Overview and technical details of a 3-dimensional bioplotter for production of intestinal crypt organoid models.
Epithelial Cell-ECM interactions are critical to promote cell survival. Various bioprinting techniques have been developed to recapitulate 3D tissue constructs, including light-assisted, droplet-based, and extrusion-based systems.
While Collagen IV is essential in polarizing the intestinal stem cell niche for Lgr5+ differentiation, Collagen IV is not well suited for bioplotting as it has intrinsic limitations in mechanical properties and structural stability after bioprinting due to slow cross-linking. UV cross-linking via methacrylation can speed up solidification, but we decided to only coat the bottom of the Transwell insert with a layer of Collagen IV, allow it to solidify, then bioprint the fabricated crypt structures on top of the Collagen IV. This allows the stem cells to be dropped directly onto the bed of Collagen IV for polarization, and subsequently utilize the fabricated Decellularized Matrix (dECM) Hydrogel crypt for self-assembly in the z direction.
A May 2020 review article titled “Polymer Hydrogels to Guide Organtypic and Organoid Cultures” brings to light the potential hydrogel-based approaches to address challenges that need to be overcome in organoid development across systems. Here they suggest a bioengineered decellularized matrix hydrogel to provide local support for cell proliferation and crypt formation. Why decellularized? Because these matrices isolate the ECM from inhabiting cells while preserving the post-translational modifications of the molecular components and tissue-specific structural features. Decellularized organs can be chopped up, ground into bits, turned into a powder, and dissolved into a liquid solution for extrusion-based bioprinting. This tissue-specific network of ECM biopolymers is suitable for embedding cells and bioprinting because the ECM fibers self-assemble upon neutralization at 37 Degrees Celsius and provide localized adhesion, allowing for proper metabolic activity, proliferation, morphology, and differentiation. Furthermore, the deECM undergoes enzymatic degradation and matrix assembly in tune with proteolytic remodeling.
If decellularizing tissue proves to be unsuccessful, our fall back plan is the ever-reliable Fibrin-Matrigel Biopolymer. Matrigel is a heterogenous mixture of many ECM components such as: laminin, collagen IV, nidogen, proteoglycans, and growth factors. However, it seems the decellularized ECM hydrogel would be more physiologically-relevant due to Matrigel being a permissive murine sarcoma-derived gel. The Fibrin-Matrigel Biopolymer is an integrated polymer network formation of laminin within the fibrin scaffold for differentaion. Fibrin hydrogels between 3 and 4.5 mg mL−1 (77 ± 25 Pa and 140 ± 47 Pa) provide the best physical properties for organoid expansion.
The ECM undergoes high remodeling during disease. To recapitulate the physiological relevance of this, tissue stiffness can be modified via increased fibrillar collagen COLL I and III with crosslinking via lysyl oxidase enzymes (LOX) and metalloproteinase degradation (MMP) to disrupt basement membrane homeostasis.