Biomaterials for microfluidic technology

Micro/nanomaterial-based drug and cell delivery systems play an important role in biomedical fields for their injectability and targeting. Microfluidics is a rapidly developing technology and has become a robust tool for preparing biomaterial micro/nanocarriers with precise structural control and high reproducibility. By flexibly designing microfluidic channels and manipulating fluid behavior, various forms of biomaterial carriers can be fabricated using microfluidics, including microspheres, nanoparticles and microfibers. In this review, recent advances in biomaterials for designing functional microfluidic vehicles are summarized. We introduce the application of natural materials such as polysaccharides and proteins as well as synthetic polymers in the production of microfluidic carriers. How the material properties determine the manufacture of carriers and the type of cargoes to be encapsulated is highlighted. Furthermore, the current limitations of microfluidic biomaterial carriers and perspectives on its future developments are presented.


Introduction
For disease treatment and tissue regeneration, the efficient delivery of therapeutic substances such as drugs, cells and genes to biological systems is essential.The ideal delivery system is supposed to achieve high therapeutic efficacy Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
while minimizing side effects.However, conventional delivery methods, including oral administration and hypodermic injections, face a number of challenges in achieving efficient treatment.For example, the bioavailability of oral administration is quite low because the drug molecules cannot penetrate the wall of the gastrointestinal tract, while hypodermic injection can lead to a burst release of the drug and reduce therapeutic effects.Hypodermic injection of cells also fails to achieve high cell retention and engraftment because of the lack of a suitable microenvironment for cell migration and proliferation [1].
Biomaterials are robust tools for advanced therapy and tissue regeneration, serving as efficient drug and cell carriers.Biomaterial carriers maintain the bioavailability of therapeutic

Future perspectives
Microfluidic technology has significantly facilitated the development of micro/nanomaterial preparation.Through flexibly controlling the fluid behavior in microchannels, various forms of micro/nanomaterials can be produced and show great potential for advanced drug and cell delivery.We reviewed advances in microfluidic-based biomaterial carriers, such as microspheres, nanoparticles and microfibers, focusing on their generation mechanism.And we highlighted biomaterials for microfluidic processing, including natural materials and synthetic polymers.
substances and control drug release, thus preventing potential toxicity induced by repeated administration [2,3].Micro-or nano-materials have aroused much attention in the field of biomaterials for their minimally invasive and injectable administration.Compared to macroscopic materials, micro/nano biomaterials exhibit improved loading efficiency owing to their small size and enhanced specific surface area.Nanomaterialbased therapies are becoming increasingly popular because of their ability to conjugate with specific ligands for targeted delivery and precise treatment [4].In addition, microand nano-materials can be assembled into macroscopic materials using bottom-up methods and incorporated into other advanced manufacturing technologies, such as 3D printing [5].Conventional methods for producing micro/nano materials include emulsion agitation and solvent evaporation [6,7].However, the lack of tight control over reaction conditions in these strategies leads to low batch reproducibility and wide size distribution of micro/nanomaterials, which hinders the controlled release of encapsulated cargos.
To overcome the above-mentioned limitations of conventional approaches, an increasing amount of research is focused on the preparation of micro/nanomaterials using microfluidic technique.Microfluidics is an emerging interdisciplinary technique that manipulates single-phase or multiple-phase fluids at the microscale [8].Through the precise control of microfluid in a homogeneous reaction environment, microfluidics allows the fabrication of a variety of micro-and nano-materials, such as microspheres, nanoparticles and microfibers with controllable size and high batch reproducibility.Moreover, sealed microfluidic channels isolate fluids from bacteria and small molecules in the atmosphere, allowing simultaneous encapsulation of cells while manipulating microfluids.Beyond the production of isotropic biomaterials, biocarriers with various morphologies and complex structures such as multicompartment and core/shell can also be manufactured via the flexible design and modification of microfluidic devices [9].
Nowadays, a broad range of biomaterials has been used in microfluidic manufacturing.They are soluble in water or specific solvents and can therefore be prepared as microfluids to fabricate advanced micro-or nano-materials (scheme 1).In this paper, we review recent advances in the applications of microfluidics to the manufacture of biomaterial carriers.First, the generation mechanism of representative microfluidic biomaterial carriers, including microspheres, nanoparticles and microfibers is introduced.The channel geometry and fluid behavior are highlighted.Then, a systematic review of materials used for microfluidic fabrication is presented.The application of natural materials (i.e.polysaccharides and proteins) and synthetic materials in drug and cellular carrier design are described.We emphasize the relationship between the properties of the material and cargoes being encapsulated.Finally, we summarize the characteristics and limitations of current microfluidic materials and discuss the future developments in designing microfluidic biomaterial carriers.

Representative microfluidic carriers and generation mechanisms
Microfluidics manipulates single or multiple fluids in microchannels to produce various forms of biomaterial carriers at scales ranging from micron to nanoscale.By adjusting the geometry of microfluidic channels and flow rates, the structure and size of the biomaterial carriers can be easily controlled.This section describes representative applications of microfluidic carriers, including microspheres, nanoparticles, and microfibers.We discuss the advantages of microfluidic carriers over those fabricated by conventional approaches and their current limitations.The mechanisms by which these biocarriers are generated in microfluidic devices are also presented.

Microfluidic droplet/microsphere
The development of microfluidics greatly benefits spherical biomaterial carriers, as microspheres or microparticles can be obtained directly from microfluidic droplets.For example, hydrogel microspheres are derived from crosslinked precursor droplets, and synthetic polymer microparticles such as polylactic acid (PLA) and poly(ε-caprolactone) (PCL) can be produced from emulsions by solvent evaporation [10,11].Microspheres are effective biomaterial carriers that can prolong the release of drugs in the body through injectable administration.In addition to loading drugs, microsphere carriers are robust tools for cell delivery by encapsulation or physical absorption due to their expanded specific surface area [12].Droplet microfluidic is the most representative subcategory of microfluidic technology, generating and manipulating droplets from an emulsion template composed of two immiscible liquids.Using droplet microfluidic technique, various engineered microparticles with controlled size and geometry have been developed as robust biomedical vehicles for drug and cell delivery.
Conventional emulsion stirring method for droplet generation lacks the control of the monodispersity of the droplets.In most cases, the droplets prepared by agitation are highly polydisperse.Microfluidic technology manipulates fluids in a microchannl, and when the scale of the fluid is miniaturized to the micron level, the inertial forces can be neglected (Reynold number is very low).In this case, viscous force and interfacial force dominate the fluid behavior, ensuring that the fluid properties remain constant.Therefore, microfluidic droplets show higher batch-to-batch reproducibility and a narrower size distribution compared to conventional methods, which facilitates precise controlled release of the encapsulated cargoes.The size and generation rate of droplets depend on the flow rate and microchannel geometry.In droplet microfluidics, two immiscible fluids are injected into microscale channels and form an immiscible interface when they meet at the junction.Once the viscous shear force overcomes the interfacial tension, one disperse fluid is squeezed and broken by the other immiscible fluid, thus enabling the precise generation of one droplet at a time [13].Microfluidic droplets can then be formed into microsphere carriers via various solidification strategies.
In order to produce microsphere carriers, droplet generation junctions need to be formed on the microfluidic device for generating emulsion templates.The most common droplet generators include flow-focusing, T-junction (also called crossflow, and co-flow [14] (figure 1(A)).Water/oil (W/O) and oil/water (O/W) systems are representative emulsion templates for preparing microfluidic droplets.Carriers for hydrophilic drugs and cells can be easily obtained from W/O emulsions while O/W emulsions are robust tools for generating hydrophobic drug vehicles.Water/water (W/W) templates using two incompatible aqueous solutions and gasshearing microfluidics have emerged in recent years and can also be generated and manipulated in conventional droplet generators.As oil-free strategies, they showed great advantages in the production of biocarriers as cell vehicles because of the absence of oil washing processes that can damage the cells [15,16].PDMS chips and coaxial glass capillaries are the two most common microfluidic devices used to produce droplet templates.Moreover, by extending the single emulsion template to double or multiply emulsion templates, droplets with more complex structures, such as core/shell and multicompartmental microspheres, can be manufactured to simultaneously deliver cargoes with different properties [17].
However, it must be pointed out that droplet microfluidics is limited by low production rate and its yields are far from comparable to conventional methods.Fortunately, parallelized microfluidic devices with multiply droplet generators and outlets have been developed to achieve high-throughput production and are expected to realize the translation of microfluidic microspheres from lab to bed [18].

