Journal: Molecular Systems Biology

Article Title: Engineering mammalian cells for detection and treatment of cardiac injury

doi: 10.1038/s44320-025-00161-x

Figure Lengend Snippet: ( A ) Designer cells engineered for cTnI-triggered TNK secretion. HEK-293 cells were transfected with constitutive expression vectors for TropR FGFR2b , TetR-Elk1 and different variants of TetR-driven TNK expression units (pSYQ267, P hCMV*-1 -TNK-pA; pSYQ294, P hCMV*-1 -NanoLuc-P2A-TNK-pA; pSYQ326, P hCMV*-1 -TNK-P2A-NanoLuc-pA), and incubated in cell culture medium containing 100 ng/mL cTnI. TNK levels in culture supernatants were quantified at 48 h post transfection by ELISA. Data were presented as mean ± SD, n = 3 individual experiments. ( B ) Morphological analysis of fibrin clots generated by coagulation of platelet-poor plasma in vitro. At 2 h after the addition of 10 mM CaCl 2 to platelet-poor plasma in a glass cuvette, gel-like fibrin clots were profiled (scale bars in green) and analyzed by scanning electron microscopy (SEM) at x3000 magnification. ( C ) Fiber size distribution of fibrin clots. Five 10000x images were obtained from the SEM analysis data shown in ( B ), and the diameters of 50 randomly picked fibers were estimated by ImageJ ( n = 250 fibers). ( D , E ) Validation of dose-dependent fibrinolysis by mammalian cell-secreted TNK. Aliquots (20 µL) of conditioned medium of HEK-293 cells transfected with a constitutive TNK expression vector (P hCMV -TNK-pA; pSYQ198) were serially diluted and added to freshly formed fibrin clots prepared as described above in ( B ). Microscopic analysis ( D ) and D-dimer amounts in the reaction system ( E ) were quantified after 24 h. Data in ( E ) are presented as mean ± SD of D-dimer levels of 2 replicate clot samples per group. .

Article Snippet: Human D-dimer ELISA Kit , Elabscience , Cat. No. E-OSEL-H0010.

Techniques: Transfection, Expressing, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Generated, Coagulation, Clinical Proteomics, In Vitro, Electron Microscopy, Biomarker Discovery, Plasmid Preparation

Journal: Molecular Systems Biology

Article Title: Engineering mammalian cells for detection and treatment of cardiac injury

doi: 10.1038/s44320-025-00161-x

Figure Lengend Snippet: ( A ) Selection of monoclonal CardioProtect cell lines. Representative single cell clones stably expressing TropR FGFR2b , TetR-Elk1, and a TetR-driven TNK expression unit (pSYQ367; ITR SB -P hCMV*-1 -TNK-pA-ITR SB ) were harvested and cultivated in medium containing 0 or 100 ng/mL human cTnI, and TNK levels in culture supernatants were quantified after 24 h. Data are presented as mean ± SD, n = 2 individual ELISA measurements. ( B ) cTnI-inducible doxycycline-repressible TNK production by monoclonal CardioProtect. One CardioProtect cell clone selected from ( A ) was harvested and encapsulated into alginate-poly- L -lysine-alginate microbeads with a density of 500 cells per capsule and a pore size of around 400 nm. About ~100 capsules (encapsulated) or 5 × 10 4 CardioProtect cells (native) were seeded per well for cultivation in cTnI (0 or 100 ng/mL) and/or doxycycline-containing medium (100 ng/mL). Alternatively, cells were pre-treated with 100 ng/mL doxycycline prior to the encapsulation process. Secreted TNK levels in cell culture medium were quantified after 24 h by ELISA. ( C ) Dose-dependent TNK production by encapsulated CardioProtect. 6.5 × 10 4 native or encapsulated CardioProtect cells were stimulated with different cTnI doses in culture and TNK levels in supernatants were quantified after 24 h by ELISA. Data were presented as the mean ± SEM; n = 3 independent experiments. ( D ) Potential mode of action of CardioProtect capsules during acute myocardial infarction (AMI). During AMI, fibrin clots (e.g., formed after rupture of atherosclerotic plaques) can cause cardiac injury and concomitant cTnI release into the bloodstream. Encapsulated CardioProtect cells designed to self-sufficiently detect systemic cTnI surges and release thrombolytic agents into the circulation should induce clot lysis and reduce further heart damage. ( E , F ) cTnI-dependent fibrinolysis by encapsulated CardioProtect cells. ( E ) In a 24-well plate, encapsulated CardioProtect cells were cultivated in human whole blood pre-mixed with an equal volume of RPMI 1640 medium, then a freshly prepared fibrin clot (prepared as described in Fig. ) was added together with inducer solutions of different concentrations (cTnI, 0 or 100 ng/mL; Dox, 0 or 100 ng/mL). Time-dependent clot lysis was monitored at the 60 h experimental endpoint. Co-culture of fibrin clots with empty capsules was included as negative control. ( F ) D-dimer levels in the blood system at 60 h post cTnI exposure. Data in ( B ) are presented as mean ± SD, n = 3 independent experiments. Data in ( C , F ) are presented as mean ± SEM, n = 3 independent experiments, statistical significance was determined with Student’s two-tailed t -test, *( p < 0.05), p = 0.0409 for Fig. 5F. .

Article Snippet: Human D-dimer ELISA Kit , Elabscience , Cat. No. E-OSEL-H0010.

Techniques: Selection, Clone Assay, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Pore Size, Capsules, Encapsulation, Cell Culture, Lysis, Co-Culture Assay, Negative Control, Two Tailed Test