Poor Cell Retention Research Articles

Challenges in scaling up a perfusion process

Development and scale-up of a perfusion process has challenges due to the complex nature of the process and unavailability of direct scale-up of the perfusion equipment. The initial scale-up to production scale resulted in poor cell growth profile. Upon investigation, the reason for low cell concentration was attributed to poor cell retention by the perfusion device. Changes were introduced in the type of mesh used for filter construction and perfusion pumps to improve retention. These modifications helped in better cell culture profiles and yields. However, the product quality was impacted because of these changes. Further changes in the perfusion flow rates were done to address the product quality differences.

Nitrogen‐Rich Plasma‐Polymerized Coatings on PET and PTFE Surfaces Improve Endothelial Cell Attachment and Resistance to Shear Flow

Low seeding efficiency and poor cell retention under flow-induced shear stress limit the effectiveness of in vitro endothelialization strategies for small-diameter vascular grafts. Primary-amine-rich plasma-polymerized coatings (PPE:N) deposited using low- and atmospheric-pressure plasma discharges on PET and PTFE are evaluated for their ability to improve endothelial cells' kinetics and strength of attachment. PPE:N coatings increase cell adhesion and adhesion rate, spreading, focal adhesion, and resistance to flow-induced shear compared with bare and gelatin-coated PET and PTFE. In particular, about 90% of the cells remain on coated surfaces after 1 h exposure to shear. These coatings, therefore, appear as a promising versatile approach to improve cell seeding strategies for vascular grafts.

Comparative analysis of adherence, viability, proliferation and morphology of umbilical cord tissue-derived mesenchymal stem cells seeded on different titanium-coated expanded polytetrafluoroethylene scaffolds

Umbilical cord tissue comprises an attractive new source for mesenchymal stem cells. Umbilical cord tissue-derived mesenchymal stem cells (UCMSC) exhibit self-renewal, multipotency and immunological naivity, and they can be obtained without medical intervention. The transfer of UCMSC to the ischemic region of the heart may have a favorable impact on tissue regeneration. Benefit from typical cell delivery by injection to the infarcted area is often limited due to poor cell retention and survival. Another route of administration is to use populated scaffolds implanted into the infarcted zone. In this paper, the seeding efficiency of UCMSC on uncoated and titanium-coated expanded polytetrafluoroethylene (ePTFE) scaffolds with different surface structures was determined. Dualmesh® (DM) offers a corduroy-like surface in contrast to the comparatively planar surface of cardiovascular patch (CVP). The investigation of adherence, viability and proliferation of UCMSC demonstrates that titanium-coated scaffolds are superior to uncoated scaffolds, independent of the surface structure. Microscopic images reveal spherical UCMSC seeded on uncoated scaffolds. In contrast, UCMSC on titanium-coated scaffolds display their characteristic spindle-shaped morphology and a homogeneous coverage of CVP. In summary, titanium coating of clinically approved CVP enhances the retention of UCMSC and thus offers a potential cell delivery system for the repair of the damaged myocardium.

Hydrogels as a Platform for Stem Cell Delivery to the Heart

Stem cell therapy offers great promise to repair the injured or failing heart. The outcomes of clinical trials to date, however, have shown that the actual benefit realized falls far short of the promise. A number of factors may explain why that is the case, but poor stem cell retention and engraftment in the hostile environment of the injured heart would seem to be a major factor. Improving stem cell retention and longevity once delivered would seem a logical means to enhance their reparative function. One way to accomplish this goal may be injectable hydrogels, which would serve to fix stem cells in place while providing a sheltering environment. Hydrogels also provide a means to allow for the paracrine factors produced by encapsulated stem cells to diffuse into the injured myocardium. Alternatively, hydrogels themselves can be used for the sustained delivery of reparative factors. Here the authors discuss chitosan-based hydrogels.

Cardiac progenitor cell sheet regenerates myocardium and renews hope for translation

This editorial refers to ‘Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function’ by L. Zakharova et al ., pp. 40–49, this issue. The goal of myocardial regeneration strategies is to transform dead scar tissue into viable functional muscle. If achieved, we could eradicate congestive heart failure. Early work using cell suspensions with delivery by injection showed promise, but substantial regeneration has remained elusive for our patients, mostly due to poor viability and donor cell retention after transplantation. The enthusiasm for this strategy is fading. The innovative work of Zakharova et al .1 could restore hope and help guide us in the right direction. The authors assembled cardiac progenitor cells into a tissue-like sheet of cells and then delivered the construct onto the epicardial surface of an acute rat infarction. This unique approach enhanced stem cell engraftment and survival, stimulated a substantial cardiogenesis, and preserved left ventricular function to an extent that could prevent the transition to overt heart failure. Importantly, the donor cells did more than survive in the host myocardium; after engraftment, they showed an encouraging combination of controlled proliferation, purposeful migration, and targeted differentiation. The authors then extended, confirmed, and reproduced these results using human progenitor cells obtained from surgical biopsy. The potential for translation to our patients using this strategy is exciting. Progress in using cell …