Microfluidic nanoparticle
Nanoparticles are fascinating biomaterial carriers that can efficiently deliver cargo by minimizing nonspecific targeting and maximizing specific interactions [19].Conventional fabrication methods for nanoparticles include nanoprecipitation and solvent evaporation from emulsions.However, these schemes lack control over particle size and distribution as well as batchto-batch inconsistency, because the inertial and viscous effects that govern mass transport in fluids result in nonlinearities and uncontrollable experimental variables [20].Nanoparticles prepared by conventional methods are large in size and commonly have a wide size distribution, which will limit the efficiency of drug delivery.For example, larger nanoparticles (>200 nm) have short circulation times as they are easily recognized and cleared by the immune system [21].
Microfluidics is known for producing highly uniform nanoparticles, whose sizes can be easily controlled by varying the flow rate.Unlike droplet microfluidics based on phase immiscibility, microfluidics for fabricating nanoparticles is based on the rapid mixing of two miscible phases.In the microchannel, the inertial effect of the fluid is negligible, so the nanoparticles are generated by molecular interdiffusion of the two miscible fluids, rather than by the shear force at the phase interface [22].In addition to the applications in drug delivery, microfluidic nanoparticles are also robust carriers with high DNA loading efficiency due to their larger surface area per unit mass [23].
To fabricate microfluidic nanoparticles, polymers dissolved in organic solvents and aqueous solution are injected into a microfluidic mixer, producing millisecond mixing and rapid solvent exchange.Polymer nanoparticles are obtained from nanoprecipitation via self-assembly.Microfluidic geometries commonly used for nanoparticle synthesis includes hydrodynamic flow focusing (figure 1(B(i))) and Y-junction (figure 1(B(ii))).When the polymer solution encounters a miscible antisolvent in the mixing unit, changes in solute concentration can trigger the supersaturation, leading to high Gibbs free energy of the system.When it reaches a critical nucleation concentration, molecular and atomic clusters in the polymer solution form polymer nuclei.Subsequently, the precipitated nuclei begin to grow with the accumulation of polymer molecules [24].
However, it should also be noted that microfluidic nanoprecipitation is limited by low production rates due to the slow molecular diffusion and is more likely to lead to channel blockage at a high flow rate.As mixing efficiency determines the yield of nanoparticles, spiral mixers (figure 1(B(iii))) and Tesla mixers (figure 1(B(iv))) that can generate vortexes in the microchannel are introduced to improve phase mixing [25].Unlike the laminar flow characteristics of fluids in conventional microchannels, vortex mixers induce turbulent mixing downstream the channel at a high flow rate (Reynolds number is very high).In addition, external stimulation modules have also been incorporated into microfluidic mixers in recent years to achieve faster mixing and high-throughput nanoparticle synthesis, such as acoustofluidics, magnetofluidics and electrofluidics [26,27].

Microfluidic fiber
Functional fibers are attractive biomaterials in drug delivery and tissue regeneration due to their tunable mechanical property, expanded specific surface area and high plasticity of morphology [28].A broad range of materials, including natural and synthetic polymers have been successfully processed into micro/nano fibers.The common spinning approaches include electrospinning, melt spinning and microfluidic spinning [29].
Compared with other spinning approaches, microfluidics allows for producing fibers under a comparable mild condition.For example, electrospinning has been widely used in fabricating polymer fibers, however, polymers dissolved in volatile organic solvents are formed into fibers under a high voltage and shear [30].The harsh working environment restricts the sensitive drug molecules and cells from being loaded onto electrospinning fiber.Moreover, as the electrospinning fibers are deposited and collected on a rolling electrode, they are typically anisotropic.Melt spinning requires a high temperature to fuse polymer solids and also does not allow for the encapsulation of proteins and cells during the preparation process.Therefore, microfluidic fibers have emerged as a promising candidate in producing biomaterial fibers in recent years because they maximize the retention of drug activity while allowing simultaneous loading of cells even during fiber formation.
In addition to droplet generation, flow-focusing (figure 1(C(i))) and co-flow (figure 1(C(ii))) geometry are also widely used for the production of microfluidic fibers.In the fabrication of microfluidic fibers, polymer precursors are injected into the inner channel and form a core flow under extrusion of immiscible sheath phases.The core flow exhibits non-Newtonian fluid behavior and its viscosity depends on the shear rate [31].Due to the low Reynolds number in the microchannel, both fluids remain laminar and the mixing between the two phases is limited to a slow diffusion at the interface [32].The jets are then solidified via different deposition or crosslinking methods (such as physical deposition, chemical crosslinking and ionic crosslinking) to preserve the designed shapes [33].Moreover, the fiber diameter can be adjusted by varying the channel diameter and flow rates.Another type of microfluidic fiber generator is a single-phase system, which possesses a progressively narrowing channel that mimics the natural silk gland (figure 1(C(iii))).This microfluidic geometry is mainly used for biomaterials based on protein assembly.When the protein solution flows through the contracted chamber, the hydrodynamic shear along the microfluidic channel induces the aggregation of protein fibrils.When the difference in flow velocity between the inner and outer phases rises to a threshold, the coiling instability is triggered and microfibers with a helical structure are generated (figure 1(C(iv))).Moreover, by varying the flow rate of the fluid, the amplitude and helical pitch of microfibers can be flexibly adapted to the tissue properties [34].Beyond producing conventional cylindrical fibers, microfluidic techniques allow the fabrication of microfibers with complex architected structures by modifying microfluidic devices, such as core/shell and Janus.
On the other hand, the formation of microfluidic fibers depends on rapid cross-linking or solidification downstream of the microchannel and have to be ejected quickly.This requires strict control of manufacturing conditions because premature aggradation will lead to flow disruption and even channel blockage.Also, the low productivity of microfluidic fibers restricts its application in industrial fields.

Polysaccharides for microfluidic technology
Polysaccharides, a class of naturally derived hydrophilic polymers, consists of monosaccharide units that are linked by glycosidic bonds.Polysaccharides are widely available, and can be easily obtained from plants, animals, bacteria, and other sources.The excellent biocompatibility, biodegradability and immunomodulatory ability of polysaccharides make them promising agents for preparing biomaterial carriers [35].The excellent water solubility of polysaccharides makes them candidates for microfluidics.The most commonly used polysaccharide materials for biomedical applications include hyaluronic acid, alginate and chitosan.Moreover, diverse modifications have been made to polysaccharides to improve their properties.This section will introduce microfluidic biomaterials based on polysaccharides and their derivatives, as well as their applications in delivering drugs or cells.Table 1 shows in detail the differences in the form and the contents of the various polysaccharide carriers.

