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Cardiac Tissue Engineering


  • ECM Gels for cardiac repair
  • Cardiac ECM for cardiac repair
Cardiac Extracellular Matrix (C-ECM) may promote faster myocardial reconstruction of functional tissue due to similar structure and function.

Using retrograde perfusion multiple types of decellularization solutions are pumped through the heart.  This method takes advantage of the high degree of vascularization to decellularize the densely cellular tissue.  Further by using a combination of enzymatic, anionic, ionic, hypertonic, and hypotonic solutions along with the pump pressure, damage to the structure and composition of the tissue as well as time to decellularize the heart is minimized.

      Heart before Decellularization                                       Trypsin/0.05% Trypsin/EPTA

           Triton x 100                                Sodium Deoxycholate                            Peracetic acid

In vitro work has shown that the ECM scaffolds are capable of supporting cardiomyocyte growth

Monoclonal Anti-α Actinin (EA-53) [Sigma] stains green Monoclonal to β-Tubulin (TU-06) [Abcam] stains red.

CMs are identified by positive α-actinin staining and positive β-tubulin staining (green-yellow) Non-CMs are recognized by negative α-actinin staining and positive β-tubulin staining (red).

      Fig. 6: CMs cultured on ECM for 4 days (600x) C-ECM top left,
      UBM top right, UBM gel bottom left, and C-ECM gel bottom right

The C-ECM will be used as a ventricular patch and tested verse Dacron and UBM.

ECM to improve cell growth.
Atrial septal defect (ASD) repair.

An ASD is a hole between the right and left atrium.  By combining UBM with a NiTi frame the hole can be closed.  This hybrid device has advantages over the current devices by creating functional tissue instead of scar tissue.  Further, the amount of foreign material can be minimized.




Arteriovenous (AV) shunt.

An AV shunt is used as an access point for dialysis patients. The basement membrane of UBM is quickly endothelialized.  Therefore, UBM makes a good vessel substrate.  The graft will connect a carotid artery to the contralateral jugular vein.  A multilayer device will be made to withstand the arterial pressure.


Cardiomyocyte Seeding of a Decellularized Rat Heart


Biologic scaffolds composed of extracellular matrix (ECM) have been successfully used clinically in over 2 million patients.  The ECM has been derived from different organs and tissues including intestine, bladder, pericardium, and skin and typically is manufactured as a 2-dimensional sheet.  While 3-dimensional constructs can be made from these sheets, the shape of such construct is generally limited to multi-laminate patches or tubular structures.  By decellularizing an entire organ most of the complex structure of the native tissue ECM can be maintained.

It has been shown that ECM structure and composition are tissue specific [1].  It has also been shown that ECM can maintain a differentiated state of homologous cells in vitro (e.g., hepatic sinusoidal endothelial cells) [2].  It is logical therefore, tht decellularization of an intact heart may provide a favorable 3-dimensional template of scaffold for whole organ reconstruction.

In the present study, an entire rat heart was decellularized and the remaining ECM sterilized.  The cardiac ECM (C-ECM) was then seeded with rat neonatal cardiomyocytes (RNCs) or human fetal cardiomyocytes (HFCs) and maintained in culture for up to 14 days.


  • The entire rat heart was decellularized by retrograde perfusion with:  0.02% trypsin/0.05% EDTA/0.05% Sodium Azide, 3% Triton X-100, and 4% sodium deoxycholic acid each for one hour with hypotonic and hypertonic washes in-between.
  • The C-ECM was then disinfected with 0.1% (v/v) peracetic acid (PAA) for one hour.

  • H&E and DAPI were used to confirm decellularization.

Decellularized Rat Heart ECM

Decellularized Rat Heart ECM H&E 100x and DAPI 200x


  • Decellularized rat heart ECM was sterilized by gamma irradiated with 2 million Rads.

  • Ten million RNCs or HFCs were antegrade injected into the coronaries through the aorta of separate decellularized hearts in 1 ml of media.

  • Media was perfused through the aorta during the culture period.

  • Human Fetal Cardiomyocytes were cultured within the rat heart ECM for 1 or 2 weeks then fixed and sectioned for imaging.

  • H&E and DAPI were used to image cross sectional slices of the ventricle.


HFC seeded in Rat Heart ECM after 2 weeks
HFC seeded in Rat Heart ECM after 1 week H&E and DAPI 200x

  •   Rhodamine Phalloidin was used to show local alignment of the f-actin fibers.


    Combined Image 400x                           DAPI Channel Removed 400x

  • DAPI for Nuclei: Blue

  • Anti-α-actin [Sigma] for muscle specific actinin: Red

  • Histone H3 [Upstate] for proliferation/mitosis: Green [3]


Primary Rat CM Isolation

  • Rat CMs were isolated with serial digestion with 3 mg/ml collagenase for 15 min then multiple 0.1% trypsin for 5 min in a 50 ml conical with a stir bar at 1 rev/sec (37%C).

  • Rat CMs were plated on nitrocellulose coated wells and were beating for 7 days.

  • Media with 200  uM BrdU had a spontaneous beat rate three times higher than without BrdU.

  • 10 million rat CMs in 1 lm of media were injected into the intact aorta of the C-ECM construct over 2 min.

  • Intact construct was cultured for 7 days and showed noticeable color change toward reddish brown.

  • A Langendorff preparation of a fresh heart was performed to validate the pressure, perfusion, and stimulation systems.

              Rat CMs in 12 well plate at 5 days                                Rat heart Langendorff preparation

  • By using retrograde perfusion, complete decellularization of the intact rat heart was possible.

  • The intact rat C-ECM supported the in-vitro growth of CMs for up to 2 weeks.

  • HFCs were present across the full thickness of the rat C-ECM.

  • Local alignment of the HFCs actin fibers was observed.

  • HFCs showed evidence of proliferation of 1 week.

  • The results show the potential for a vascularized 3D C-ECM construct as part of an

  • in vitro bioreactor for cell maintenance and proliferation.



    1. Brown, B., et al., The Basement Membrane Component of Biologic Scaffolds Derived from Extracellular Matrix.  Tissue Engineering, 2006.  12(3):  p. 519-526.
    2. Sellato, T., et al., Maintenance of Hepatic Sinusoidal Endothelial Cell Phenotype in Vitro Using Organ-Specific Extracellular Matrix Scaffolds.  Tissue Engineering, 2007.  13(9):  p. 2301-2310.
    3. Brenner, R.M., et al., Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium.  Hum Reprod, 2003.  18(6):  p. 1185-93.



Partially supported by the NIH-NHLBI training grant (T32-HL76124) entitled
“Cardiovascular Bioengineering Training Program.”


Updated 16-May-2011