<p>Supplementary Material</p><p>Cell-laden Polymeric Microspheres for Biomedical Applications Wenyan Leong and Dong-An Wang Division of Bioengineering, School of Chemical & Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore 637457 Corresponding author: Wang. D.-A. ([email protected])</p><p>Figure S1. Schematic diagrams of commonly used microsphere fabrication techniques. (A) Emulsion formation by stirring a polymer solution in an immiscible continuous phase, after which polymerization occurs; (B) Extrusion of polymer solution through a nozzle tip into a collecting bath, where polymerization then occurs; (C & D) Atomization of polymer droplets into various collectors depending on setting mechanism. Setting by solvent extraction requires air drying (C) while temperature-responsive, photocrosslinkable or ionic-responsive polymers can be collected in the collecting bath where polymerization occurs (D); and (E) microfluidics of T-junction configuration where both phases flow at different rates and polymerization can occur in the downstream channel or in a collecting bath. Table S1 Properties of common polymers for microsphere fabrication</p><p>Polymers Type, source Common Reversible? Advantages Disadvantages Ref crosslinking mechanism Natural Typically - Biodegradable - Batch-to-batch polymers physical - Low toxicity variation crosslinked - Low cost, widely - Presence of impurities, available requires stringent - Typically mild purification crosslinking, allowing - Usually weak direct cell encapsulation mechanical properties, suitable for mimicking soft tissue only - Uncontrollable degradation, unstable</p><p>- Alginate Polysaccharide, Ionic: Yes. - Suitable for direct cell - Non-cell adhesive; [S1] brown algae Introduce Immersion in encapsulation - Susceptible to divalent ions for chelating agents - Mild crosslinking uncontrollable sol-gel transition (sodium citrate) - High water content dissolution in presence of monovalent ions - Agarose Polysaccharide, Temperature: No. - Suitable for direct cell - Non-cell adhesive [S2] seaweed Decrease encapsulation - Brittle temperature for - High water content sol-gel transition</p><p>- Collagen Protein, Self-assembly Yes. - Cell-adhesive - Allogenic or [S3, connective Enzymatic - ECM xenogeneic source, S4] tissue of animal degradation by - Good mechanical may induce host sources MMPs properties immune response - Suitable for direct cell when transplanted encapsulation</p><p>- Chitosan Polysaccharide, pH: Yes. - Cell-adhesive - Not able to encapsulate [S5, deacylation of increase pH for Enzymatic - Good mechanical cells directly; cells can S6] chitin sol-gel transition degradation by properties be seeded after lysozyme - Possess amino groups fabrication for functionalization - Degree of deacylation is not precise - Low hydrophilicity - Slow degradation</p><p>- Fibrin Protein, blood Self-assembly: Yes. - Cell-adhesive - Allogenic or [S7] plasma Fibrinogen Enzymatic - Highly compliant xenogeneic source, cleavage by degradation by - Suitable for direct cell may induce host thrombin to plasmin encapsulation immune response form fibrin when transplanted monomers, self- - Chance of viral assembly to transmission (prepared form fibrous from blood) network</p><p>- Gellan Polysaccharide, Ionic: Yes. Increase in - FDA-approved - Lack of cell-adhesive [S8, fermentation of Introduce temperature. - Good mechanical moieties; requires S9] Spinghomonas multivalent properties functionalization elodea cations for sol- - High acyl content gives gel transition; or compliant and elastic Temperature gels - Suitable for direct cell encapsulation</p><p>- Gelatin Protein, Temperature: Yes. - Cell-adhesive - Quick dissolution [S10, denatured Decrease Increase in - ECM-like under physiological S11] collagen temperature for temperature or - Suitable for direct cell temperature; require sol-gel transition enzymatic encapsulation covalent crosslinking degradation by