*3.3. Fibrin*

Fibrin is a blood-derived fibrous protein formed by fibrinogen polymerization under the catalysis of thrombin, calcium ions, and/or factor XIII. Fibrinogen powder can dissolve in water to form a solution with low viscosity. Like gelatin, fibrin has excellent biocompatibility and biodegradability for various biomedical applications, such as hemostasia, wound healing, pharmacy, microencapsulated cell delivery, tissue engineering, and 3D bioprinting [104].

As stated above, layer-by-layer bioprinting is a logical choice for the manufacture of stratified organs, such as the liver, heart, kidney, and lung, containing multiple cell types. 3D bioprinting of stratified organ replacements depends on bioinks with appropriate rheological and cytocompatible properties. An obvious shortcoming of the fibrinogen solution for 3D organ bioprinting is that its viscosity is too low to be piled up in layers even in very high concentration (e.g., 4% w/v) [55]. After polymerization, the 3D printed constructs undergo further contraction or shrinkage for homogeneous or heterogeneous tissue formation. Structural stability is the major concern for pure fibrinogen as a bioink for 3D organ bioprinting.

The first pertinent fibrin 3D bioprinting was reported in 2007, in which fibrinogen was used as an additive of gelatin-based bioinks [105,106]. By blending fibrinogen with thermoresponsive gelatin solutions, high-resolution 3D constructs with excellent cell viability (i.e., ≈ 100%) have been obtained. During the 3D printing process, gelatin molecules provide fast gelation and immediate postprinting structural fidelity, while fibrinogen molecules ensure long-term mechanical stability upon thrombin polymerization. Physical blending and chemical crosslinking of fibrinogen/gelatin solutions can prevent breakdown and shrinkage of 3D printed constructs for a relatively longer period under physiological conditions (e.g., 37 ◦C).

More than 10 years later, Hinton and colleagues printed a Ca2+-mediated alginate/fibrinogen hydrogel into a gelatin slurry bath containing thrombin using a similar extrusion-based 3D printer and working principle [16,17]. When the alginate/fibinogen hydrogel was extruded from the 3D printer nozzle, it met the thrombin molecules in the bath and rapidly polymerized to solidify the Ca2+-mediated alginate/fibrinogen hydrogel. This is another double crosslinking or gelling path for 3D bioprinting with fibrinogen-containing composite bioinks and is termed as "bioplotting" (Figure 3). With different gelling paths, cells in the fibrinogen-containing hydrogel can be assembled in layers according to the predesigned CAD models.

Similarly, Burmeister and colleagues delivered ASCs via PEG–fibrin, i.e., PEGylated fibrin (FPEG) hydrogel, as an adjunct to meshed split-thickness skin grafts in a porcine model [107]. They showed that ASCs delivered in FPEG could enlarge the blood vessel size in a dose-dependent way, wherein FPEG acts as both a porous scaffold to prevent contraction and an ASC-delivery vehicle to accelerate angiogenesis.

For 3D organ bioprinting, the printing properties of fibrinogen solutions can be easily tailored by varying the proportion of its component in the composite bioinks [108–111]. Thermosensitive polymers, such as gelatin and agar, support extrusion-based 3D bioprinting of a wide range of natural polymers, including fibrinogen, chitosan, alginate, and hyaluronate. Succeeding (or tandem) chemical crosslinking and polymerization of fibrinogen-containing composite bioinks facilitates the printing results of the stratified cell-laden 3D constructs with increased stiffness and stabilization. For example, double gelation of gelatin/alginate/fibrinogen bioinks using both CaCl2 (for alginate ion crosslinking) and thrombin (for fibrinogen polymerization) is the most popular approach today for large-scale organ manufacturing (Figure 8) [30,36].

**Figure 8.** 3D bioprinting of ASC-laden gelatin/alginate/fibrin hydrogel for organ manufacturing in Prof. Wang's laboratory at Tsinghua University: (**a**) a pioneering double-nozzle 3D bioprinter made in this laboratory; (**b**) schematic description of the cell-laden gelatin/alginate/fibrin hydrogel and pancreatic islets being printed into a grid construct using the 3D bioprinter; (**c**) a large-scale 3D printed grid construct containing ASC-laden gelatin/alginate/fibrin hydrogel cultured in a plate; (**d**) a grid ASC-laden gelatin/alginate/fibrin construct after being cultured for one month; (**e**) a multicellular construct after three weeks of culture, containing both ASCs encapsulated in the gelatin/alginate/fibrin hydrogel before epidermal growth factor (EGF) engagemen<sup>t</sup> and relatively integrated pancreatic islets seeding in the predefined channels (immunostaining with anti-insulin in green); (**f**) some envelopes of the islets were broken after one month of culture; (**g**) immunostaining of the 3D construct with mAbs for CD31+ cells (i.e., mature endothelial cells from the ASC differentiation after three days of culture with EGF added in the culture medium) in green, having a fully confluent layer of endothelial cells (i.e.,

endothelium) on the surface of the predefined channels; (**h**) a vertical image of the 3D construct showing the fully confluent endothelium (formed from endothelial cells) and the predefined go-through channels; (**i**) immunostaining of the 3D construct with mAbs for CD34+ cells (i.e., endothelial cells) in green and pyrindine (PI) for cell nuclei (nucleus) in red; (**j**) immunostaining of the 3D construct with mAbs for CD34+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; (**k**) immunostaining of the 3D construct with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (**l**) a control of (k), immunostaining of the 3D construct with mAbs for CD31+ endothelial cells di fferentiated from the ASCs in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; ( **m**) immunostaining of the 3D construct with mAbs for CD31+ cells in green and Oil Red O staining for adipocytes in red, showing both the heterogeneous tissues coming from the ASC di fferentiation after a cocktail growth factor engagemen<sup>t</sup> (i.e., on the surface of the channels, the endothelium coming from the ASCs di fferentiation after being treated with EGF for 3 days; deep inside the gelatin/alginate/fibrin hydrogel, the adipose tissue coming from the ASC di fferentiation after being subsequently treated with insulin, dexamethasone, and isobutylmethylxanthine (IBMX) for another three days); (**n**) a control of (m), showing all the ASCs in the 3D construct di fferentiated into target adipose tissue after three days of treatment with insulin, dexamethasone, and IBMX but no EGF; (**o**) immunostaining of two-dimensional (2D) cultured ASCs with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (**p**) immunostaining of 2D cultured ASCs with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium. Images reproduced with permission from [36].