Hyaluronic acid (HA)
HA is one of the most popular derivatives of polysaccharides in the medical field and is widely distributed in human tissues, such as synovial fluid and vitreous of the eye, as well as in the extracellular matrix (ECM).HA is comprised of glucuronic acid and N-acetylglucosamine, connected repeatedly by β-1-3 and β-1-4 glycosidic bonds.The molecular weight (M w ) of HA ranges between 120 to 2500 kDa, with HA of high M w (greater than 1000 kDa) [36] exhibiting the capability of inhibiting the production of pro-inflammatory mediators, making it a promising agent for treating inflammatory diseases, such as osteoarthritis (OA) [37] and intervertebral disk degeneration (IDD) [38].HA is highly soluble in water and can encapsulate hydrophilic cargoes by forming hydrogels through molecular interactions.Owing to its properties of natural materials, there has been a long history of using HA for tissue engineering and drug delivery.More importantly, since HA is negatively charged under a neutral-pH environment, oppositely-charged drugs and proteins can easily bind to HA via electrostatic interactions [39].However, the physical crosslinking of HA hydrogel is rather weak and invertible, which makes the use of unmodified HA for microfluidic manufacturing challenging.HA also faces the drawbacks of low mechanical strength, which may lead to collapse of the HA scaffold after implantation and even to premature release of the encapsulated cargo.In addition, the rapid degradation of HA is unexpected since it cannot sustain the long-term release of the loaded drug.
To overcome above-mentioned shortcomings, various improvement strategies have been proposed to enhance the performance of HA hydrogels for use in microfluidics.Due to its abundant side groups, such as carboxyl, hydroxyl, and acetamido, HA provides various sites for chemical reactions and modifications [40].For instance, Li et al [41] synthesized functionalized HA by grafting dopamine (DA) onto HA via amidation reaction between the carboxyl group of HA and the amine group of DA, and then fabricated microfluidic HA-DA droplets in W/O emulsions.The hydrogel droplets were then mixed with sodium periodate so that they could be crosslinked via oxidation of the catechol groups.Hyaluronic acid methacrylate (HAMA) is one kind of photo-crosslinking hydrogel, prepared by grafting methacrylate onto the HA through esterification reaction [42].The properties of HAMA, including stiffness and degradation, also can be modulated by altering the degree of methacrylation.In a previous study, our group took advantage of the negative charge of HAMA microspheres to bind positively charged liposomes to them, and modified PDA on the surface of HAMA to construct adhesion microspheres (figure 2(A)) [43].Microsphere/liposome compositive system could penetrate the dense cartilage matrix to deliver drugs for OA treatment.Kim et al [44] encapsulated 131 I, a β-and γ-emitting radioisotope, into the HAMA microfluidic microspheres to target tissue for radiotherapy. 131I-labeled HAMA was prepared by introducing N-(3-Aminopropyl)imidazole (API) groups into HAMA polymeric chains via amidation reaction, and iodinating API moiety in HAMA via chloramine T-catalyzed electrophilic aromatic substitution reaction. 131I-labled HAMA aqueous solution and oil phase were injected into a centrifugal microfluidic device to generate hydrogel droplets, which were subsequently crosslinked under UV radiation.HAMA microsphere carriers were demonstrated to prolong the local retention of radioisotopes and reduce the distribution of radioisotopes in non-targeted organs, and therefore had the potential for targeted cancer radiotherapy.In another study, Busatto et al [45] used microfluidic technique to construct an oil-in-microgel container that allowed hydrophobic drugs to be encapsulated in HAMA microspheres.Oil-in-water nano-emulsions were used as the disperse phase, prepared by mixing the drug-laden oil droplets with HAMA precursors.HAMA microspheres encapsulating oil nanodroplets were obtained from the O/W/O emulsion in a conventional co-flow microfluidic device.Progesterone, chosen as a hydrophobic model drug, was encapsulated in the oil droplets and exhibited long-term retention and uniform distribution within the HAMA hydrogel network.In addition to HAMA, ethylsulfated hyaluronic acid (VS-HA) is another photo-crosslinked hydrogel that has been applied in the manufacture of microfluidic microspheres encapsulating neural stem cells (NSCs) as delivery vehicles for immunomodulation [46].Mendes et al [47] developed modified HA microspheres that mimicked the hierarchical organization of natural ECM and were crosslinked via thiol-norbornene click chemistry.
A microfluidic device with dual input channels was used, in which HA modified with norbornenes (NorHA) and dithiothreitol crosslinker were mixed together as the first inner phase, while platelet lysate (PL) solution was used as the other internal phase.Thrombin was added into NorHA solution to induce fibrinogen polymerization within the PL.The two internal phases were injected into a Y-shaped channel and remained in laminar flow until they were squeezed into droplets by the oil phase at the junction, thus avoiding blockage of the channel.The coagulation cascade was triggered during the mixing of the two internal phases in the downstream channel after droplet generation, leading to the selfassembly of fibrin fibers, while the fiber structure was stabilized after the photo-crosslinking of NorHA microspheres.
The hierarchical fibrin structure of microspheres offered numerous cellar anchorage sites that facilitated cell adhesion and proliferation, and therefore were efficient cell carriers.
In other cases, HA is prepared as microfibers for drug and cell delivery.It should be noted that conventional electrospinning methods are not allowed to be applied to HA due to the high viscosity and surface tension of the HA solution [48].Therefore, more and more attention is now being focused on microfluidic HA fibers.HAMA fibers encapsulating human tendon-derived cells (hTDCs) have been developed using microfluidics and the ability of tendon cells to deposit ECM is preserved, indicating the potential of HAMA microfluidic fibers for designing artificial fibrous structures [49].Giammona et al [50] synthesized a derivative of HA (HA-EDA-C 18 ) that could be physically crosslinked via selfassembling in saline solutions by introducing octadecylamine (C 18 -NH 2 ) and ethylenediamine (EDA) to the HA backbone.More importantly, the hydrophobic modification of HA achieved high loading efficiency of hydrophobic drug molecules.HA fibers were generated when polymer solution was extruded from the chip and collected in a PBS bath.Metal ions in PBS solution induced the crosslinking and solidification of HA-EDA-C 18 fibers.In addition, HA solution was mixed with fibronectin and received cyclic RGD functionalization for cell vehicle preparation.The composite HA fibers exhibited improved cell adhesion and oriented cell distribution [51].

Alginate
Alginate is a naturally derived anionic polysaccharide that is easily available by the extraction of natural brown algae and bacterial synthesis.As an FDA-approved high molecular compound, alginate possesses a long history of use in biomedical applications for its biocompatibility and biodegradability.In particular, the carboxyl group of alginate chains can be crosslinked with multivalent cations to form hydrogel networks.Among these cations, C a is the most popular cation used for crosslinking alginate hydrogels because it is nontoxic and alginate exhibits a moderate affinity to C a caution [52].Considering its water-solubility, alginate can be easily prepared into various scaffolds for loading hydrophilic drugs and cells.Via microfluidic technology, advanced alginate-based micro/nanocarriers have been developed for delivering cargoes.
Droplet microfluidics allows the production of alginate hydrogel microspheres from a W/O emulsion template.Typically, alginate droplets are generated under the shearing force of the oil phase, and then collected in a C a 2+ -containing bath to complete the crosslinking.For instance, Liu et al [53] fabricated microfluidic Na-alginate droplets in a W/O emulsion and crosslinked them in C a Cl 2 solutions.However, oil-containing emulsion templates have intrinsic drawbacks.As oil is chosen as the continuous phase, an additional washing process is necessary to remove the oil adhering to the surface of the alginate hydrogel microspheres.The organic solvents are always used in the washing process, and may lead to leakage of the encapsulated drugs.An oil-free method was proposed by Zhang et al [54] using dimethyl carbonate (DMC) as the continuous phase.DMC could not only ensure the construction of a stable two-phase immiscible system, but reduce the risk of channel blockage.Moreover, as water was slightly soluble in DMC, alginate droplets generated in the DMC fluid would have shrinkage because of the water extraction by DMC.The droplets were then collected in a DMC-containing bath and crosslinked in C a Cl 2 solution, obtaining alginate microspheres with sizes smaller than 50 µm.However, when the alginate droplets are solidified outside the microfluidic device, the gelatin always occurs on the outer surface of the alginate droplets, leading to inhomogeneous crosslinking of the hydrogel microspheres.Another strategy for crosslinking alginate droplets is internal gelatin.Typically, divalent cation salts that are poorly solvable in water are mixed in the alginate solution, and will release divalent cations under external stimulus, inducing the crosslinking of alginate droplets.For example, An et al [55] used a one-step microfluidic strategy to fabricate MSCs-laden alginate microspheres.To prepare the disperse phase, MSC suspension and C a -EDTA were added to the alginate solution.Acetic acid was mixed into the continuous oil phase to create a low-PH environment, triggering the release of divalent cation from C a -EDTA complex.The alginate droplets were generated at a flow focusing junction, and subsequently solidified via ionic crosslinking with acetic acid diffusing into the droplets.The MSCs encapsulated in the alginate microspheres exhibited good viability and proliferation.Moreover, alginate microspheres provided a microenvironment with a desirable stiffness to support the osteogenesis differentiation of the encapsulated MSCs.A W/W system was built to avoid possible damage to the encapsulated cells during the oil washing process [56].Dextran and polyethylene glycol (PEG) solutions were chosen as the disperse and continuous phase, because they became incompatible when the concentrations were above a critical value.To prepare mild cell-laden microspheres, alginate and pancreatic islet (β-TC6) cells suspensions were added into the dextran phase to form droplets, which were then crosslinked in C a Cl 2 bath.
In addition to microfluidic alginate microspheres, in recent years, microfluidic alginate fibers have also aroused much attention.Due to the high surface tension of alginate solutions, traditional methods for fiber fabrication (i.e.electrospinning) are unable to produce pure alginate fibers [57].Therefore, the development of microfluidic technology has created the possibility of manufacturing pure alginate fibers and achieved certain progress.For example, alginate microfibers were fabricated in one study using a flow-focusing device [58].C a Cl 2 solution acting as a shearing phase squeezed the alginate fluid to form a thin jet, and with the diffusion of calcium ions, alginate jets transformed into fibers.Owing to the mild gel conditions, alginate fibers can be easily prepared as cell carriers.Liu et al [59] used T-junction geometry to fabricate MSCladen alginate fibers.Na-alginate solution was used as the core phase, extruded by the sheath phase made up of C a Cl 2 solution.Once these two fluids merged at the junction, gelation occurred with the diffusion of C a 2+ in the alginate fluids.C a Cl 2 solution could also serve a lubricant to avoid the clogging caused by the rapid crosslinking of alginate fibers.Furthermore, MSCs were suspended in the alginate solution to manufacture cellencapsulated scaffolds and exhibited great proliferation and viability.Chaurasia et al [60] further developed alginate fibers with a more complex structure using a coaxial capillary microfluidics.The inner capillary was filled with the oil phase while the alginate solution was injected into the outer capillary.Under the shearing force of the alginate liquid, oil droplets were formed and encapsulated in alginate fibers, which were collected and solidified in a C a Cl 2 bath (figure 2(B)).The low interfacial tension at oil/water interface ensured stable encapsulation of the oil droplets.
However, ionically crosslinked alginate carriers are difficult to maintain long-term stabilization under physiological conditions since divalent ions in alginate can be exchanged by other ions in the surrounding media, resulting in dissolution of the alginate carrier [61].Photoresponsive alginate materials based on covalent crosslinking have emerged as a new choice for alginate carriers owing to their enhanced mechanical property and structural stabilization.Methacrylated alginate (AlgMA) is synthesized by introducing MA groups into the alginate main chains and can form a stable crosslinking network under UV irradiation while maintaining excellent biocompatibility.Oxidized methacrylated alginate microgels generated in conventional O/W emulsions were used as cell carriers for chondrocyte delivery, presenting higher chondrocyte encapsulation survival rates compared to ionic crosslinking methods [62].Through the partial substitution of alginate hydroxyl groups by MA, AlgMA retains ionic crosslinking properties.In the fabrication of AlgMA microfibers, C a Cl 2 solution is still used as the shear phase to extrude and solidify the AlgMA jets.The ionically crosslinked fibers are subsequently exposed to UV light to form dual crosslinked hydrogel fibers.In one study, AlgMA microfluidic fibers were used to encapsulate human umbilical vein endothelial cells (HUVECs) and exhibited high cell viability and proliferation [63].

