions, and enzymes; it is particularly made of mucin vesicles and silk-like IF keratin threads which dominantly form the hagfish exudate and originate the slime's properties [36]. These threads are made of three main intermediate filament proteins of α, β and γ; protein β is a post-translational modification of γ protein. High contents of threonine (13%) and phosphorylation of it change the ultrastructure of threads over maturation [37].

Thread production begins with a small bundle of IFs and continues with adding a huge number of them and binding microtubules (MTs) through ionic and hydrogen connections to raise the length and width of the thread [38]. A 12-nm IF spirals along the forming IF structure, after which the spiral rind is disappeared at the condensation step while a fluffy frame is created. Finally, as discrete IFs are adjoined, a single IF superstructure is formed. MTs and the fluffy frame of surface disappear and the empty space occupied by MTs is replaced by IFs, as a result, a single-stranded, continuous, non-branched slime thread is formed (Fig. 1a) [39]. The threads, with ~15 cm resting length and breaking point at 34 cm and a diameter of 1–3 μm, are exquisitely packaged in a skein of exudate (Fig. 1b) [37].

Experiments have shown that all mucin vesicles in hagfish exudate are swollen and ruptured in the presence of ≥3 mM calcium ions. 40% of them are ruptured in a hyperosmotic solution of sodium chloride that exists in seawater as well as in DMEM (Dulbecco's modified eagle medium). We used the latter solution to mix with the hagfish exudate in order to create the hagfish exudate scaffold [40]. A rupture of the mucin vesicles releases the elongated strands of mucus glycoproteins [41].

On the surface of skeins, there are adhesive proteins which resembles S-type lectins or galectins. Galectins bind with glycan moieties containing galactose and its derivatives that exist in mucin glycoproteins. These adhesive proteins facilitate the binding of threads with mucin strands and forge cross-links between the threads and mucous. The entanglement of threads and mucin strands transfer the hydrodynamics forces to the skeins and trigger unraveling the skeins. This process forms the thread fibrous network after suctioning and mixing with DMEM or seawater (Fig. 2) [42].

In the present study, we create a keratin hydrogel scaffold from hagfish exudate and slime using a cost/time effective method with no considerable extraction, purification, processing, functionalization or blending with other materials. After extracting the exudate from the fish and sterilization using chloroform vapor, DMEM is used to swell the mucin vesicles, unravel the skeins, and form a fibrous scaffold. A variety of cells, including Hela-FUCCI, NMuMG-FUCCI, 10T1/2 and C2C12, are cultivated on the hagfish biomaterial to assay the interaction of cells with this natural scaffold. The results show that the cells are successfully grown on the scaffold and formed organoid shape structures due to the high proliferation of cells. Softness, beading, fibrosity, porosity and biocompatibility of hagfish exudate can make it a promising scaffold for 3D cell culture in tissue engineering and regenerative medicine.