Graduate Student University of Washington, Seattle Seattle, Washington, United States
Introduction:: Coronary artery disease, the most prevalent form of heart disease, can be characterized by occluded blood vessels which supply oxygen and nutrients to the heart. This blockage commonly causes downstream cardiac ischemic death, referred to as a myocardial infarction (MI), or heart attack. Cardiac stem cell therapy is a clinically emerging treatment strategy which offers substantial promise toward the goal of heart regeneration post-MI. Several studies conducted in large animal models have shown restoration of heart function using stem cell-derived cardiomyocytes (hPSC-CM) delivered via a standard hypodermic needle. However, one setback is that there are major losses of injected hPSC-CMs due to poor retention and low control of cell distribution in the infarcted region. To address this, we have designed a novel microneedle array device (MNA) to be used for cardiac stem cell therapy. Our approach aims to increase cell retention and improve control of cell distribution while streamlining the technique to be rapid and consistent. Testing the MNA in naïve rat heart models yielded promising preliminary retention data both acutely and after a 2-week engraftment period. Additionally, confocal imaging of injected ex vivo pig hearts revealed the high controllability of retained cell islets. Our results support the therapeutic feasibility of the MNA and demonstrate the importance of considering delivery methods in investigating new approaches for stem cell injections. Ultimately, we hope that this research will result in overall improved clinical outcomes for millions of patients suffering from MI.
Materials and Methods:: To quantify retention, In vivo work to date has been conducted in naïve rat hearts in both acute and 2-week engraftment studies. The current MNA design features 4 needles spaced 2 mm apart in a square pattern in order to meet the size requirements for rat hearts while remaining able to puncture the myocardium. In acute injection studies, 1 x 107 C2C12s (skeletal myoblasts) were injected into the hearts of Sprague-Dawley rats using either the MNA or a hypodermic needle, which is the standard method, and then excised after 5 minutes. Hearts were then fixed and sectioned for immunohistochemistry. For the 2-week engraftment studies, lactate-purified hPSC-CMs were injected into the hearts of athymic rats with the MNA or hypodermic needle and excised after 2 weeks. Harvested tissue in both studies were stained for human slow skeletal troponin I to identify C2C12s and hPSC-CMs and measure total graft area as a proxy for retention. In an additional study, CellTracker-dyed C2C12 cells were injected into ex vivo porcine heart sections using the MNA and then processed using a tissue clearing protocol. Volumetric renderings were then generated using confocal microscopy at low z-stacking intervals, which were analyzed for total cell volume and distribution.
Results, Conclusions, and Discussions:: The results of these preliminary studies have shown promise for establishing the MNA approach. Acute injections with the MNA yielded similar, albeit lower retention of cells by percent area of heart section when compared to the hypodermic needles. For the 2-week engraftment studies, the MNA had a cell engraftment area that was nearly half that of the hypodermic needles. We attribute this underperformance to the short length of the needles in the MNA. To this end, we have iterated upon our design by vastly increasing the sharpness and length of the needles (from 800 µm to 2 mm).
Ex vivo injections into porcine heart sections displayed an even distribution of cell islets. We observed that smaller needle length resulted in immediate backflow of product out of the injection site, supporting the hypothesis that longer needles indeed improve retention. Additionally, volumetric estimations showed that higher amounts of cells remained in the tissue after being injected with these longer devices. Confocal imaging of these tissues revealed very distinct, punctate cell islets at each needle’s injection site, which supports that cell distributions can be controlled using the MNA approach. The significance of this finding is in the implications of improved targeting of therapeutic delivery to infarcted zones of the heart.
This project will provide key insights into how delivery methods impact the efficacy of cardiac stem cell therapy. Within the field of regenerative medicine, delivery methods are seldom considered, and far less often improved upon, so this research will be pivotal not only for the cardiac niche, but for other targets of stem cell therapies such as the liver and lung. The results posed here will act as a foundation for future studies aiming to optimize stem cell delivery as a whole, and by extension, clinical outcomes in patients.