Chitosan (CS)
CS, a linear polysaccharide obtained from the shells of crustaceans, is the second most abundant naturally derived polymer.CS is composed of glucosamine and N-acetylglucosamine linked in a β (1-4) manner.Unlike other negatively charged polysaccharides, CS is an alkaline polysaccharide with positive charge [64].CS is a biocompatible, and antibacterial polymer that can be biodegraded in vivo by several enzymes [65,66].CS could self-assemble with anionic molecules to form nanoscale complexes or hydrogels, and thus has a wide range of applications in delivering negatively charged nucleic acids, such as siRNA and miRNA [67].Owing to its hydrophilic property, CS can be easily processed into microfluidic carriers in W/O or O/W systems.
However, the insolubility of CS in neutral pH environment restricts its applications as biomaterial carriers.Therefore, in most cases, CS needs to be dissolved in an acidic aqueous solution for microfluidics.Tripolyphosphate (TPP) is a polyanion that is widely used to prepare CS biomaterials, including CS nanoparticles and microspheres, because its small anionic molecules can physically crosslink CS by electrostatic interaction [68].The use of microfluidic mixing devices allows for further optimization of the particle preparation process.Moradikhah et al [69] produced Alendronate (ALN) loaded CS nanoparticle by a flow-focusing device.ALN dissolved in CS solution was prepared as a core flow, sheared by lateral flows composed of TPP.The size distribution of CS particles could be adjusted by changing the flow ratio of the lateral flow to the core flow.On the other hand, due to the immediate electrostatic interaction between the two streams when they met at the junction, some of the formed CS particles might stagnate and aggregate throughout the microchannel, leading to channel blockage.A modified approach, also based on flowfocusing geometry, used acidified water as the core stream while the CS and ATT solutions were injected into the side channel [70].The central acidified water stream delayed the molecular contact between CS and ATT, because the crosslinking occurred as the CS and ATT molecules gradually diffused into the core water stream.Moreover, TPP crosslinked CS nanoparticles have also shown the ability to encapsulate hydrophobic drugs in microfluidic mixing units [71].
Alternatively, CS carriers can be produced by polyelectrolyte associations with other polyanionic polysaccharides, such as HA, alginate and sodium carboxymethyl cellulose (CMC) [72,73].For example, composite hydrogel fibers were fabricated in Y-shaped microchannels by mixing CS with HA solutions [47].Fibril microstructures generated within the CS fiber matrix were found to further improve the ECM deposition of encapsulated tendon cells.Wang et al [74] used gasshearing microfluidics to fabricate multilayer CS microcapsules via layer-by-layer assembly (figure 3(A)).The CMC solution generated droplets under gas shear, which formed single-layer capsules when collected in the CS solution.Subsequently, the CS solution containing the single-layer capsules could be injected again into the microfluidic device and solidified in the CMC solution, forming bilayer capsules that exhibited the ability to encapsulate different drugs within separate layer cavities.
However, CS biomaterial carriers formed by polyelectrolyte complexation always possess poor hydrolytic stabilization.Chemically crosslinking strategies have also been applied to microfluidic CS carriers.For example, He et al [75] dissolved CS in acetic acid solution to prepare the disperse phase, and fabricated CS droplets in an O/W emulsion.These droplets were then crosslinked in a collecting bath containing glutaraldehyde (GTA).The use of GTA was intended to produce a chemical crosslinking network within the CS microspheres, in which the aldehydic of GTA formed Schiff base linkages with the amino groups of chitosan.5-Fluorouracil (5-Fu), a hydrophilic anticancer drug, was loaded on CS microspheres and achieved high encapsulation efficiency.More importantly, the loaded drug presented a responsive release behavior to the tumor microenvironment due to the pH sensitivity of SF microspheres.In addition to the application in hydrophilic drug delivery, CS microfluidic microcapsules are another robust vehicle for loading hydrophobic drug molecules.Mu et al [76] used co-flow capillary devices to produce O/W emulsion template, where hydrophobic drug dissolved in the oil phase acted as the disperse phase and was sheared by the CS aqueous solution to form drug-laden microcapsules.Terephthalaldehyde was chosen as the chemical crosslinker to form a stable capsule membrane outside the oil core.

Others
Dextran is a natural biological macromolecule consisting of α-1, 6 glycosidic linkages between glucose monomers and is readily available from the natural fermentation process [77].Dextran and its derivatives have been widely used in preparing nanoparticle, microgel and micelle carrier for precise drug delivery [78].In view of the immiscibility of dextran and PEG solution, dextran biocarriers can be easily produced using microfluidics based on phase immiscibility [79].Furthermore, various dextran derivatives have also been used in microfluidic fabrication.Kamperman et al [80] synthesized dextrantyramine (Dex-TA) to fabricate enzymatically crosslinked dextran hydrogel microspheres in a PDMS device.A mixture of DEX-TA and horseradish peroxidase (HRP) enzyme was used as the precursor solution, generating droplets in an O/W emulsion.Additionally, MSCs were suspended in the dextran solution to produce single-cell encapsulated droplets.In particular, two aligning channels containing H 2 O 2 solution were assembled adjacent to the outlet channel to trigger enzymatic crosslinking.Using the perm-selectivity of PDMS toward H 2 O 2 , dextran droplets underwent a delayed enzymatic crosslinking via tyramine-tyramine bonds and achieved the localization of single cells in the center of the microsphere, thus preventing cell escape and enabling efficient cell delivery (figure 3(B)).Liu et al [81] modified vicinal diols of dextran with 2-methoxypropene to obtain acetalated dextran (AcDX), a water-insoluble dextran derivative.An O/W emulsion template was built in capillary devices to produce AcDX microspheres.Moreover, AcDX microspheres exhibited a strong adsorption capability for glutamate and calcium ions in cerebrospinal fluid, thereby reducing glutamate-induced excitotoxicity and protecting the traumatic spinal cord.
Heparin is a polysulfated glycosaminoglycan and has a long history of use in the preparation of hydrogel materials [82].The greatest advantage of heparin is its high affinity for growth factors, so much attention has been paid to designing heparin carriers to deliver growth factors.In a previous study, our group synthesized methacrylate heparin (HepMA) for loading platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta3 (TGF-β3), and further covalently conjugated HepMA onto HAMA to prepare stem cellrecruiting hydrogel microspheres for the treatment of OA [83].Husman et al [84] developed cell-laden functionalized heparin microspheres from a W/O emulsion.To prepare the disperse water phase, heparin functionalized with six maleimide groups was mixed with the peptide-functionalized four-armed PEG.The droplets were crosslinked via Michael-type addition reaction between heparin and PEG and gelation time could be adjusted within 3 minutes by changing the pH of the four-armed PEG solution.Also, the mild crosslinking conditions of heparin microsphere made it suitable for loading cells.The potential of heparin microspheres on MSC differentiation has also been studied [85].A mixture of eight-arm PEG-thiol and HepMA was prepared as the water phase, and poly(ethylene glycol) diacrylate (PEGDA) was added as a crosslinker to solidify heparin microspheres via Michael addition.Embryonic stem cells (ESCs) were encapsulated in heparin microspheres and differentiated into definitive endoderm with the addition of growth factors Nodal, Activin A and fibroblast growth factor-2.
Recently, a large number of studies have been conducted to extract polysaccharides from other natural substances for microfluidic production.Pectins extracted from plants are another polysaccharide with promising clinical applications and, like alginate, they can be ionically crosslinked by divalent metal ions to prepare hydrogels.Using a flow-focusing microfluidic device, pectin hydrogel microspheres and microfibers were fabricated in a W/O emulsion [86].Konjac glucomannan (KGM) is a liner macromolecular polysaccharide from the corm of amorphophallus konjac.When KGM is dissolved in water, it can be physically crosslinked by hydrogen bonding to form a hydrogel [87].Mu et al [88] demonstrated the feasibility of using plant polysaccharides to prepare microfluidic fibers by developing KGM microfibers loaded with ofloxacin.Akhter et al [89] isolated ginseng polysaccharide from North American ginseng root and prepared it into nanoparticles using T-junction microfluidic devices.The aqueous solution of ginseng polysaccharide was sheared by the acetone in the junction.Acetone was used as an antisolvent to control the nanoprecipitation process in the microfluidic device, so that ginseng polysaccharide nanoparticles with a narrow size distribution could be obtained.More importantly, microfluidic ginseng polysaccharide nanoparticles were more effective in immune stimulation than those prepared by conventional nanoprecipitation and microemulsion methods.Zykwinska et al [90] synthesized marine exopolysaccharide (EPS) GY785 from the deep-sea hydrothermal vent bacterium for encapsulation of TGF-β1.EPS was a slightly sulfated anionic heteropolysaccharide that could also be physically gelated by divalent cations to generate drug-loading microspheres for cartilage regeneration.

