Functionalized Microgel Rods Interlinked into Soft Macroporous Structures for 3D Cell Culture

Functionalized Microgel Rods Interlinked into Soft Macroporous Structures for 3D Cell Culture

1 Introduction

To understand interactions between living matter and artificial materials, tissue engineering research groups focus on the development of 3D scaffolds with increasingly controllable properties, reaching from biochemical interactions at molecular scale to nanoscale hydrogel networks, micrometer-scale pores, and macroscopic architectures.[1] Soft hydrogel scaffolds are conventionally formed by crosslinking a precursor solution, resulting in soft, tissue mimetic constructs. Cells are encapsulated in the precursor before crosslinking, while the formed hydrogel allows for cells to spread and proliferate by degrading and/or remodeling the network.[2] As an alternative to bulk hydrogel applications, macroporous 3D scaffolds are fabricated by several techniques like electrospinning, solvent casting, or gas foaming/particle leaching, with the advantage that no degradation is required for cell migration and proliferation, which enhances the rate of cell infiltration.[13] Control over the porous structure in 3D cell scaffolds is of particular importance as different cell types and their subunits, like neurites, migrate in a limited yet specific manner.[34]

To provide a soft environment with micrometer-scale pores in contrast to bulk hydrogels or stiff scaffolds, microporous annealed particle (MAP) scaffolds were developed by the Segura group.[5] MAPs are injectable and are formed by interlinking microgels together instead of nanoscale precursor molecules, enhancing cell invasion and migration with simultaneous control over the composition of the scaffold building blocks.[156] The macroporous void, referred to as percolated interstitium, can be varied depending on the diameter of the microgels. The position of attached cells to micro- and macroporous 3D hydrogel structures in contrast to embedded cells inside bulk-based 3D hydrogels can provide further insight for proprioception studies to form 3D functional tissue and restore damaged tissue.[37]

In the first MAP report, spherical microgel building blocks were produced via microfluidics by Michael-type addition, while transglutaminase peptide substrates were coupled into the microgels to interlink them together with activated Factor XIII.[58] Spherical microgels in the range of 30–150 µm diameter resulted in pore sizes between ≈10 and 45 µm, respectively, which are in a relevant range for cell migration.[134] An increase in microgel stiffness and RGD concentration inside the microgel networks both result in enhanced cell spreading and better proliferation.[6] Cell spreading differences were also observed depending on the microgel dimensions, favoring larger microgels likely due to the larger pore sizes available. Further employment of another biocompatible chemical interlinking mechanism, based on tetrazine norbornene cycloaddition click reaction, reduced inflammation and astrogliosis levels when MAP scaffolds were injected in the brain after a prior initiated photothrombotic stroke.[69]

Alternatively, to covalent bonds between the microgels, supramolecular guest–host (β-cyclodextrin and adamantane) interactions have been employed to interlink complementary functionalized polyethylene glycol (PEG)-based spherical microgels with diameters ranging from 10 to 100 µm. This led to porous interlinked networks with interparticle distances ranging from ≈5 to 25 µm (after centrifugal filtration) that supported the growth of THP-1 monocyte cells, which have a migratory phenotype with adhesion-independent characteristics, enabling the investigation of rapid and dynamic cell behavior at the tissue scale.[10]

2 Results

2.1 Microgel Rod Production via Continuous Plug-Flow On-Chip Gelation

Primary amine and epoxy microgel rods are produced via continuous on-chip gelation in microfluidics. The free-radical polymerization (FRP) crosslinking reaction is triggered by controlled UV-irradiation inside the straight section of the microfluidic channel before the outlet utilizing lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator. To apply UV-radiation (λ = 365 nm) dose appropriate for crosslinking on-chip, different irradiance settings ranging from 128 to 957 mW cm−2 are tested with an irradiation time of ≈2.3 s. This revealed that for higher initial AMA concentrations, a higher light intensity is needed to crosslink the microgels sufficiently on-chip to avoid deformation after leaving the device. If 957 mW cm−2 is applied, all microgels retain their aspect ratio and no further flow changes are observed at higher irradiation power (Figure S1, Supporting Information).

To enable continuous collection of all types of rod-shaped microgels, the transport of the product out of the outlet required a second flow-focusing oil-stream (Figure 1). This oil stream avoids potential jamming of the anisometric microgels at the outlet chamber caused by the higher aspect ratio compared to spherical microgels. The chip design facilitates flushing out the crosslinked microgels at the microfluidic outlet, as the resistance is reduced (Figure S1, Supporting Information). The production rate of microgel rods employed in this study is ≈11 300 microgels per hour.

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