Combination of melt-electrospun poly-ε-caprolactone scaffolds and hepatocyte-like cells from footprint-free hiPSCs to create 3D biohybrid constructs for liver tissue engineering.

The liver is a vital organ with numerous functions, including metabolic functions, detoxification, and the synthesis of secretory proteins. The increasing prevalence of liver diseases requires the development of effective treatments, models, and regenerative approaches. The field of liver tissue engineering represents a significant advance in overcoming these challenges. In this study, 3D biohybrid constructs were created by combining hepatocyte-like cells (HLCs) derived from patient-specific footprint-free human induced pluripotent stem cells (hiPSCs) and 3D melt-electrospun poly-ε-caprolactone (PCL) scaffolds. First, a differentiation procedure was established to obtain autologous HCLs from hiPSCs reprogrammed from renal epithelial cells using self-replicating mRNA. The obtained cells expressed hepatocyte-specific markers and exhibited important hepatocyte functions, such as albumin synthesis, cytochrome P450 activity, glycogen storage, and indocyanine green metabolism. Biocompatible PCL scaffolds were fabricated by melt-electrospinning and seeded with pre-differentiated hepatoblasts, which uniformly attached to the fibers of the scaffolds and successfully matured into HLCs. The use of patient-specific, footprint-free hiPSC-derived HLCs represents a promising cell source for personalized liver regeneration strategies. In combination with biocompatible 3D scaffolds, this innovative approach has a broader range of applications spanning liver tissue engineering, drug testing and discovery, and disease modeling.

Standardized in vitro bleeding tests in a non-coated novel hybrid prosthesis for frozen elephant trunk demonstrates minimal oozing during full heparinization, supported by clinical data.

Introduction: Recent reports have questioned the blood impermeability of the novel frozen elephant trunk (FET) device E-vita Open NEO© (EO-NEO). Therefore, standardized in vitro bleeding tests using porcine heparinized blood were performed, as well as stress testing on the blood tightness of the collar suture line, to investigate this observation. Material and methods: EO-NEO prostheses were examined in vitro for blood permeability in three test series. Initially, antegrade perfusion with heparinized porcine blood [activated clotting time (ACT) of 500 s, with a 60 min duration] was performed, followed by ante/retrograde testing via the EO-NEO side port. Testing of the collar suture line under a tension of 10 Newton (N) within a suspension device (blood pressure 120 mmHg, ACT of 560 s, 1 min duration) was carried out with the suture material force fiber white (FFWs) yarn, using standard fixation (5 stitches/cm), FFWh yarn in hemostatic fixation (15 stitches/cm), and flow weave yarn (FWYh). Results: Blood permeability testing of EO-NEO through the prosthetic lumen or via the side port demonstrated minor leakage without statistical difference between the standard and hemostatic suture lines or suture materials used, or positioning on the crimped or tapered portion (p > 0.05). The specific collar anastomosis testing demonstrated leakage volumes of 140 ml/min for FFWs vs. 16 ml/min for FFWh (p = 0.02), vs. 9 ml/min with the FWYh (p = 0.01). Conclusion: Different blood leakage tests showed minimal oozing and no difference in blood loss through the fabric and different collar suture lines, but unphysiological pressurized retrograde perfusion of the collar region showed significantly less leakage using FWYh and FFWh, prompting production modification of EO-NEO. Clinical results confirmed low blood loss using this novel FET device.

Effects of Different Container Types on (1→3)-ß-D-glucan Recovery.

It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1→3)-ß-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1→3)-ß-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1→3)-ß-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1→3)-ß-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1→3)-ß-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1→3)-ß-D-glucans.

Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA.

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 µg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 µg of native TE mRNA with a me1 Ψ modification or 10 and 30 µg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 µg of a codon-optimized TE mRNA variant with the me1 Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 µg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

Development of an In Vitro Blood Vessel Model Using Autologous Endothelial Cells Generated from Footprint-Free hiPSCs to Analyze Interactions of the Endothelium with Blood Cell Components and Vascular Implants.

Cardiovascular diseases are the leading cause of death globally. Vascular implants, such as stents, are required to treat arterial stenosis or dilatation. The development of innovative stent materials and coatings, as well as novel preclinical testing strategies, is needed to improve the bio- and hemocompatibility of current stents. In this study, a blood vessel-like polydimethylsiloxane (PDMS) model was established to analyze the interaction of an endothelium with vascular implants, as well as blood-derived cells, in vitro. Using footprint-free human induced pluripotent stem cells (hiPSCs) and subsequent differentiation, functional endothelial cells (ECs) expressing specific markers were generated and used to endothelialize an artificial PDMS lumen. The established model was used to demonstrate the interaction of the created endothelium with blood-derived immune cells, which also allowed for real-time imaging. In addition, a stent was inserted into the endothelialized lumen to analyze the surface endothelialization of stents. In the future, this blood vessel-like model could serve as an in vitro platform to test the influence of vascular implants and coatings on endothelialization and to analyze the interaction of the endothelium with blood cell components.