Proteins for microfluidic technology
Proteins are naturally derived functional macromolecules, regulating various biological processes and pathways [91].Protein-based biomaterials have aroused much attention due to their low cost, sustainability and biodegradability.Using the self-assembly properties of proteins, proteins can be engineered to generate microfluidic functional complexes via nanofibril formation within protein fluid [92].In this section, protein materials for microfluidics including silk fibroin (SF), collagen, gelatin and their derivatives are introduced, and their representative applications as efficient biomedical vehicles are listed in table 2.

SF
SF is a naturally derived protein polymer obtained mainly from silkworm cocoons.SF has been used in biomedical field for many decades due to its good biological compatibility and proteolytic degradability.Unlike other natural polymers with poor mechanical property, SF exhibits a highly aligned structure, which results in a significantly enhanced mechanical strength.As one kind of material with high solubility in aqueous salt solutions [93], SF has been tailored to diverse biocarriers, such as film, scaffold, electrospun fiber and hydrogel [94].In addition, owing to its amphiphilic property, the hydrophilic and hydrophobic chain segments of SF allow the formation of micelles via the self-assembly in an aqueous environment.Thus, hydrophobic drugs can be incorporated into the hydrophobic core of the micelles during the assembly process.
A typical method for preparing SF nanospheres is to mix SF aqueous solution with organic solvent and form inhomogeneous SF spheres through self-assembly under stirring.Via precise control of microfluid, microfluidics fabricates SF nanocarrier with controlled characteristics via micromixing.Wongpinyochit et al [95] injected SF solution and organic solvent into two separate microfluidic channels, and then these two phases met and mixed at the junction, inducing the selfassembly of SF spheres.Microfluidic mixing was proved to not only allow the sphere size to be tuned by varying the flow Gelatin [116] Microsphere Physical crosslinking --Gelatin [117] Microsphere Enzymatic crosslinking --Gel-SH [119] Microsphere Chemical crosslinking BMSCs Cell BP-grafted GelMA [121] Microsphere Photo-crosslinking -- GelMA [122] Microsphere Photo-crosslinking DS Hydrophobic drug GelMA [128] Microsphere Photo-crosslinking BMSCs Cell rate ratio, but also to ensure a high degree of homogeneity of the obtained SF nanospheres with a polydispersity range of 0.1-0.25.Another microfluidic method for SF biomaterials is based on manipulating two immiscible liquid phases.Classical droplet microfluidics is a robust tool to obtain SF microgels.Briefly speaking, the SF solution is used as the disperse phase, squeezed by the oil phase to generate droplets.The shear forces in the microfluidic channel can induce protein aggregation, resulting in the solidification of SF droplets [96].Drug-laden SF microspheres are constructed by dissolving drug molecules in the SF solution and have been demonstrated to stabilize the encapsulated cargo [97].Liu et al [98] conducted an in-depth study of the drug-carrying capacity of SF microspheres by adjusting the composition of the water phase, where the disperse phase was made up of the SF dissolved in ethanol.After incubation, the SF droplets self-assembled and turned into microgels.Furthermore, the release behavior of the laden drugs was controlled by the amount of SF or ethanol in the polymer solution.In another study, W/O/W and O/W/O double emulsion templates were made to generate core/shell SF microspheres, where SF could act not only as the inner core, but as the outer shell [99].In addition to using oil as the continuous phase, PVA solution can also be chosen as the shear phase [100].After the generated SF droplets flowed through the downstream channel, the interaction between SF and PVA induced the silk self-assembly, forming cured SF microsphere.
However, the high viscosity of DF solution allows only very limited drug loading.To overcome this difficulty, Zhao et al [101] established a mesoporous silica composite SF microfluidic microsphere to carry dimethyloxaloylglycine, one kind of small-molecule drug motivating neovascularization for vascularization and abdominal wall defects.Composite SF microspheres were produced by dispersing hollow mesoporous silica nanocarriers (HMSNs) in SF polymer solutions followed by the formation of homogeneous component droplets in microfluidic channels.After emulsion drying, solidified SF microspheres were obtained.HMSNs allowed the encapsulation of both hydrophilic and hydrophobic drug molecule via physical absorption, and its further coupling with the SF microsphere scaffold could achieve controlled release of the laden drug.
SF fibers have been widely used in biomedical applications for their mimicry of the natural ECM and anisotropic structure.The conventional strategy for manufacturing SF fibers requires dissolving SF in volatile solvents and then evaporating or freezing the solution to harvest SF fibers.However, it is challenging to obtain oriented and single fiber using classical methods.The advantage of microfluidics is its ability to fabricate fibers with aligned microstructures.Microfluidic technique for SF fibers mainly includes two categories: (a) microfluidic dry spinning.(b) Microfluidic wet spinning [102].For dry spinning strategy, nanofiber fragments in SF solution are transformed into solid with the water evaporation, thus forming SF fibers.Typically, the microfluidic device for dry spinning possesses channels with a contracting geometry that mimicks the natural silk glands [103].For example, regenerated silk fibroin (RSF) fibers with aligned hierarchical structures have been fabricated [104].Silk nanofibers had self-assembly in an aqueous solution and formed nanofiber hydrogels, which were lyophilized and dissolved in formic acid for preparing SF solution.When SF solution was injected in the microchannel and flowed through the shrinkage area, SF nanofibers had prealignment and solidified in ethanol-containing bath.The microfluidic fibers exhibited enhanced mechanical property due to the aligned structure while the hierarchical feature stimulated cell spreading and migration.In another study, cellulose nanofibers were mixed with RSF solutions to manufacture microfluidic fibers [105].Due to the high orientation of RSF molecules and cellulose nanofibers in the microchannel as well as the hydrogen bonds, the composite fibers showed significant increase in mechanical strength.A more advanced biomimetic strategy that mimics the metal ion transport in natural arthropod silk spinning has been proposed using a flow-focusing device [106].RSF solution mixed with C a Cl 2 was injected into the inner channel while KCl and HCl were added to the shear phase containing polyethylene oxide to construct ion gradients in dry-spinning RSF fibers.The extruded RSF fibers were then submerged in methanol baths for dehydration and solidification.The ion gradient promoted β-sheet formation from hydrophobic sequence in RSF, which significantly improved the mechanical strength of the fibers.
Microfluidic wet spinning is typically based on phase immiscibility, using an immiscible shear phase to squeeze the SF solution and generate pre-polymer jet.For the solidification of SF fibers, additional coagulants or crosslink agents are required.Luken et al [58] performed wet spinning of SF fibers using flow-focusing microfluidic chips (figure 4(A)).The silk solution was prepared by immersing SF into hexafluoroisopropanol, while ethanol in PEG was used as the shear phase.SF solutions were injected into the top and bottom channels, and squeezed by PEG fluid at the flow-focusing junction, forming SF fibers after protein precipitation.Moreover, both the diameter and mechanical properties of the microfibers could be freely adjusted by modifying the fluid parameters.When co-cultured with cells, SF microfluidic fiber exhibited excellent cell adhesion property and was a promising cell carrier.

Collagen
Collagen is the main component of ECM proteins in the body.Owing to its low antigenicity and inflammatory response, collagen is considered to be an ideal biomaterial [107].Furthermore, Collagen-based biomaterials exhibit the ability to improve cell adhesion and proliferation and therefore have been widely used for tissue regeneration.Among all types of collagens, collagen type I has attracted the most attention and interest in the biomedical field because it is easily available from animal tissues, such as skin and tendons, and can be prepared into a variety of biocarriers, such as hydrogels and 3D printed scaffolds [108].Collagen is soluble in water and can self-assemble to form fibrous hydrogels via noncovalent interactions, including hydrogen bonding and electrostatic or hydrophobic interactions [109].A classical method of preparing collagen hydrogels is to neutralize an acidic collagen solution and incubate it at 37 • C, inducing fibrillogenic to self-assemble into hydrogels [110].This allows for producing collagen microspheres using droplet microfluidic devices.In addition, the gentle thermal gelation maximizes the retention of cellular activity, making collagen an ideal r carrier for cell capsulation.For example, BMSC-laden collagen microspheres have been developed in a W/O emulsion [111].Hong et al [112] constructed microtissue spheroids with hepatic-lobule structures on collagen microfluidic microspheres to develop an in vitro 3D cellular model that could mimic the human liver (figure 4(B)).The microspheres were designed to have multiple independent compartments using a precursor cartridge.HepG2/C3A and EA.hy926, HUVEC lines, were encapsulated in separate compartments for generating the parenchymal and vascular fractions of the microtissue spheroid.Collagen microspheres exhibited a hepatic-lobulelike structure upon incubation and formed more functional vessels in vivo.
Collagen possesses a natural hierarchical structure, making collagen hydrogel fibers the most popular type of biocarrier for collagen.More importantly, collagen microfibers fabricated using microfluidics allow for the aligned orientation of collagen fibrils within the fibers, which is particularly suitable for designing nerve conduits or scaffolds.Wu et al [113] reported an electroconductive hydrogel fiber that could promote the neuronal functional expression and stimulate neurogenesis.Electroconductive polypyrrole nanoparticles were mixed into the acetic collagen solution for preparing the inner phase, which was squeezed by the PEG-containing shear phase.It was also found collagen microfluidic fibers with oriented collagen fibrils supported the aligned elongation and neurogenesis of encapsulated PC12 cells.
In general, the pure collagen fibers that are physically crosslinked have insufficient mechanical strength, thereby various modification strategies and crosslinking methods have been proposed to overcome this drawback.Dasgupta et al [114] systematically investigated the mechanical properties and biocompatibility of microfluidic collagen microfibers crosslinked by different methods.Fibers were obtained using coaxial needle microfluidics in which the acidic collagen solution was squeezed as an internal phase by an external phase consisting of a neutralizing alkaline solution.For producing chemically crosslinked fibers, the extruded fibers were further submerged into a solution of crosslinkers.It was found Glyoxal-crosslinked collagen fibers presented the highest mechanical strength and excellent biological compatibility, suggesting them as a potential alternative to surgical sutures and tissue-engineered implants.