MMPs</p><p>Synthetic Typically No. - Easily tunable - Lack of cell-adhesive polymers chemically Require mechanical and moieties; requires crosslinked modification chemical properties functionalization e.g. - Stable - Usually harsh photodegradable - Consistent quality (no/ fabrication conditions crosslinkers little batch-to-batch - Expensive variation) - Non-degradable/ toxic degradation products</p><p>- Poly(ethylen Polyester Covalent No - FDA approved; - Lack of cell-adhesive [S12, e glycol) biocompatible moieties S13] (PEG) - Suitable for direct cell encapsulation</p><p>- Poly(lactic- Polyester Solvent No - FDA approved; - Hydrophobic [S14- co-glycolic extraction/ biocompatible - Lack of cell-adhesive S16] acid) evaporation - Non-toxic moieties (PLGA) - Bioresorbable - - Poly(caprola Polyester Solvent No - FDA approved; - Lack of cell-adhesive [S17, ctone) (PCL) extraction/ biocompatible moieties S18] evaporation - Bioresorbable - Slower degradation than PLGA - Ease of blending with other polymers Table S2 Common microsphere fabrication techniques </p><p>Technique Principles Advantages Disadvantages Factors affecting microsphere size Example ref Emulsion - Two immiscible - No specialized - Large size - Size of vessel Collagen [S3], phases. equipment required distribution - Viscosities of phases Fibrin [S7], Dispersed phase - Scalable - Washing steps - Stirring rate Gelatin [S11], contains - Multiple emulsions required - Presence and concentration of PLGA [S16], polymer achievable - Batch-wise surfactant Chitosan [S19] solution. - High throughput process - Mechanical agitation/ stirring to break up polymer solution into droplets</p><p>Extrusion - Polymer - Uniformly sized - Specialized - Flow rate Chitosan [S5, solution is microspheres equipment - Viscosity of polymer solution S6], extruded - Continuous process required - Diameter of nozzle Alginate [S1, through a nozzle achievable - Low throughput S20] and fall into (1 bead at a time) collector</p><p>Atomization - Atomized - Small microspheres - Specialized - Flow rate PVA [S21], droplets fall into with narrow size equipment - Viscosity of polymer solution alginate [S22] a collector distribution required - Frequency - High throughput - Height of collector and air flow - Continuous process direction (for spray drying) achievable - Temperature - Scalable Microfluidics - Two immiscible - Uniformly sized - Washing steps - Flow rates Agarose [S2], phases flowing microspheres required - Viscosities of phases PEG [S23], continuously, - Easy to customize - Not scalable - Diameter of nozzle Poly(NIPAAm) with continuous - Small platform - Low throughput - Dimension of setup [S24] phase pinching - Continuous process (1 bead at a time) - Presence and concentration of off beads of achievable surfactant dispersed phase - Multiple emulsions (polymer achievable solution) Table S3 List of commercially available microcarriers, data obtained from respective companies. Unless stated as macroporous, the microcarriers are microporous.</p><p>Material Commercial name/ Density Coating/ Feature Mean Company (g/ cm3) Diameter (µm) Dextran Cytodex 1 1.03 - Positively charged 190 (GE Healthcare) Cytodex 3 1.04 - Porcine gelatin- 175 (GE Healthcare) coated - Gelatin Cultispher S 1.02-1.04 - High thermal 130-380 (Sigma Aldrich) stability - Macroporous Cultispher G 1.02-1.04 - Macroporous 130-380 (Sigma Aldrich)</p><p>Collagen Spherecol 1.02-1.03 - Human type I 125-212 (Advanced collagen-coated Biomatrix)</p><p>Cellulose Cytopore 1 1.1 - Positively charged 230 (GE Healthcare) - Macroporous; increases surface area Cytopore 2 1.1 - Highly positively 230 (GE Healthcare) charged - Macroporous - Polystyrene BB-MF 3092 1.02 - Porcine gelatin- 125-212 (SoloHill, Sigma) coated Synthemax II 1.026 - Collagen 125-212 (Corning) References S1 Chayosumrit, M., et al. (2010) Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm. 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