The cytoprotective capacity of processed human cardiac extracellular matrix

Abstract

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 μm (15-157 μm) by laser diffraction, and 57 μm (20-182 μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, -72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Collagen Fibrils Mechanically Contribute to Tissue Contraction in an In Vitro Wound Healing Scenario

“…Consequently, tissue stability and tensional state would be independent of active cell forces. [38] In total 21 different proteins were detected (Tables S1 and S2, Supporting Information) and collagens including type I, III, VI, and XII were identified resembling almost 80% of all detected peptides (Figure 5c,d). The depletion was validated by histology and confocal microscopy (Actin, Nuclei) as well as residual DNA quantification after decellularization (Figure 5a, Figure S5a, Supporting Information).…”

Decellularized Extracellular Matrix Materials for Cardiac Repair and Regeneration

“…Another novel direction for using milled dECM is through the generation of dECM microparticles, which may retain ECM structure and protein composition better than soluble ECM, while allowing for injection into the myocardium . Kappler et al developed a method of generating uniform cECM particles derived from human left ventricle . The particles had native‐like fibrous structures, contained a large variety of ECM proteins, and were easily resuspended in buffer and gelatin solutions.…”

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane

“…Therefore, ECM homogenization is a critical additional processing step that could open up new possibilities in regenerative medicine [ 34 ]. Additionally, we have already been able to show that processing the human cardiac ECM in this way does not impact its cytoprotective capacity [ 15 ].…”

“…We have previously succeeded in isolating Extracellular Matrix (ECM) from human myocardium (hcECM) with a more preserved protein composition compared to protocols reported by other groups [ 12 ], for example, with regards to glycosaminoglycans (GAG) [ 13 ], and demonstrated its capacity to support cell viability and induce cardiac lineage differentiation of pluripotent stem cells [ 14 ]. Furthermore, we demonstrated that the additional processing to microparticles and subsequent reconstitution as a hydrogel does not affect the cytoprotective capacity of the hcECM [ 15 ].…”