Improving Bioactive Characteristics of Small Diameter Polytetrafluoroethylene Stent Grafts by Electrospinning: A Comparative Hemocompatibility Study.

Polytetrafluoroethylene (PTFE) is a commonly used biomaterial for the manufacturing of vascular grafts and several strategies, such as coatings, have been explored to improve the hemocompatibility of small-diameter prostheses. In this study, the hemocompatibility properties of novel stent grafts covered with electrospun PTFE (LimFlow Gen-1 and LimFlow Gen-2) were compared with uncoated and heparin-coated PTFE grafts (Gore Viabahn®) using fresh human blood in a Chandler closed-loop system. After 60 min of incubation, the blood samples were examined hematologically and activation of coagulation, platelets, and the complement system were analyzed. In addition, the adsorbed fibrinogen on the stent grafts was measured and the thrombogenicity was assessed by SEM. Significantly lower adsorption of fibrinogen was measured on the surface of heparin-coated Viabahn than on the surface of the uncoated Viabahn. Furthermore, LimFlow Gen-1 stent grafts showed lower fibrinogen adsorption than the uncoated Viabahn®, and the LimFlow Gen-2 stent grafts showed comparable fibrinogen adsorption as the heparin-coated Viabahn®. SEM analysis revealed no sign of thrombus formation on any of the stent surfaces. LimFlow Gen-2 stent grafts covered with electrospun PTFE exhibited bioactive characteristics and revealed improved hemocompatibility in terms of reduced adhesion of fibrinogen, activation of platelets, and coagulation (assessed by ß-TG and TAT levels) similar to heparin-coated ePTFE prostheses. Thus, this study demonstrated improved hemocompatibility of electrospun PTFE. The next step is to conduct in vivo studies to confirm whether electrospinning-induced changes to the PTFE surface can reduce the risk of thrombus formation and provide clinical benefits.

Improving hemocompatibility of artificial lungs by click conjugation of glycoengineered endothelial cells onto blood-contacting surfaces.

Artificial lungs, also known as oxygenators, allow adequate oxygenation of the blood in patients with severe respiratory failure and enable patient survival. However, the insufficient hemocompatibility of the current of artificial lungs hampers their long-term use. Therefore, in this study, a novel strategy was developed to efficiently endothelialize blood-contacting surfaces to improve their hemocompatibility. Hollow fiber membranes (HFMs) were functionalized with dibenzylcyclooctyne (DBCO), and endothelial cells were glycoengineered for covalent conjugation to DBCO by a copper-free click reaction. Metabolic glycoengineering using azidoacetylmannosamine-tetraacylated (Ac4ManNAz) resulted in highly efficient functionalization of endothelial cells with azide (N3) molecules on the cell surface without negative impact on cell viability. After 48 h, significantly improved endothelialization was detected on the HFM surfaces functionalized with DBCO compared to unmodified HFMs. Endothelial cells were responsive to inflammatory stimulus and expressed adhesion-promoting molecules (E-selectin, VCAM-1, and ICAM-1). Furthermore, the hemocompatibility of HFMs was analyzed by dynamic incubation with fresh human blood. DBCO-coated and uncoated HFMs showed a comparable hemocompatibility, but the endothelialization of HFMs significantly reduced the activation of blood coagulation and platelets. Interestingly, the incubation of endothelialized HFMs with human blood further reduced the expression of E-selectin and VCAM-1 in endothelial cells. In this study, a highly efficient, cell-compatible method for endothelialization of artificial lungs was established. This click chemistry-based method can be also applied for the endothelialization of other artificial surfaces for tissue engineering and regenerative medicine applications.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

Profiling of time-dependent human plasma protein adsorption on non-coated and heparin-coated oxygenator membranes.