Gelation
Gelatin is the product after partial hydrolysis of collagen and is considered a substitute for collagen because it is highly soluble at physiological pH, overcoming the drawback of the acidic-soluble nature of collagen [115].As one class of natural material, gelatin also possesses excellent biocompatibility and degradability, and has been widely used in biomedical applications.Gelatin hydrogels are among the most popular thermosensitive hydrogels and can therefore be used to deliver drugs and cells by injectable administration.Moreover, gelatin exhibits advantages over other naturally derived materials due to its arginine-glycine-aspartic acid (RGD) peptide sequence, which provides favorable cell adhesion sites.Gelatin can be used not only as drug or cell carriers alone, but also in couple with other materials to enhance its cell adhesion capacity.Therefore, more and more studies are using gelatin as a delivery vehicle to load both cells and drugs.
As one of the most popular thermosensitive hydrogels, gelatin assumes a solvated state when heated, and will gradually cure as the temperature decreases.In most cases, to produce microfluidic gelatin microspheres, a heating plate is placed at the inlet to liquefy the gelatin for injection into the channel, and then a cooling module is installed at the outlet to solidify the gelatin droplets [116].Due to the weak physical crosslinking as well as the reversible phase transition of gelatin, the stabilization of gelatin microspheres is poor, limiting its application in cargo delivery.To overcome this problem, novel crosslinking methods and manufacturing techniques have been proposed for producing gelatin microspheres.Enzymatic crosslinking has been introduced to prepare gelatin microfluidic microgels with higher resistance to temperature [117,118].The microfluidic device possessed two inlets for the disperse phase, injected with gelatin and transglutaminase solutions, respectively.The enzymatic crosslinking proved to maintain the morphology of the gelatin microspheres even at high temperatures because enzyme induced the formation of covalent bonds between the gelatin strands and generated a permanent network.
On the other hand, the amino and carbonyl groups of gelation make it easy to have chemical modification.For example, thiol-modified gelatin (Gel-SH) was synthesized and could be rapidly crosslinked with HA-VS via thiol-Michael addition reaction at room temperature [119].Using Gel-SH and HA-VS as dispersed phases, respectively, Gel-SH microspheres had spontaneous crosslinking once the liquid phases met without additional external stimulation.Moreover, mild reaction conditions made microfluidic Gel-SH microspheres excellent cell carriers.Among the derivatives of gelation, methacrylated gelatin (GelMA) has aroused much attention in recent years.GelMA is one kind of photo-crosslinking hydrogel, synthesized by modifying gelation with methacrylamide (MA), in which the amino groups of gelation are partially substituted by methacrylate groups [120].Compared to unmodified gelatin, GelMA presents strengthened mechanical properties as well as prolonged degradation process.Moreover, the properties of GelMA can be freely adjusted by controlling the degree of substitution of amino groups, radiation duration and polymer concentration, thus it can be processed into various forms of biomaterial carriers, including bulk hydrogels, hydrogel microspheres, hydrogel fibers and 3D printed scaffolds.To fabricate microfluidic GelMA microspheres, GelMA and photoinitiator are dissolved in water together to prepare the disperse phase solutions.Next, the GelMA hydrogel droplets are formed in the microfluidic device and crosslinked under UV radiation.In this process, the primary issue is the concentration of the dispersed phase solution.A GelMA concentration between 5 wt% and 15 wt% is generally considered to be a suitable choice for the preparation of GelMA microspheres, which enables the hydrogel droplets to solidify without causing channel blockage.
Our group has produced GelMA hydrogel microspheres via microfluidic technology for the delivery of a wide range of drugs.Due to the gelatin surface rich in moieties, GelMA microspheres can be loaded with drugs through both physical absorption and chemical grafting.In previous studies, we grafted bisphosphonates (BP) onto GelMA through the Schiff alkali reactivity and coordinate bond to construct functionalized microspheres that could capture magnesium ions, which shown the capability of stimulating cancellous bone regeneration (figure 5) [121].We also produced lubricated microspheres through modifying GelMA microgels for OA treatment [122].Poly (dopaminemethacrylamide-tosulfobetaine methacrylate) (DMA-SBMA) was grafted onto the surface of GelMA microspheres to achieve enhanced lubrication.Furthermore, the presence of the lubricating coating allowed the drug to be loaded through physical absorption.In this study, diclofenac sodium (DS) was successfully carried to the GelMA microsphere and sustained release was achieved for nearly one month.Another mussel inspired superlubricated microsphere was fabricated by dip coating selfadhesive copolymer (DMA-MPC) on GelMA, showing therapeutic effects against OA by strengthening lubrication and alleviating inflammation [11].
GelMA microspheres have also been demonstrated to be effective scaffolds for loading various nanoparticles.Our group constructed composite microsphere systems by combining GelMA microspheres with liposomes [123,124] and fullerol nanocrystals [125].Typically, composite systems were easily established by mixing nanoparticles into the precrosslinking GelMA solution.An alternative approach is to immerse the freeze-dried microspheres in a solution containing nanoparticles, which are adsorbed on the surface of the microspheres.GelMA microspheres exhibited the ability to improve the stabilization of the encapsulated nanoparticles and further extend the drug release kinetics.Moreover, the composite microspheres presented mechanical enhancement because of the formation of non-covalent bonds between the liposome groups and GelMA hydrogel network [126].Yan et al [127] constructed a light-controlled composite therapeutic system to treat osteosarcoma, which was made up of mesoporous silica-coated gold nanorods (AuRN@SiO 2 ) and GelMA microspheres.AuRN@SiO 2 loaded with anti-cancer drugs had a photothermal response and was promising in treating osteosarcoma, however, it cannot be retained in the tumor for a long time after injection.After anchored into GelMA microsphere scaffold, AuRN@SiO 2 achieved long-term stabilization and enhanced drug release.More importantly, composite microsphere system enabled multiple rounds of light responsive therapy even through a single administration.
In addition to delivering drug molecules, GelMA microfluidic microspheres have also emerged as effective tools for loading cells.In a previous study, we fabricated BMSCladen GelMA microspheres by re-suspending stem cells in GelMA solution [128].Using microfluidic technology, homogeneous stem cell-laden hydrogel droplets were formed, which could be rapidly crosslinked within 20 s.Due to the excellent biocompatibility of GelMA, this method ensured the longterm survival of cells encapsulated in microspheres.Furthermore, the improved osteogenesis of BMSCs made cell-laden GelMA microspheres a powerful tool for the treatment of bone defects.Another strategy for loading cells is to immerse freeze-dried crosslinked GelMA microspheres into the cell suspension for physical absorption.Since GelMA provides RGD cell adhesion sites cells are able to adhere to the surface of GelMA microspheres without adding additional growth factors.More importantly, it enables the high variability of the laden cells since the cells are not involved in the photodependent crosslinking process of hydrogel microspheres.We have proved the feasibility of loading BMSC on the surface of porous GelMA microspheres for cancellous bone regeneration [12].The concentration of GelMA determined the pore size of the microsphere and microspheres made of 7 wt% GelMA exhibited the best cell adsorption capacity because they possessed moderate mechanical property and offered suitable space for cell adhesion.Three-dimensional tissue models are potential clinical drug screening tools due to their simulation of the ECM microenvironment.Lee et al [129,130] encapsulated breast adenocarcinoma cells into GelMA microspheres to develop tumor spheroids.The mechanical properties of the microspheres could be controlled by varying the polymer concentration to investigate the effect of the mechanical microenvironment on the formation of tumor spheres.

