Development The development of biocompatible cellular constructs in non-diffusion limit size can provide suitable physicochemical and biological properties to support cell viability and functions, including cell adhesion, proliferation and differentiation (39, 40). The SF is one of the valuable and applicable natural polymers for its high strength (1, 7), good biological compatibility, and low immunogenicity that resulted in a wide application for various biomedical purposes (4-6). It can be physically and chemically modified to amend the biophysical and biochemical properties of SF-based scaffolds and constructs for tissue engineering and drug delivery purposes (1, 10). SF-based constructs can be fabricated through different techniques and shapes of architectures such as films, sponges, nano/micro-particles, fibers/tubes, bulk hydrogel, and conduit (1, 8, 10).
We prepared the scaffolds using extracted SF. The cellular vehicle should mimic an extracellular matrix and possess a 3D structure (39, 41). We applied modified SF with phenol moieties to fabricate the new composite scaffolds with good microstructure and suitable biocompatibility and mechanical properties for various biomedical applications, including tissue engineering and drug or cellular delivery. The spherical-shaped micro vehicles were obtained through conjugation of Ph moieties to the backbone of SF and in following using the microfluidic technique, which performed HRP-catalysed crosslinking reaction during particle fabrication.
3.1 SF-Ph synthesis and characterization
As depicted in Fig. 2A, SF polymer was modified by conjugation of Ph moieties deriving from tyramine hydrochloride in the presence of ethyl(dimethylaminopropyl) carbodiimide (EDC) and NHS (7). As shown in Fig. 2, 1H-NMR test elucidated peaks from 6.8 to 7.2 ppm, referring to the aromatic protons of the phenolic group based on reports (37, 42). In the mentioned region, striking differences were noticed between SF and SF-Ph peaks, prove successful synthesis. The Ph content introduced to modified SF was measured by UV visible spectrophotometer, which was 1.24 × 10-4 mol-Ph /g-SF-Ph. Since the reported Ph content was close to related works (35, 43), which have been utilized other polymers to fabricate microparticles, this synthesized polymer would also be suitable for that.
3.2 Preparation of SF-Ph microparticles
We evaluated the possibility of SF-Ph microparticle formation by the designed coaxial microfluidic device and enzyme-mediated crosslinking reaction using HRP in the presence of H2O2 as an electron donor. As microphotographs show, microparticles were successfully produced via the HRP-mediated enzymatic reaction, which was in agreement with other reports related to microparticles fabrication with other polymers which were modified with Ph moieties and used for enzymatic reactions such as hyaluronic acid (HA), alginate, carboxymethyl cellulose, and amylopectin, and gelatin (35) . As here liquid paraffin solution as a continuous phase meets the SF-Ph solution as a dispersed phase, shear stress causes the SF-Ph solution to pinch-off and form droplets. In fact, shear stress is caused by a reduction in the size of the dispersed phase channel at the orifice, at which hydrodynamic flow-focusing occurs. For further study, the microparticles size dependency was investigated by changing the flow rate of paraffin at the constant SF-Ph solution flow rate of 75 µL/min with the optical microscopic images, in which SF concentration was 4% (w/v) in the final solution. Image J was used to analyze resultant images, showing that paraffin velocity changes from 0.75 to 4.2 mL/min gave rise to changes in microparticles diameters approximately from 300 to 80 µm, as illustrated in Fig. 3 A and B. It was evident that an increase in liquid paraffin solution flow rate decreased microparticles diameter, as was proved in various reports (35) , confirming microparticles diameter dependency on liquid paraffin flow rate as a volumetric-driven flow force. The uniformity in size distribution and high cell viability are critical parameters for cell encapsulation, and also, there is size diffusion limit for cellular constructs. The size below 200 µm is highly recommended for cellular vehicles (40, 44) . As shown in Fig. 3 C, the mean diameter of microparticles was considered about 150 µm for the following experiments to minimize the formation of necrotic regions in the microparticles' canter (44) . Microphotograph and narrow size distribution for fabricated microparticles confirm that specimens were in uniform size and spheroid shape. These properties give us this opportunity to utilized SF based microparticles for cell or drug delivery in specified density or concentration, respectively. The average size of microparticles follows a Poisson-like distribution and can be finely controlled by flow velocity. We did not observe any structural instability, including coalescence of SF-Ph hydrogel in micron size, which means proper concentration of SF-Ph polymer, reactants, volumetric flow of solutions in a microfluidic device, and cellular density. It is reported that these parameters can cause breakage of particles as well as their coalesce into a bigger size or non-size uniformity such as pear, oval, and spindle shape vehicles even if the surfactant concentration in the collection solution be quite high (45) .
3.3 Cellular behaviour
The probable side effects of enclosing cells can be associated to lack of culture medium which means lack of oxygenation and nutrition combined with shear stresses causes in the microencapsulation process, including mixing cells with viscus polymeric solution of SF-Ph, the existence of chemical reactants, and hydrogelation as well as shear stress through perfusion flow in microfluidic channels (35, 38). Cellular viability and activity were evaluated by both microscopic images and MTT assay at two-time points just and 12 days after cell enclosing (35). Morphologically, the spread up of cells in the initial hours of seeding, which were harvested from the encapsulation process and even after 12 days, were similar and did not find any difference among their morphologies and growth ability (Fig. 4A-F). Moreover, cellular growth shows harvested cells from encapsulation in different time-lapse (Fig. 4G) proliferated equally and mitochondrial activity of cells which indicate lack of any cytotoxicity of encapsulation process and extension time of incubation for encapsulated cells.
3.4 Enclosed cells activity assay
The viability and proliferation of enclosed cells are the deterministic factors in considering a suitable cell carrier (24). Hence, cell growth in microparticles was evaluated by microscopic images and MTT assay on different days after microencapsulation. Until the 15th day, embedded L929 cells vividly proliferated and occupied all regions of microparticles, illustrated in Fig. 5A-F. In addition, the quantitative analysis of cellular growth has proceeded through the MTT assay. Fig. 5. indicated that cell mitochondrial activity elevated until the 18 days of microencapsulation, which can be attested to denser microstructures (46). The cellular growth and proliferation in SF-Ph microparticles would be due to the existence of cell interactive moieties in SF structure, such as cell adhesive motifs (10, 47). Meanwhile, cellular growth on hydrogel surfaces for most polysaccharides and synthetic polymers hindered and obtained a low proliferation rate. Protein-based hydrogels such as gelatin, silk, and collagen could promote cellular growth even 3D cell culture system for encapsulated cells which microscopic stress surrounding cells have a negative impact on cell growth (41, 48, 49).
These results supported that microstructures lent themselves in favour of cell growth without specific harmful effects on it and efficiently transported oxygen and nutrition to enclosed cells. However, further following of mitochondrial activity showed that it reached a plateau after the 21st day, which means microparticles possibly suppressed cell growth (44).
3.5 Spherical microtissue fabrication
By the time the gradual aggregation of enclosed cells powerfully confirmed the presence of cell-cell interaction, which is crucial for the preservation of cell viability (24). These microparticles provided cell-cell and cell-matrix interactions resulted in strong adhesive spherical microtissue with a more sophisticated microenvironment than the single cell (50). As it was shown in (Fig. 5I), the remaining spherical microtissue of L929 cells after microparticles digestion using collagenase supported the mentioned claim, making this procedure convenient to apply for spherical tissue formation to use in disease modeling (28), drug discovery (29), and tissue engineering purposes (30, 31). Moreover, collagenase used to digest microparticles is naturally exists in the body tissues (51), so microparticles can be used as cell and drug delivery vehicles in vivo (35).