Patients with severe lung diseases are highly dependent on lung support systems. Despite many improvements, long-term use is not possible, mainly because of the strong body defence reactions (e.g. coagulation, complement system, inflammation and cell activation). The systematic characterization of adsorbed proteins on the gas exchange membrane of the lung system over time can provide insights into the course of various defence reactions and identify possible targets for surface modifications. Using comprehensive mass spectrometry analyses of desorbed proteins, we were able to identify for the first time binding profiles of over 500 proteins over a period of six hours on non-coated and heparin-coated PMP hollow fiber membranes. We observed a higher degree of remodeling of the protein layer on the non-coated membrane than on the coated membrane. In general, there was a higher protein binding on the coated membrane with exception of proteins with a heparin-binding site. Focusing on the most important pathways showed that almost all coagulation factors bound in higher amounts to the non-coated membranes. Furthermore, we could show that the initiator proteins of the complement system bound stronger to the heparinized membranes, but the subsequently activated proteins bound stronger to the non-coated membranes, thus complement activation on heparinized surfaces is mainly due to the alternative complement pathway. Our results provide a comprehensive insight into plasma protein adsorption on oxygenator membranes over time and point to new ways to better understand the processes on the membranes and to develop new specific surface modifications.

Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium.

Endothelial progenitor cells (EPCs) are one of the most important stem cells for the neovascularization of tissues damaged by ischemic diseases such as myocardial infarction, ischemic stroke, or critical limb ischemia. However, their low homing efficiency in the treatment of ischemic tissues limits their potential clinical applications. The use of synthetic messenger RNA (mRNA) for cell engineering represents a novel and promising technology for the modulation of cell behavior and tissue regeneration. To improve the therapeutic potential of EPCs, in this study, murine EPCs were engineered with synthetic mRNAs encoding C-X-C chemokine receptor 4 (CXCR4) and P-selectin glycoprotein ligand 1 (PSGL-1) to increase the homing and migration efficiency of EPCs to inflamed endothelium. Flow cytometric measurements revealed that the transfection of EPCs with CXCR4 and PSGL-1 mRNA resulted in increased expressions of CXCR4 and PSGL-1 on the cell surface compared with the unmodified EPCs. The transfection of EPCs with mRNAs did not affect cell viability. CXCR4-mRNA-modified EPCs showed significantly higher migration potential than unmodified cells in a chemotactic migration assay. The binding strength of the EPCs to inflamed endothelium was determined with single-cell atomic force microscopy (AFM). This showed that the mRNA-modified EPCs required a three-fold higher detachment force to be released from the TNF-α-activated endothelium than unmodified EPCs. Furthermore, in a dynamic flow model, significantly increased binding of the mRNA-modified EPCs to inflamed endothelium was detected. This study showed that the engineering of EPCs with homing factors encoding synthetic mRNAs increases the homing and migration potentials of these stem cells to inflamed endothelium. Thus, this strategy represents a promising strategy to increase the therapeutic potential of EPCs for the treatment of ischemic tissues.

Delivery of synthetic mRNAs for tissue regeneration.

In recent years, nucleic acid-based therapeutics have gained increasing importance as novel treatment options for disease prevention and treatment. Synthetic messenger RNAs (mRNAs) are promising nucleic acid-based drugs to transiently express desired proteins that are missing or defective. Recently, synthetic mRNA-based vaccines encoding viral proteins have been approved for emergency use against COVID-19. Various types of vehicles, such as lipid nanoparticles (LNPs) and liposomes, are being investigated to enable the efficient uptake of mRNA molecules into desired cells. In addition, the introduction of novel chemical modifications into mRNAs increased the stability, enabled the modulation of nucleic acid-based drugs, and increased the efficiency of mRNA-based therapeutic approaches. In this review, novel and innovative strategies for the delivery of synthetic mRNA-based therapeutics for tissue regeneration are discussed. Moreover, with this review, we aim to highlight the versatility of synthetic mRNA molecules for various applications in the field of regenerative medicine and also discuss translational challenges and required improvements for mRNA-based drugs.

Influence of Human Jaw Periosteal Cells Seeded ß-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.

Synthetic Material Abdominal Swabs Reduce Activation of Platelets and Leukocytes Compared to Cotton Materials.

During surgical procedures, cotton abdominal swabs with their high absorptive capacity and malleability are used to retain organs and absorb blood or other body fluids. Such properties of the natural material cotton are advantageous for most operations, but in cardiopulmonary bypass (CPB) surgery, a high blood volume can accumulate in the thoracic cavity that is quickly retransfused via the heart-lung machine (HLM). This common practice is supposed to be safe due to the high anticoagulation. However, in vitro analyses showed that blood cells and plasma proteins were activated despite a high anticoagulation, which can propagate especially an inflammatory response in the patient. Thus, we investigated patients' blood during CPB surgery for inflammatory and coagulation-associated activation after contact to the HLM and either cotton or synthetic abdominal swabs. Contact with cotton significantly increased thrombocyte and neutrophil activation measured as ß-thromboglobulin and PMN-elastase secretion, respectively, compared to synthetic abdominal swabs. Both inflammatory cytokines, interleukin (IL) 1ß and IL6, were also significantly increased in the cotton over the synthetic patient group, while SDF-1α was significantly lower in the synthetic group. Our data show for the first time that cotton materials can activate platelets and leukocytes despite a high anticoagulation and that this activation is lower with synthetic materials. This additional activation due to the material on top of the activation exerted by the tissue contact that blood is exposed to during CPB surgery can propagate further reactions in patients after surgery, which poses a risk for this already vulnerable patient group.