Synthetic polymers for microfluidic technology
Synthetic polymers always possess a higher mechanical property and design flexibility compared with natural materials.Most synthetic biomaterials have a long degradation cycle, which can be precisely controlled by chemical alteration of the polymer chain.In addition, synthetic materials avoid the risks associated with the immunogenicity of natural materials.However, synthetic polymers lack critical bioactivity and cellinteraction moieties, restricting application in cellular delivery [131].This section describes advances in the fabrication of microfluidic carriers from synthetic polymers such as PEGDA and aliphatic polyesters that include poly(lactic acidco-glycolic acid) (PLGA), PCL, PLA and poly(l-lactic acid) (PLLA).Table 3 lists the applications of synthetic polymerbased carriers for cell and drug delivery.

PEGDA
PEG is a hydrophilic and biocompatible synthetic polymer that has been widely used in biomedical applications.PEGDA is one kind of photo-crosslinking hydrogel, obtained by substituting the hydroxyl group of PEG with acrylates.Compared with other photo-crosslinking hydrogels from modified natural materials, such as GelMA and HAMA, PEGDA hydrogels exhibit a slow degradation rate and are therefore promising biocarriers for long-term drug release [132].
The solubility of PEGDA depends on its M w and PEGDA with high M w always has better water solubility.Using this property, PEGDA microfluidic carriers can be fabricated in a W/O emulsion.In one study, PEGDA microspheres were designed as cell carriers and loaded single cells and cell clusters exhibited a high post-encapsulation viability of over 97% after UV crosslinking [133].In addition, PEGDA fibers can also be fabricated in microfluidic devices.Sharifi et al [134] used PEG solution as the sheath fluid to extrude PEGDA fibers (figure 6(A)).MSCs and adult rat hippocampal stem/progenitor cells were cultured on the surface of PEGDA fibers, and showed good cell adhesion and proliferation, as well as ECM deposition.Based on the laminar flow characteristics of PEGDA and PEG in microfluidic devices, Aykar et al [135] further broadened the function of PEGDA fibers by developing hollow microfluidic microvessels.To form the hollow structure, the PEG solution was injected into the core and sheath flow channels, while PEGDA was used as the shell flow.After UV crosslinking, the PEGDA shell was solidified and the core solution was removed by washing, resulting in fibers with hollow structures.
In addition, take advantage of the insolubility of PEGDA with low M n in certain solvents, PEGDA microcapsules with core reservoirs have emerged as advanced drug carriers.For example, Dinh et al [136] fabricated microfluidic microcapsules with a liquid dextran core and PEGDA hydrogel shell from W/W/O emulsions via phase separation of PEGDA and dextran.Dextran and PEGDA solution were selected as the inner and middle phases to form a stable laminar flow, while the fluorocarbon oil was used as the outer phase to generated droplets.Growth factors consisting of VEGF and PDGF-BB was added to the dextran liquid core and showed slow release from the PEGDA microcapsules.More importantly, the sustained release of growth factors improved long-term blood vessel regeneration in vivo.Nam et al [137] leveraged the insolubility PEGDA 250 (M n = 250 g mol -1 ) in both water and oil to develop PEGDA microcapsules capable of encapsulating both hydrophilic and hydrophobic cargoes.Using a capillary device, water/PEGDA 250/water (W/P/W) and oil/PEGDA 250/water (O/P/W) double emulsions were built.Due to the insoluble nature of PEGDA 250 in water and oil, it was prepared as the middle fluid that could easily form a shell structure to encapsulate the inner phase of water or oil under the shear force exerted by the external aqueous phase.

PLGA
PLGA is a biocompatible copolymer that has been approved by FDA as a biomedical product.Owing to its biodegradability and easy availability via chemical synthesis, PLGA has a long history of use for carrying drugs, cells and DNAs [23].The degradation of PLGA is ascribed to hydrolysis, and enzymes have no obvious influence on PLGA degradation [138].The lactate generated during PLGA degradation stimulates angiogenesis, thus PLGA can be used to improve wound healing, even without the loading of drugs [139].However, it also has to be noted that the acidic byproducts during PLGA degradation are harmful in some specific tissue repair processes.As a hydrophobic synthetic material, PLGA is widely used as a carrier for hydrophobic agents.Moreover, it can be deeply customized according to the property of the laden drug and clinical demands.For example, through changing the molecular weight and the ratio of monomer LA and GA, PLGA with different degradability and drug loading capability is synthesized.The mechanism of preparing PLGA-based biomaterials includes both phase miscibility and immiscibility.
A typical strategy for preparing PLGA nanoparticles is to use a microfluidic mixer [140], where PLGA is dissolved Photo-crosslinking VEGF and PDGF Hydrophilic drug Microcapsule [137] Photo-crosslinking Fibrinogen and mineral oil Hydrophilic and hydrophobic drug PLGA Nanoparticle [141] Physical assembly PFC Hydrophobic drug Microsphere [142] Solvent evaporation Levofloxacin Hydrophobic drug Microsphere [145] Solvent evaporation SDF-1 and kartogenin Hydrophilic and hydrophobic drug PCL Microfiber [147] Solvent extraction --PLA Microsphere [150] Solvent evaporation Ibuprofen Hydrophobic drug PLA Microcapsule [151] Solvent evaporation Yeast cells Cell PLLA Microsphere [153] Solvent evaporation rhsTNFRII Hydrophilic drug in a water-soluble organic solvent and then mixed with an aqueous solution.After nanoprecipitation caused by solvent displacement, PLGA can be assembled into nanoparticles.Microfluidic techniques are increasingly used in the fabrication of PLGA nanoparticles due to their ability to control the structural parameters of nanoparticles.Hoogendijk et al [141] loaded perfluorocarbon (PFC), an insoluble drug that exhibited hydrophobic and lipophobic properties simultaneously, onto PLGA nanoparticles using microfluidic technology.PFC and PLGA were dissolved in the organic solvent, and then mixed with the PVA aqueous solution in the micromixer to generate a preliminary macroemulsion.A sonication flow unit was assembled at the downstream of the microfluidic channel to further reduce the particle size, and the radius of the PLGA nanoparticles exhibited a distribution within 100-200 nm.Moreover, PFC-laden nanoparticles could not only be used for medical imaging, but also for conveying therapeutic oxygen, showing potential for treating hypoxia-related diseases, such as cancer and stroke.On the other hand, in order to prepare PLGA microspheres, droplet microfluidics is in need to construct O/W emulsion templates.Typically, PLGA dissolved in the dichloromethane (DCM) solution is used as the disperse phase while PVA aqueous solution acts as the continuous phase.The formed PLGA droplets are subsequently collected in a PVA bath and solidified.The hydrophobic drug is easily incorporated into the PLGA solution due to the use of organic solutions as the dispersed phase in the microfluidic fabrication process.Based on this approach, diverse PLGA microfluidic carriers can be fabricated.Agnolotti et al [142] proposed lung-targeting PLGA microfluidic microspheres to deliver levofloxacin for the treatment of bacterial lung infection (figure 6(B)).The obtained microspheres exhibited a high monodispersity with an average diameter of 12 µm and coefficient variation (CV) of less than 5.2%, achieving a high selectivity for lung targeting.Lim et al [143] developed MgO-loaded PLGA microspheres to release magnesium ions for bone tissue regeneration.To fabricate ionic composite microspheres, a composite O/W emulsion template was built by mixing MgO nanoparticles into PLGA solution.Due to the sponge-like porous structure of PLGA microspheres, the release of magnesium ions can be precisely controlled by adjusting the microsphere size and porosity.In another study, ultraporous PLGA microspheres were produced for the treatment of pulmonary arterial hypertension [144].Considering that pulmonary delivery required deposition of drug carriers into small airways and alveoli, ammonium bicarbonate (AB) was added to PLGA solution as a porogenic agent to generate ultraporous structures, thus providing PLGA microspheres with long-term retention and lung targeting capability.S-aspirin (ACS14), a synthetic H 2 S donor, was encapsulated in PLGA microspheres for sustained release of H 2 S for pulmonary arterial hypertension therapy.
Several studies have also proposed microfluidic PLGA microspheres with complex structures.Wu et al [145] reported a microfluidic PLGA core/shell microspheres delivering both SDF-1 (stromal cell-derived factor-1) and KGN (Kartogenin).The core/shell structure was built from a double-emulsion template using a capillary device, in which KGN dissolved in DMSO was used as middle oil phase while SDF-1 aqueous solution and PLGA dissolved in DCM were used as core and outer phases, respectively.After evaporation of DCM in the double-emulsion droplets, the core and shell of PLGA microspheres were encapsulated with SDF-1 and KGN, respectively.Co-encapsulation of the two drugs realized coordinated delivery to repair articular cartilage defect, where SDF-1could recruit BSMCs to the microspheres and then, with the release of KGN, BMSCs were guided to differentiate into chondrocytes.

