A Versatile Biosynthetic Hydrogel Platform for Engineering of Tissue Analogues

Abstract

For creating functional tissue analogues in tissue engineering, stem cells require very specific 3D microenvironments to thrive and mature. Demanding (stem) cell types that are used nowadays can find such an environment in a heterogeneous protein mixture with the trade name Matrigel. Several variations of synthetic hydrogel platforms composed of poly(ethylene glycol) (PEG), which are spiked with peptides, have been recently developed and shown equivalence to Matrigel for stem cell differentiation. Here a clinically relevant hydrogel platform, based on PEG and gelatin, which even outperforms Matrigel when targeting 3D prevascularized bone and liver organoid tissue engineering models is presented. The hybrid hydrogel with natural and synthetic components stimulates efficient cell differentiation, superior to Matrigel models. Furthermore, the strength of this hydrogel lies in the option to covalently incorporate unmodified proteins. These results demonstrate how a hybrid hydrogel platform with intermediate biological complexity, when compared to existing biological materials and synthetic PEG-peptide approaches, can efficiently support tissue development from human primary cells.

Keywords: FXIII; Matrigel; gelatin; liver organoids; osteogenesis; polyethylene glycol; vasculogenesis.

Figure 1

Enzymatic crosslinking reaction between PEG and gelatin. The FXIIIa-specific amino acid sequence for glutamine (gln), NQEQVSPL, was conjugated to 8 arm PEG (PEG-Gln), which can be crosslinked with lysine (lys) residues that are naturally occurring on gelatin. The reaction takes place under physiological conditions and cells can be encapsulated in the same step. It is also possible to immobilize other lysine-containing proteins in the hydrogel network for enhanced tissue specificity (see Figure S2, Supporting Information).

Figure 2

Physical fine-tuning and characterization of gelPEG hydrogels. a) Hydrogels with the lowest swelling ratio were obtained at a molar ratio (gelatin)Lys/(PEG)Gln of 2:1 for 3% w/v gelPEG. b) Decreasing gelPEG concentration resulted in higher swelling ratios. c) Compressive moduli were significantly increased from 2% to 3% gelPEG. n.d., not determined; lys: lysine; gln: glutamine; data are depicted as mean + SD; n = 5.

Figure 3

Vasculogenesis in gelPEG and Matrigel characterized by stabilized capillary-like structures with lumen. Human GFP-ECFCs and MSCs were cocultured for 10 d in EGM-2. a,e) Projections of 150 μm confocal stacks through a hydrogel. b,f) Stabilization of vascular networks by pericyte-like cells (red). c,g) Capillary-like structures were composed of multiple fused endothelial cells indicated by cell-cell contact by VE-cadherin junctions. d,h) Capillary-like structures in gelPEG and Matrigel were characterized by lumenization (dotted lines indicate cutting sections). i) Capillary-like structures with flopodia at a site of active angiogenesis, here in a gelPEG hydrogel. j,k) Total vessel length and average vessel thickness in gelPEG hydrogels and Matrigel. Data are depicted as mean + SD; N = 3, n = 3.

Figure 4

Development of prevascularized bone-like tissue analogues in Matrigel and gelPEG-based hydrogels. ECFC-MSC cocultures were cultured for 2 weeks under osteogenic culture conditions in the different hydrogels before analysis of osteogenesis, vasculogenesis, and presence of pericyte-like cells. Expression of BCLAP and SPP1 as osteogenic markers, CDH5 and PECAM1 were measured as endothelial markers and CSPG4 and ACTA2 as pericyte markers. a,e,i) Von Kossa staining was positive for gelPEG and gelPEG+LN111 hydrogels highlighting mineralization. b,f,j) Sections of hydrogels highlighted the presence of the osteogenic marker osteonectin in all groups. c,g,k) 100 μm z-projections of vascular-like networks (green) in the centre of the hydrogels. d,h,l) Vascular-like structures, stabilized by αSMA-positive pericyte-like cells (red). m) Total vessel length was equal in Matrigel and gelPEG+LN111, and significantly longer than in gelPEG hydrogels. n,o) mRNA expression for osteogenic genes encoding for osteocalcin and osteopontin were comparably expressed in Matrigel, gelPEG, and gelPEG+LN111. p,q) Vasculogenesis-associated genes encoding for CD31 and VE-cadherin were expressed in all hydrogels, with highest VE-cadherin expression in Matrigel, followed by gelPEG and gelPEG+LN111. r,s) Pericyte-associated genes encoding for αSMA and NG2 were equally expressed in all hydrogels. Data are depicted as mean + SD; N = 3, n = 3.

Figure 5

Development of liver-like tissue analogues in Matrigel (MG) and gelPEG-based hydrogels. A functional read-out of protein levels and enzyme activities was performed after 9 d culture of liver organoids. H&E staining of liver organoids in a) Matrigel, b) gelPEG, c) gelPEG+LN111, and d) gelPEG+LN521. e) Albumin protein levels in cultured organoid cell lysates. f,g) ALAT an ASAT enzyme activities of liver organoid cell lysates. h,i) LDH and GLDH enzyme levels of liver organoid cell lysates. Data are depicted as mean + SD; N = 3, n = 3.