Current Strategies for the Regeneration of Skeletal Muscle Tissue.

Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.

Selection of aptamers against triple negative breast cancer cells using high throughput sequencing.

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with [Formula: see text] values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

Tissue Adhesives: From Research to Clinical Translation.

Sutures, staples, clips and skin closure strips are used as the gold standard to close wounds after an injury. In spite of being the present standard of care, the utilization of these conventional methods is precarious amid complicated and sensitive surgeries such as vascular anastomosis, ocular surgeries, nerve repair, or due to the high-risk components included. Tissue adhesives function as an interface to connect the surfaces of wound edges and prevent them from separation. They are fluid or semi-fluid mixtures that can be easily used to seal any wound of any morphology - uniform or irregular. As such, they provide alternatives to new and novel platforms for wound closure methods. In this review, we offer a background on the improvement of distinctive tissue adhesives focusing on the chemistry of some of these products that have been a commercial success from the clinical application perspective. This review is aimed to provide a guide toward innovation of tissue bioadhesive materials and their associated biomedical applications.

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs.

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient's somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells' pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

Hemocompatibility of new magnetically-levitated centrifugal pump technology compared to the CentriMag adult pump.

The specific hemocompatibility properties of mechanical-circulatory-support (MCS)-pump technologies have not previously been described in a comparable manner. We thus investigated the hemocompatibility-indicating marker of a new magnetically-levitated (MagLev) centrifugal pump (MT-Mag) in a human, whole-blood mock-loop for 360 min using the MCS devices as a driving component. We compared those results with the CentriMag adult (C-Mag) device under the same conditions according to ISO10993-4. Blood samples were analyzed via enzyme-linked-immunosorbent-assay (ELISA) for markers of coagulation, complement system, and the inflammatory response. The time-dependent activation of the coagulation system was measured by detecting thrombin-anti-thrombin complexes (TAT). The activation of the complement system was determined by increased SC5b-9 levels in both groups. A significant activation of neutrophils (PMN-elastase) was detected within the C-Mag group, but not in the MT-Mag group. However, the amount of PMN-elastase at 360 min did not differ significantly between groups. The activation of the complement and coagulation system was found to be significantly time-dependent in both devices. However, coagulation activation as determined by the TAT level was lower in the MT-Mag group than in the C-Mag group. This slight disparity could have been achieved by the optimized secondary flow paths and surface coating, which reduces the interaction of the surface with blood.

Hydrojet-based delivery of footprint-free iPSC-derived cardiomyocytes into porcine myocardium.

The reprogramming of patient´s somatic cells into induced pluripotent stem cells (iPSCs) and the consecutive differentiation into cardiomyocytes enables new options for the treatment of infarcted myocardium. In this study, the applicability of a hydrojet-based method to deliver footprint-free iPSC-derived cardiomyocytes into the myocardium was analyzed. A new hydrojet system enabling a rapid and accurate change between high tissue penetration pressures and low cell injection pressures was developed. Iron oxide-coated microparticles were ex vivo injected into porcine hearts to establish the application parameters and the distribution was analyzed using magnetic resonance imaging. The influence of different hydrojet pressure settings on the viability of cardiomyocytes was analyzed. Subsequently, cardiomyocytes were delivered into the porcine myocardium and analyzed by an in vivo imaging system. The delivery of microparticles or cardiomyocytes into porcine myocardium resulted in a widespread three-dimensional distribution. In vitro, 7 days post-injection, only cardiomyocytes applied with a hydrojet pressure setting of E20 (79.57 ± 1.44%) showed a significantly reduced cell viability in comparison to the cells applied with 27G needle (98.35 ± 5.15%). Furthermore, significantly less undesired distribution of the cells via blood vessels was detected compared to 27G needle injection. This study demonstrated the applicability of the hydrojet-based method for the intramyocardial delivery of iPSC-derived cardiomyocytes. The efficient delivery of cardiomyocytes into infarcted myocardium could significantly improve the regeneration.