Engineering hydrogels as extracellular matrix mimics

Abstract

Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell-cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine.

Figure 1. The total number of publications with ‘tissue engineering’ and ‘hydrogel’ or ‘hydrogels’ in the title

The exact numerical values are provided at 5-year intervals (Source: Science Citation Index Expanded [SCI-EXPANDED]). The interest in hydrogels has significantly increased since 1990.

Figure 2. Fabrication of a 3D cell-laden microscale hydrogels using micromolding

(A) Liquid prepolymer is deposited onto a hydrophilic poly(dimethyl siloxane) (PDMS) pattern. (B) A hydrophobic PDMS cover slip is placed on top of the prepolymer, forming a reversible seal. (C) Polymer liquid is crosslinked using UV light or heat. (D) The PDMS cover slip is removed. (E) Hydrogels are washed from pattern. (F) Hydrogels are free of the pattern. (G) Flourescent image of microscale tissue constructs comprised of cell-laden hydrogels containing hepatocytes and fibroblasts. Scale bar: 400 μm. Reproduced with permission from [131].

Figure 3. Fabrication of a 3D cell-laden microscale hydrogel using optofluidic maskless lithography method

(A) The 3D-Optofluidic maskless lithography (OFML). OFML is performed in a two-layered PDMS microfluidic channel with UV curable resin placed in the bottom. The height of the bottom channel is controlled via deformation of the PDMS membrane under pneumatic pressure of the top chamber. Projection patterns are sequentially launched and projected onto the channel. (B) The 3D-OFML system is composed of two parts; the height-tunable PDMS microfluidic channel and the optical system. Pneumatic pressure is applied with a syringe pump and the UV projection for each layer is computer controlled. (C) Microfluidic control methods for exchanging resin materials to generate hybrid microstructures. Solutions can be injected sequentially through the same inlet (top) or multilaminar flow can be exploited (bottom). (D, F) Generation of 3D hydrogel tissue-like microstructures containing different living cells in each part of the structure. HeLa cells stained with red and blue fluorescence (D) are used for heterogeneous cell patterning. (F) Confocal microscope images of the hydrogel block containing cells. Differential interference contrast transmission image shows the circular and the rectangular pattern of hydrogel structure (left). (E) An array of patterned hydrogel with two different fluorescent microspheres. First layer is patterned in cross-shape and the other beads in the square-shape. Images were taken from the bottom of the structure with an inverted optical microscope. Scale bars in (E) and (F) indicate 100 μm. DMD: Digital micromirror-array device; PDMS: Poly(dimethyl siloxane). Reproduced with permission from [218].

Figure 4. Fabrication of cellular microfluidic hydrogels using a molding method

(A) The fabrication process. The dotted line indicates PEI coating. (B) Cell viability results using live/dead staining. H: Height (mm in thickness); PEI: polyethylenimine. Reproduced with permission from [35]

Figure 5. A schematic perfusion bioreactor

(A) 2D microchannel perfusion/viability model: single- and dual-channeled hydrogel constructs were prepared using cell-encapsulating agarose. (B) Fractional cell viability with single- and dual-channel set-ups taken at day 1, 2 and 3, respectively, after the channel constructs were built. 3T3: Mouse embryonic fibroblast cell line; o.d.: outer diameter; PDMS: Poly(dimethyl siloxane). Reproduced with permission from [170].

Figure 6. Printing cellular microfluidic hydrogels

(A) Design template for building branching tubular structures by depositing agarose rods and multicellular spheroids of the same diameter. (B) Fusion patterns of multicellular spheroids (300 μm HSF spheroids) assembled into tubular structures after 6 days of deposition. (C–F) Building a double-layered vascular wall by assembling human umbilical vein smooth muscle cells and HSF multicellular cylinders according to specific patterns (C). (D–F) show the results of H&E (D), smooth muscle α-actin (E) and caspase-3 (F) straining of the structure after 3 days of fusion. Reproduced with permission from [219].

Figure 7. Fabrication of cellular microfluidic hydrogels using printing

(A) Proliferation-ready endothelial cells aligned inside the fibrin channels using a thermal inkjet printer. The technology may enable building human microvasculature. (B & C) Channel structure of printed microvasculature cultured for 21 days. (B) Printed ring shaped microvasculature cultured for 21 days (C) Integrity of printed structure cultured for 21 days. HMVEC: Human microvascular endothelial cells. Reproduced with permission from [186].