Engineered Matrices Enable the Culture of Human Patient‐Derived Intestinal Organoids

Abstract

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t 1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10 −3 -1 × 10 −3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

Hyaluronan and Elastin-Like Protein (HELP) Gels Significantly Improve Cargo Retention in the Myocardium

“…When comparing the two bio-conjugated formulations, HA-A vs . HA-B, we find that the stress-relaxation rate is dependent on the exchange kinetics of the hydrazone bond, with aldehydes having a faster exchange rate than benzaldehyde and, therefore, a faster stress- relaxation time, consistent with other reported literature [39, 53]. For context, our measured shear stiffness moduli are less than what has been reported for cardiac tissue, 5 – 50 kPa [54, 55]; however, direct comparisons are difficult due to variations in testing methodology.…”

“…The hyaluronan and elastin-like protein (HELP) gel system presented here has several benefits compared to other currently available, injectable hydrogel systems. These include: (1) independent tuning of biochemical and biomechanical cues [38], (2) secondary crosslinking mechanisms induced at physiological temperatures [37], (3) high degree of cell-compatibility [37–39], and (4) cell membrane protection during injection [37]. Additionally, hyaluronan (HA) has been shown to promote angiogenesis, suppress fibrous tissue formation, and natively bind water to the heart muscle, which improves mechanical and electrophysiological properties of the heart muscle [69–71].…”

“…The ELP used presently contains a central lysine (K) group within the elastin-like region. These lysine moieties were functionalized with hydrazine groups according to the following protocol, as has been reported on previously [37, 39]. First, 180 mg of ELP is dissolved in 6 mL of room-temperature anhydrous dimethyl sulfoxide (DMSO) in a 25-mL round-bottom flask.…”

“…The procedure for alkyne modification has been described elsewhere [39]. Briefly, 200 mg of 100 kDa HA (sodium salt) was dissolved in 20 mL MES buffer (0.2 M MES hydrate, 0.15 M NaCl in DI water; pH 4.5) at a concentration of 1% (w/v).…”

“…Our lab has recently developed a two-part, hyaluronan and elastin-like protein (HELP) hydrogel system ( Figure 1B ) that is injectable, cell-compatible, and has independently tunable mechanical and biochemical properties [37–39]. Briefly, the hyaluronan (also termed hyaluronic acid, HA) component can be functionalized either through oxidation or conjugation chemistry to have a pendant aldehyde or benzaldehyde moiety.…”