Others
PCL is a semicrystalline polymer, obtained from degraded aliphatic polyester.PCL is an economical synthetic material approved by FDA for biomedical use, and various types of PCL biocarriers have been developed, such as 3D printed scaffolds and electrospinning fibers [2,146].Compared with PLGA, the degradation of PLC does not cause acidification of the microenvironment.PCL is soluble in a wide range of organic solvents, making it suitable for microfluidic control manufacturing.For example, Sharifi et al [147] prepared core stream by dissolving PCL in 2,2,2-trifluoroethanol (TFE), which was extruded by a sheath stream consisting of aqueous PEG solution and cured by solvent extraction to produce PCL microfluidic fibers.Compared with the electrospinning technique, the tensile strain at break of microfluidic PCL fibers significantly increased.
However, the hydrophobic nature of PCL inhibits cell adhesion and proliferation, restricting its application in regeneration medicine.Therefore, additional bioactive components are always incorporated into PCL to enhance its biological characteristics.Cui et al [148] used microfluidic blow-spinning technology to nanofiber PCL scaffold for repairing large-area skin damage.The microfluidic nanofibers possessed a core/shell structure, with PCL/SF as the core segment and fibrinogen as the shell.SF and fibrinogen were introduced to improve the cell attachment and differentiation of PCL fibers.The core/shell jets were formed in a T-junction microfluidic chip and extruded by air flow to generate nanoscale fibers that were subsequently solidified via spraying thrombin on the fibrinogen shell to induce in situ crosslinking.As conventional microfluidics were restricted to the production of micro-scale fibers, microfluidic blow-spinning was able to fabricate nanofibers with a mean diameter of 65 nm, whose expanded specific surface area increased cell attachment, proliferation and migration.
PLA, an aliphatic thermoplastic polyester, is a class of biodegradable polymers derived from renewable resources such as cornstarch and sugarcane, and has therefore attracted a lot of attention in the biomedical field [149].PLA is soluble in various organic solvents for microfluidic manufacture.For instance, PLA dissolved in DCM can be used as the disperse phase to obtain PLA microfluidic microspheres from an O/W emulsion template, which allows for one-step loading of hydrophobic drugs [150].Furthermore, more complex emulsion templates are constructed to produce anisotropic PLA microspheres based on the incompatibility of DCM with water.Ekanem et al [151] used DCM as the middle phase to produce W/O/W emulsions for the fabrication of PLA microcapsules with an aqueous core and a PLA shell.The thickness of the shell was controlled by the flow rate and the water core could be used to load hydrophilic drugs and cells.
PLLA is a semi-crystalline PLA homopolymer that has the slowest degradation rate of all PLA materials [152].Porous PLLA microfluidic microspheres were developed in our previous study (figure 7) [153].Similar to common microfluidic methods for preparing synthetic material-based microspheres, we constructed an O/W emulsion in which PLLA was dissolved in DCM as the disperse phase, while the PVA solution was used as the continuous phase.Additionally, to form a highly porous structure, gelatin was mixed into the PLLA solution as a porogen.After incubation in a warm water bath, the gelatin was removed and left a macroporous structure.Porous PLLA microsphere subsequently received alkali hydrolysis modification, so that it could act as scaffolds for loading soluble TNF receptor type II (sTNFRII)encapsulated bovine serum albumin (BSA) nanoparticles via chemical bonds.Due to its excellent mechanical property, PLLA microsphere exhibited the ability to buffer tissue stress and stabilize BSA nanoparticles for sustained drug release to treat IDD.

Conclusions and outlook
This review summarizes recent progress in biomaterials used to fabricate microfluidic carriers for drug and cell delivery.Starting with a comparison of advanced microfluidic technology and conventional fabricating methods in the fabrication of micro/nanomaterials, we introduce the generation mechanisms of different forms of carriers, such as spherical microparticles, nanoparticles and microfibers in microfluidic channels.A broad range of materials, including natural materials and synthetic polymers, have been chosen for microfluidic production and designed as different carriers depending on their properties.
Although microfluidics for biomaterial carriers has become increasingly sophisticated, many challenges remain to be addressed in improving the performance of microfluidic biocarriers.First, despite the achievements in fabricating carriers with high batch-to-batch reproducibility using microfluidics, intra-batch reproducibility of cell-laden vehicles has remained a major problem so far.Since most cell carriers are prepared by suspending cells in a polymer precursor, the number of cells encapsulated varies at different time points as cells are gradually deposited.However, most of the current studies on microfluidic cell carriers have ignored this issue.Therefore, it requires additional measures to keep the cells uniformly suspended in the polymer solution for a long time, ensuring high homogeneity of the cell carriers within the same batch.We recommend the addition of an auxiliary vibration module to the syringe pump to ensure uniform distribution of cells in the polymer precursor.Secondly, conventional manufacturing methods are still dominant today due to their ability to achieve mass production.However, as microfluidic biomaterial carriers are generated from tiny PDMS microfluidic chips and glass capillaries, it is challenging to realize high throughput production of microfluidic materials.Currently, a common strategy for scaling up production is to use parallelized or multilayer microfluidic channels, which requires sophisticated fabrication techniques.In most cases, higher flow rates are needed to increase productivity, which leads to a high pressure inside the microchannels.High shear forces may lead to deformation or even damage of the microchannels.It is particularly unfavorable during the production of cell carriers, as high fluid shear can affect the viability of cells suspended in microfluids and reduce the efficiency of cell delivery.External incentives, such as electrical and magnetic stimulations have also been utilized to generate droplets or nanoparticles as a non-pressure driven strategy.Their potential for the construction of cellular carriers remains to be explored.In general, how to achieve mass production of microfluidic carriers while maintaining high cell encapsulation rates is a long-standing problem that needs to be addressed in future research.Third, material properties greatly influence the production of microfluidic carriers.For instance, most aqueous polysaccharide solutions are highly viscous, and their viscosity increases with the polysaccharide concentration.Normally, the polysaccharide content in aqueous solutions should not be too high to prevent microchannel clogging and decrease in carrier homogeneity.The polysaccharide concentrations in most studies were set between 1 wt% and 4 wt%.During the processing of protein materials, the ambient temperature and pH of the solution need to be strictly controlled in case protein is deposited and crosslinked in the channel.For naturally derived materials, the poor flowability of their aqueous solutions is a key factor limiting their processing flexibility.Photografting appears as an ideal scheme because it allows for the crosslinking of carriers at a low polymer concentration, which significantly reduces the viscosity limits of the polymer solution for microfluidic processing.In fact, most of the natural materials used for microfluidic processing have received photo-modifications, such as HAMA, AlgMA and GelMA.Rapid solidification is another merit of photocrosslinking method, and short-term exposure to UV light does not cause damage to the encapsulated cells.In summary, we suggest more chemical modifications to natural materials to make them easier to use for microfluidic processing.In addition, as aliphatic polyesters can only be dissolved in organic solvents, the channels of microfluidic devices are supposed to be highly resistant to chemicals and deformation.Glass-based microfluidic devices are preferred for processing organic fluids; however, the design flexibility is restricted by the difficulty of forming complex channel geometries on glass devices.Overall, a deeper understanding of the relationship between materials and fluid behavior in microfluidic devices is demanded for developing and optimizing microfluidic biomaterial carriers.

Scheme 1 .
Scheme 1. Schematic diagram of microfluidic carriers made of various materials.

Figure 2 .
Figure 2. Microfluidics carriers made of hyaluronic acid and alginate.(A) Construction of PDA-modified liposome-HAMA microsphere.[43] John Wiley & Sons.(© 2021 Wiley-VCH GmbH).(B) Schematic diagram of oil droplet-encapsulated alginate fibers.The shape of the oil droplets and inter-droplet distance could be changed by varying the flow rates.Reprinted from [60], Copyright (2017), with permission from Elsevier.

Figure 4 .
Figure 4. Microfluidics carriers made of SF and collagen.(A) Single (i), (ii) and double (iii) SF fibers generated by flow-focusing co-extrusion.The diameter and structure of the fiber can be adjusted by varying the shear pressure and flow rate.[58] John Wiley & Sons.[© 2021 The Authors.Advanced Healthcare Materials published by Wiley-VCH GmbH].(B) (i) Schematic diagram of the fabrication of hepatic-lobule-like microtissue collagen spheroids using a precursor cartridge.(ii) Fluorescence image of the fabricated collagen microspheres with or without lobule-like microstructure.(iii) Shadowgraphic microscopy image of microspheres.[112] John Wiley & Sons.[© 2021 The Authors.Advanced Materials published by Wiley-VCH GmbH].

Figure 6 .
Figure 6.Microfluidics carriers made of PEGDA and PLGA.(A) The fabrication of microfluidic PEGDA fiber and its longitudinal SEM image.Reprinted with permission from [134].Copyright (2019) American Chemical Society.(B) (i) SEM images of PLGA microspheres and their cross section.(ii) The distribution of radiolabeled PLGA microspheres in vivo.Reprinted with permission from [142].Copyright (2020) American Chemical Society.

Table 1 .
The application of polysaccharide in preparing microfluidic carriers.

Table 2 .
The application of proteins in preparing microfluidic carriers.

Table 3 .
The application of synthetic polymers in preparing microfluidic carriers.