Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids

Abstract

Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

The physics of organoids: a biophysical approach to understanding organogenesis

“…The predilection of most organoids for Matrigel is a hurdle, but moving away from Matrigel to hydrogels, where the mechanical properties can be manipulated independently of biochemical components, will empower such approaches (Gjorevski et al, 2016;Greggio et al, 2013). In this context, intestinal organoids embedded in bovine collagen gels or elastin domain-based engineered hydrogels with varying stiffness grow with various efficiencies (DiMarco et al, 2014(DiMarco et al, , 2015. Collagen gels loaded with polydimethylsiloxane (PDMS) further enable one to control stiffness independently of pore size (Cassereau et al, 2015).…”

Bioengineering for intestinal organoid cultures

“…[10] This study showed that different matrix parameters are preferred at different stages of organoid formation; stiffer matrix was required during initial stage of ISC expansion then subsequent softening of the matrix was essential for continued expansion and organoid formation, and only laminin-111 supported budding of organoids whereas laminin-111-derived peptides did not. (Figure 1B) Interestingly, the reported optimal ECM mechanical stiffness for organoid formation is similar for both the elastin-based eECM[19] and the PEG-based synthetic ECM[10]. The use of artificial matrices with minimal components allows the deconvolution of relevant environmental cues, otherwise unachievable using naturally-derived matrices.…”

“…DiMarco et al [19] developed an elastin-based engineered-ECM (eECM), where stiffness and adhesion site density could be independently controlled. Mouse organoid formation efficiency was similar to collagen I with the best efficiency observed with an eECM of 180 Pa mechanical stiffness and 3.2 mM of Arg-Gly-Asp (RGD) cell-binding sites.…”

Innovation for hepatotoxicity in vitro research models: A review

“…3D organoid scaffold models, which contain harvested collagen matrices, provide the effects of biochemical and biomechanical cues. Decreasing mechanical stiffness and increasing cell adhesiveness were found to increase organoid yield (DiMarco, Dewi, Bernal, Kuo, & Heilshorn, ; Zhang et al, ). When HepG2 cells were seeded on scaffolds, cells could penetrate the scaffolds and proliferate.…”

“…Decreasing mechanical stiffness and increasing cell adhesiveness were found to increase organoid yield (DiMarco, Dewi, Bernal, Kuo, & Heilshorn, 2015;Zhang et al, 2016). When HepG2 cells were seeded on scaffolds, cells could penetrate the scaffolds and proliferate.…”