Mesothelial Cells vs. Skeletal Myoblasts for Myocardial Infarction

Cell transplantation for the regeneration of ischemic myocardium is limited by poor graft viability and low cell retention. Omental flaps in association with growth factors and cell sheets have recently been used to increase the vascularization of ischemic hearts. This experimental study was undertaken to evaluate the hemodynamic evolution and histological modifications of infarcted myocardium treated with mesothelial cells, and to compare the results with those of hearts treated with skeletal myoblasts. Myocardial infarction was created by surgical ligature of 2 coronary branches in 34 sheep; 6 died immediately due to ventricular fibrillation. Mesothelial cells were isolated from greater omentum, and myoblasts from skeletal muscle. After expanding the cells for 3 weeks, infarcted areas were treated with culture medium (control group), mesothelial cells, or myoblasts. After 3 months, echocardiographic studies showed significant limitation of ventricular dilatation and improved ejection fractions in both cell-treated groups compared to the controls. In the mesothelial cell group, histological studies showed significantly more angiogenesis and arteriogenesis than in the control and skeletal myoblast groups. Mesothelial cells might be useful for biological revascularization in patients with ischemic heart disease.

Labeling human embryonic stem cell-derived cardiomyocytes with indocyanine green for noninvasive tracking with optical imaging: an FDA-compatible alternative to firefly luciferase.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37 degrees C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data (p < 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Labeling Human Embryonic Stem Cell-Derived Cardiomyocytes with Indocyanine Green for Noninvasive Tracking with Optical Imaging: An FDA-Compatible Alternative to Firefly Luciferase

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37°C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data ( p &lt; 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Abstract 5372: In Vivo Myocardial Distribution of Adult Stem Cells Immediately Following Intracoronary Delivery in a Swine Model of Myocardial Infarction

Background: Cardiac cell-based therapy (CCT) has emerged as a promising therapeutic strategy. There are few data comparing the fate of different stem cell (SC) populations delivered by the intracoronary injection (ICI). Aim: Document the in vivo myocardial distribution of SC delivered by ICI following recent myocardial infarction. Methods: In a myocardial infarction swine model, near-infrared (NIR) fluorescence was used to provide high sensitivity in vivo tracking of SC in the immediate phase (&lt; 60 minutes) after ICI of 2x107 cells. After labeling with the NIR fluorophore, IR-786, the NIR signal intensities and myocardial distribution patterns of mesenchymal (MSC), bone marrow-(BMMNC) and peripheral blood-mononuclear (PBMNC) cells were compared using an open-chested fluorescence imaging system. Results: The SC populations of interest were successfully loaded with IR-786. While all SC populations readily distributed along the vascular territory of the infarct-related artery immediately after injection, there was a mean SI intensity drop of 29.8% and 14.1% for PBMNC and BMNC (p&lt;0.05), respectively, which correlated with poor cell retention. No significant SI change was found in MSC-injected swine, which was associated with less cell loss after delivery. There was also evidence of MSC-related vessel plugging in some swine (Figure ). Conclusion: Our in vivo findings suggest that immediately after injection (during the first 60min ICI), there is already evidence of poor SC retention and distribution vary depending on cell population, potentially impacting clinical efficacy and safety of CCT.

Abstract 1861: Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium (MAGNUM Clinical Trial): One Year Outcome

Objectives Cell transplantation for myocardial regeneration is limited by poor graft viability and low cell retention. In addition, in ischemic cardiomyopathy the extracellular matrix is deeply altered. Therefore it could be important to associate a procedure aiming at regenerating myocardial cells and restoring the extracellular matrix function. We evaluated intrainfarct stem cell therapy associated with a cell-seeded collagen scaffold grafted onto infarcted ventricles. Methods In 15 patients (aged 54.2±3.8 years) presenting LV postischemic myocardial scars (age of the infarcts 8.2±3.5 months) and with indication for a single OP-CABG, bone marrow was collected by aspiration from the iliac crest. The cell suspension was loaded on Ficoll-Paque density gradient and 300 ± 28 million mononuclear bone marrow cells (BMC) were implanted during surgery in the scar. A type I collagen matrix seeded with the same number of BMC (300 ± 28 million cells) was added on top of the scarred area and fixed onto the epicardium by 6 single PDS sutures and covered by a second non-cellularized matrix. Results There was no mortality and any related adverse events (follow-up 15±4.2 months), no malignant cardiac arrhythmias were reported, no patient was lost to follow-up. NYHA FC improved from 2.3±0.5 to 1.4±0.3 (p=0.005). LV end-diastolic volume evolved from 142±24 to 117±21mL (p=0.03), LV filling deceleration time improved from 162±7ms to 196±8ms (p=0.01). Scar area thickness progress from 6±1.4 to 9±1.5mm (p=0.005). EF improved from 25±7 to 33±5% (p=0.04). Conclusions Simultaneous intramyocardial injection of BMC and fixation of a cell seeded matrix onto the epicardium is feasible and safe. The cell seeded collagen matrix seems to increase the thickness of the infarct scar with viable tissues and help to normalize cardiac wall stress in injured regions, thus limiting ventricular remodelling and improving diastolic function. Functional improvements can not be conclusively related to the cells and matrix due to the association of CABG. Cardiac tissue engineering seems to be a promising way for the creation of “bioartificial myocardium”. Efficacy and safety of this approach should be evaluated in a large randomized controlled trial.

Adding Endothelium to Artificial Vascular Grafts.

The endothelium of healthy blood vessels serves anticoagulant, antiproliferative, and vasodilator functions. Prosthetic vascular grafts, widely used for reconstruction of large vessels and to provide access to blood vessels for hemodialysis, lack endothelial cells and often fail because unopposed