The cytoprotective capacity of processed human cardiac extracellular matrix.
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.
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Benjamin
B
Kappler
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Petra
P
Anic
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Matthias
M
Becker
DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany.|Deutsches Herzzentrum Berlin (DHZB), Augustenburger Platz 1, 13353, Berlin, Germany.
Andreas
A
Bader
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Kristin
K
Klose
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Oliver
O
Klein
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Barbara
B
Oberwallner
Department of Cardiac and Thoracic Surgery, Heart Center of the University, University of Cologne, Cologne, Germany.
Yeong-Hoon
YH
Choi
DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany.|Deutsches Herzzentrum Berlin (DHZB), Augustenburger Platz 1, 13353, Berlin, Germany.
Volkmar
V
Falk
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany. stamm@dhzb.de.|DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany. stamm@dhzb.de.|Deutsches Herzzentrum Berlin (DHZB), Augustenburger Platz 1, 13353, Berlin, Germany. stamm@dhzb.de.
Christof
C
Stamm
D000328:Adult; D000818:Animals; D015687:Cell Hypoxia; D019070:Cell Lineage; D049109:Cell Proliferation; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005947:Glucose / Q000737:chemistry; D016027:Heart Transplantation; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology; D013696:Temperature; D023822:Tissue Engineering / Q000379:methods*; D055815:Young Adult
D020100:Hydrogels; D005947:Glucose